CA1103153A - Vinyl polymerisate latex particles bonded to immunological material - Google Patents
Vinyl polymerisate latex particles bonded to immunological materialInfo
- Publication number
- CA1103153A CA1103153A CA307,224A CA307224A CA1103153A CA 1103153 A CA1103153 A CA 1103153A CA 307224 A CA307224 A CA 307224A CA 1103153 A CA1103153 A CA 1103153A
- Authority
- CA
- Canada
- Prior art keywords
- vinyl
- latex
- immunologically active
- inclusive
- particles
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/823—Immunogenic carrier or carrier per se
Abstract
ABSTRACT
This invention relates to a water-insoluble reagent for an immunologlcal determination having a specific weight corresponding to about that of water in tbe form of discrete latex particles, to which is bound an immuno-logically active material, wherein the latex consists of a dispersion of particles of vinyl polynerisates, which carry the end-groups groups of the formula whereby the particles are formed from a core of vinyl and/or diene polymerisate, which carries carboxyl and/or sulphonate functions, and of an outer layer of vinyl polymerisate, which carries as the end-groups groups of the formula
This invention relates to a water-insoluble reagent for an immunologlcal determination having a specific weight corresponding to about that of water in tbe form of discrete latex particles, to which is bound an immuno-logically active material, wherein the latex consists of a dispersion of particles of vinyl polynerisates, which carry the end-groups groups of the formula whereby the particles are formed from a core of vinyl and/or diene polymerisate, which carries carboxyl and/or sulphonate functions, and of an outer layer of vinyl polymerisate, which carries as the end-groups groups of the formula
Description
3~S3 The present invention relates to immunological diag-nostic reagents. More particularly, the invention is con-cerned with immunological diagnostic reagents, a process for their manufacture and diagnostic methods in which said reagents are used.
The diagnosis of pathological or other conditions in human beings and animals is often carried out using immuno-logical principles. These principles are used for the detection of antibodies or antigens in the body fluids of the living beings. An antigen is a foreign substance which, when it is applied to the living being, brings about the formation of certain substances which are soluble and known as the antibody. Any substance such as, for example, a protein, which is normally not present in a specific living being, can cause the formation of anti-bodies when it is applied to the living being under suitable conditions.
After their formation, the antibodies react with the antigens and in this way protect against infections in the case of a bacteria or virus foreign body.
Immunological test processes depend on the antigen~
-antibody reaction, which usually manifests itself by insolubility or agglutination.
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The diagnosis of pathological or other conditions in human beings and animals is often carried out using immuno-logical principles. These principles are used for the detection of antibodies or antigens in the body fluids of the living beings. An antigen is a foreign substance which, when it is applied to the living being, brings about the formation of certain substances which are soluble and known as the antibody. Any substance such as, for example, a protein, which is normally not present in a specific living being, can cause the formation of anti-bodies when it is applied to the living being under suitable conditions.
After their formation, the antibodies react with the antigens and in this way protect against infections in the case of a bacteria or virus foreign body.
Immunological test processes depend on the antigen~
-antibody reaction, which usually manifests itself by insolubility or agglutination.
Hen/9.6.78 : . : . . .
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- 2 -In general, the presence of an antigen or an antibody is confirmed or determined by adding the corresponding antibody or the corresponding antigen to a body fluid of the living being, mainly urine, blood serum or a specially S treated blood extract. However, other body fluids can also be used. The presence or the absence of the anti-body or the antigen in the body fluid of the living being is ascertained by establishing the occurrence or non--occurrence of an antigen-antibody reaction.
Because some complexes only form very slowly and have very small particle sizes, it is necessary to use carriers in order to make them visible. In a hitherto preferred method the antibody or the antigen was bound by means of a carbodiimide via an amide bond to discrete particles of carboxylated latex polymers such as, for example, car-boxylated copolymers of butadiene and styrene.
This method has, however, the disadvantage that during the coupling of the protein (antibody or antigen) to the latex particles, there occurs as the side-reaction, because of the use of carbodiimides, an undesirable lattice-like polymerisation of the protein used and, accordingly, a part of the often very expensive protein is lost for the coupling with the carrier.
The present invention is concerned with polymeric carriers, with which the above disadvantages can be avoided . .
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~3 and which can form with a wide spectrum of immunologically active materials a diagnostically useful reagent, which is stable, specific and sensitive and makes possible a readily detectable visual estimation in a ver~ short time.
More particularly, the present invention is concerned with a water-insoluble reagent for an immunological determination having a specific weight corresponding to about that of water in the form of discrete late~ particles, to which is bound an immunologically active material, wherein the latex consists of a dispersion of particles of vinyl polymerisates, which carry as the end-groups groups of the formula -S~ .
N~2 , wherein the particles are formed from a core of vinyl and/or diene polymerisate, which carries carboxyl and/or sulphonate functions, and from an outer layer of vinyl polymerisate, which car~ies as the end-groups groups of the formula r --S~
N~2 , and have an average diame~er between 0.03 ~m and 5 ~m.
The water-insoluble reagent is in the form of an aqueous suspension and has a pH in the range of about 5 to 9.
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' Further, the present invention is concerned with a process for the manufacture of such a reagent, which process comprises reacting the latex, after diazotisation or in the presence of suitable bifunctional reagents, with the immunologically active material.
As "immunologically active substances" there can be named all constituents in physiological fluids, cell extracts and tissue extracts for which there is present or can be formed an immunological reaction partner Thereto there belong amines, amino acids, peptides, proteins, lipo-proteins, glycoproteins~ sterols, steroids, lipoids, nucleic acids, enzymes, hormones, vitamins, polysaccharides and alkaloids. Preferred immunologically active substances are compiled in the Table I hereinafter:
Table I
I. Antigens produced by microorganisms . _ ~
Bacteria 1. Gram-positive cocci Streptococci (pyogenes, fecalis and viridans) Staphylococci (aureus and albus) Pneumococci ~D. pneumoniae) 2. Gram-negative cocci Neisseria (gonorrhoeae and meningitidis) :, ' r `' ' '"`
_ 5 _ 1~3~3
Because some complexes only form very slowly and have very small particle sizes, it is necessary to use carriers in order to make them visible. In a hitherto preferred method the antibody or the antigen was bound by means of a carbodiimide via an amide bond to discrete particles of carboxylated latex polymers such as, for example, car-boxylated copolymers of butadiene and styrene.
This method has, however, the disadvantage that during the coupling of the protein (antibody or antigen) to the latex particles, there occurs as the side-reaction, because of the use of carbodiimides, an undesirable lattice-like polymerisation of the protein used and, accordingly, a part of the often very expensive protein is lost for the coupling with the carrier.
The present invention is concerned with polymeric carriers, with which the above disadvantages can be avoided . .
: , .. ~ ; .
.
,. :: "
., . : ~
: , . .
~3 and which can form with a wide spectrum of immunologically active materials a diagnostically useful reagent, which is stable, specific and sensitive and makes possible a readily detectable visual estimation in a ver~ short time.
More particularly, the present invention is concerned with a water-insoluble reagent for an immunological determination having a specific weight corresponding to about that of water in the form of discrete late~ particles, to which is bound an immunologically active material, wherein the latex consists of a dispersion of particles of vinyl polymerisates, which carry as the end-groups groups of the formula -S~ .
N~2 , wherein the particles are formed from a core of vinyl and/or diene polymerisate, which carries carboxyl and/or sulphonate functions, and from an outer layer of vinyl polymerisate, which car~ies as the end-groups groups of the formula r --S~
N~2 , and have an average diame~er between 0.03 ~m and 5 ~m.
The water-insoluble reagent is in the form of an aqueous suspension and has a pH in the range of about 5 to 9.
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., , . . ~ ;
.
- ' ~
' Further, the present invention is concerned with a process for the manufacture of such a reagent, which process comprises reacting the latex, after diazotisation or in the presence of suitable bifunctional reagents, with the immunologically active material.
As "immunologically active substances" there can be named all constituents in physiological fluids, cell extracts and tissue extracts for which there is present or can be formed an immunological reaction partner Thereto there belong amines, amino acids, peptides, proteins, lipo-proteins, glycoproteins~ sterols, steroids, lipoids, nucleic acids, enzymes, hormones, vitamins, polysaccharides and alkaloids. Preferred immunologically active substances are compiled in the Table I hereinafter:
Table I
I. Antigens produced by microorganisms . _ ~
Bacteria 1. Gram-positive cocci Streptococci (pyogenes, fecalis and viridans) Staphylococci (aureus and albus) Pneumococci ~D. pneumoniae) 2. Gram-negative cocci Neisseria (gonorrhoeae and meningitidis) :, ' r `' ' '"`
_ 5 _ 1~3~3
3. Gram-positive, aerobic bacilli Bacillus anthracis Corynebacterium diphtheriae Erysipelothrix Listeria monocytogenes
4. Gram-positive, anaerobic bacilli Clostridia (botulinum, perfringens, welchii and tetani)
5. Gram-negative, anaerobic bacilli Bacteroides
6. Gram-negative, intestinal bacilli Escherichia Klebsiella Enterobacter Proteus Pseudomonas Salmonella Shigella
7. Gram-negative, non-intestinal bacilli Pasteurella (pestis and tularensis) Hemophilus influenzae Brucella (melitensis, abortus and suis) Bordetella pertussis Malleomyces
8. Spirochetae Treponema pallidum :: . . ,~. - ;
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Leptospira Borrelia
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Leptospira Borrelia
9. Mycoplasma
10. Mycobacteria
11. Vibrio
12~ Actinomyces Protozoa 1. Intestinal protozoa Am-obae 2. Flagellates Trichomonas --Leishmania Trypanosomes Toxoplasma (T. Gondii) 3. Sporozoa Plasmodia (vivaxt falciparum, malariae and ovale) Fungi 1. Sporotrichum 2. Cryptococcus 3. Blastomyces 4. Histoplasma 5. Coccidioides - - .
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6. Candida Viruses and Rickettsia l. Rickettsia 2. Viruses Canine hepatitis Shope papilloma Influenza A & B
Fowl pest Herpes simplex Adenoviruses Polyoma Rous sarcoma Vaccinia Polio virus German measles Canine distemper Leukaemia Mumps Newcastle disease (domestic fowl disease) Sendai Foot and mouth disease Psittacosis Rabies Ectromelia Arborviren ., .-.
~3~;i3 II. Foreiqn antigens Polysaccharides Hyaluronidases Tetanus toxin Egg ovalbumin Sheep serum albumin Human plasma gammaglobulin Human serum albumin III. Natural antigens 1. Hormones Insulin Glucagon Thyroid hormone Choriongonadotropin Chorion growth hormone - prolactin 2. Enzymes Pancreas chymotrypsinogens Procarboxypeptidases Deoxyribonucleases Ribonucleases Catalases Creatin phosphokinases 3. Organ-specific antigens Kidney Liver Skin Heart (myoglobin) ., .
9 ~ 3~53 Gastrointestinal tract Prostate Embryo antigens (e.g. CEA antigen) Tumour antigens 4. Connective tissue components Muscle Collagen Amyloid 5. Blood cell antigens, blood group substances and _ other isoantigens Platelets Megacaryocytes Leucocytes Erythrocytes Blood group substances Forssman antigen Histo-compatible antigens 6. Plasma proteins Fibrin and fibrinoid Plasminogen and plasmin 7. Pathological globulins Myeloma, macroglobulinaemic and dysglobulinaemic proteins Rheumatoid factor C-reactive proteins ,:.. . - - : ~ -- . ~, . ... : .
, :. . , : .-' . - " .
~ 3~3 IV. Natural antibodies 1. Natural gammaglobulins Natural antibodies - nephrotoxic antibody complement 2. Auto-antibodies Antinuclear factor Thyroid autoantibodies Adrenal autoantibodies Autoantibodies for gastric-parietal cells in the case of pernicious anaemia Autoantibodies for spermatozoa Muscle autoantibodies in the case of Myasthenia gravis Autoantibodies for nerve tissue Autoantibodies against fibre-like tissue and vascular components Autoantibodies against platelets and megacaryocytes Antibodies against trophoblasts 3. Induced antibodies against:
Immunoglobulin classes IgG, IgA, IgM or variant species V. Haptenic compounds Opium alkaloids (morphine) Antipyrine Barbituric acid ., ,~
':
3~3 Especially preferred immunologically active substances in the scope of the present invention are albumin, rheu-matoid factor, human immunoglobulin IgG and antibodies against IgG.
Under vinyl polymerisates, which form the core of the particles, there are to be understood homopolymerisates of monomers such as styrene and its derivatives: methyl-styrenes, ethylstyrenes, vinyltoluene; vinyl chloride, vinylidene chloride; vinyl acetate; acryl derivatives such as alkyl acrylates and alkyl methacrylates (the alkyl groups containing 1 to 10 carbon atoms), which are option-ally hydroxylated, such as 2-hydroxyethyl acrylate, 2--hydroxyethyl methacrylate, 2-hydroxypropyl acrylate and 2-hydroxypropyl methacrylate; and acrylonitrile and meth-acrylonitrile; as well as copolymerisates of these mono-mers with one another andtor with modified vinyl comonomers such as divinylbenzene, acrylamide and methacrylamide and their N-substituted derivatives such as, for example, methylolacrylamide; these comonomers may represent up to 5 wt.~ of the copolymerisate.
Under diene polymerisates, which form the core, there are to be understood homopolymerisates of butadiene and its derivatives: chloroprene, isoprene; as well as copoly-merisates of these monomers with one another and/or with vinyl monomers, such as those mentioned earlier, in all ratios and/or with modified acting vinyl monomers, as ,' ' . '' ~ -^ ' ' '` ' ~, ;'` ' ` ' ' ~
.
.
mentioned earlier, whose amounts may constitute up to 5 wt.% in the copolymerisate.
The vinyl polymerisates, which form the outer layer of the particle, are homopolymerisates of monomers such as styrene and its derivatives, e.g. methylstyrene, ethyl-styrene and vinyltoluene; optionally hydroxylated alkyl acrylates and alkyl methacrylates (the alkyl groups con-taining 1 to 10 carbon atoms); acrylonitrile and meth-acrylonitrile; as well as copolymerisates of these mono-mers with another and/or with modified acting vinyl comono-mers such as divinylbenzene, acrylamide and methacrylamide as well as their N-substituted derivatives such as methyl-olacrylamide, which may constitute up to 5 wt.% of the copolymerisate.
In the particles, the core polymerisate may represent 30 wt.% to 99.5 wt.%, preferably 60 wt.% to 99 wt.%, and the polymerisate of the outer layer may represent 70 wt.% to 0.5 -~`
wt.% t preferably 40 wt.% to 1 wt.%.
The polymerisate particles, whose particle dis-tribution can be wide or narrow depending on the desiredproperties of the latex and on the intended use, have an average diameter between 0.03 ~m and 5 ~m, preferably between 0.05 ~m and 1 ~m. They may represent up to 60 wt.~, preferably up to 45 wt.% of the latex. However, the latex can be diluted or concentrated.
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6. Candida Viruses and Rickettsia l. Rickettsia 2. Viruses Canine hepatitis Shope papilloma Influenza A & B
Fowl pest Herpes simplex Adenoviruses Polyoma Rous sarcoma Vaccinia Polio virus German measles Canine distemper Leukaemia Mumps Newcastle disease (domestic fowl disease) Sendai Foot and mouth disease Psittacosis Rabies Ectromelia Arborviren ., .-.
~3~;i3 II. Foreiqn antigens Polysaccharides Hyaluronidases Tetanus toxin Egg ovalbumin Sheep serum albumin Human plasma gammaglobulin Human serum albumin III. Natural antigens 1. Hormones Insulin Glucagon Thyroid hormone Choriongonadotropin Chorion growth hormone - prolactin 2. Enzymes Pancreas chymotrypsinogens Procarboxypeptidases Deoxyribonucleases Ribonucleases Catalases Creatin phosphokinases 3. Organ-specific antigens Kidney Liver Skin Heart (myoglobin) ., .
9 ~ 3~53 Gastrointestinal tract Prostate Embryo antigens (e.g. CEA antigen) Tumour antigens 4. Connective tissue components Muscle Collagen Amyloid 5. Blood cell antigens, blood group substances and _ other isoantigens Platelets Megacaryocytes Leucocytes Erythrocytes Blood group substances Forssman antigen Histo-compatible antigens 6. Plasma proteins Fibrin and fibrinoid Plasminogen and plasmin 7. Pathological globulins Myeloma, macroglobulinaemic and dysglobulinaemic proteins Rheumatoid factor C-reactive proteins ,:.. . - - : ~ -- . ~, . ... : .
, :. . , : .-' . - " .
~ 3~3 IV. Natural antibodies 1. Natural gammaglobulins Natural antibodies - nephrotoxic antibody complement 2. Auto-antibodies Antinuclear factor Thyroid autoantibodies Adrenal autoantibodies Autoantibodies for gastric-parietal cells in the case of pernicious anaemia Autoantibodies for spermatozoa Muscle autoantibodies in the case of Myasthenia gravis Autoantibodies for nerve tissue Autoantibodies against fibre-like tissue and vascular components Autoantibodies against platelets and megacaryocytes Antibodies against trophoblasts 3. Induced antibodies against:
Immunoglobulin classes IgG, IgA, IgM or variant species V. Haptenic compounds Opium alkaloids (morphine) Antipyrine Barbituric acid ., ,~
':
3~3 Especially preferred immunologically active substances in the scope of the present invention are albumin, rheu-matoid factor, human immunoglobulin IgG and antibodies against IgG.
Under vinyl polymerisates, which form the core of the particles, there are to be understood homopolymerisates of monomers such as styrene and its derivatives: methyl-styrenes, ethylstyrenes, vinyltoluene; vinyl chloride, vinylidene chloride; vinyl acetate; acryl derivatives such as alkyl acrylates and alkyl methacrylates (the alkyl groups containing 1 to 10 carbon atoms), which are option-ally hydroxylated, such as 2-hydroxyethyl acrylate, 2--hydroxyethyl methacrylate, 2-hydroxypropyl acrylate and 2-hydroxypropyl methacrylate; and acrylonitrile and meth-acrylonitrile; as well as copolymerisates of these mono-mers with one another andtor with modified vinyl comonomers such as divinylbenzene, acrylamide and methacrylamide and their N-substituted derivatives such as, for example, methylolacrylamide; these comonomers may represent up to 5 wt.~ of the copolymerisate.
Under diene polymerisates, which form the core, there are to be understood homopolymerisates of butadiene and its derivatives: chloroprene, isoprene; as well as copoly-merisates of these monomers with one another and/or with vinyl monomers, such as those mentioned earlier, in all ratios and/or with modified acting vinyl monomers, as ,' ' . '' ~ -^ ' ' '` ' ~, ;'` ' ` ' ' ~
.
.
mentioned earlier, whose amounts may constitute up to 5 wt.% in the copolymerisate.
The vinyl polymerisates, which form the outer layer of the particle, are homopolymerisates of monomers such as styrene and its derivatives, e.g. methylstyrene, ethyl-styrene and vinyltoluene; optionally hydroxylated alkyl acrylates and alkyl methacrylates (the alkyl groups con-taining 1 to 10 carbon atoms); acrylonitrile and meth-acrylonitrile; as well as copolymerisates of these mono-mers with another and/or with modified acting vinyl comono-mers such as divinylbenzene, acrylamide and methacrylamide as well as their N-substituted derivatives such as methyl-olacrylamide, which may constitute up to 5 wt.% of the copolymerisate.
In the particles, the core polymerisate may represent 30 wt.% to 99.5 wt.%, preferably 60 wt.% to 99 wt.%, and the polymerisate of the outer layer may represent 70 wt.% to 0.5 -~`
wt.% t preferably 40 wt.% to 1 wt.%.
The polymerisate particles, whose particle dis-tribution can be wide or narrow depending on the desiredproperties of the latex and on the intended use, have an average diameter between 0.03 ~m and 5 ~m, preferably between 0.05 ~m and 1 ~m. They may represent up to 60 wt.~, preferably up to 45 wt.% of the latex. However, the latex can be diluted or concentrated.
.
:, .
- 13 -The core polymerisate can be manufactured by emulsion polymerisation of the vinyl monomer~s) and/or diene mono-mer(s) in the presence of at least one ethylenic monocar-boxylic acid or polycarboxylic acid which is copolymerisable with the monomer(s) and/or at least one copolymerisable unsaturated alkali organosulphonate. Then, the poly-merisate of the outer layer is manufactured by emulsion polymerisation of the vinyl monomer(s) in the presence of the latex of the core polymerisate, manufactured as des-cribed earlier, and in the presence of a chain-propagating agent.
The monomers used in the polymerisation of the core polymerisate and in the polymerlsation of the polymerisate of the outer layer are monomers which are mentioned earlier.
They are either used all before the polymerisation or are used in part before the polymerisation, whereby the remain-ing part is added to the reaction medium in the course of the polymerisation in successive fractions or continuously, or all is added in the course of the polymerisation in successive fractions or continuously.
As copolymerisable ethylenic monocarboxylic acids or polycarboxylic acids there may be named acrylic acid, methacrylic acid, maleic acid, fumaric acid, crotonic acid, sorbic acid, cinnamic acid, itaconic acid and aconitic acid, such acids being used in an amount between 0.5 wt.%
and lS wt.%, preferably between 0.5 wt.% and 10 wt.%, based on the monomer(s).
.
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~ ~1 3~
The monomers used in the polymerisation of the core polymerisate and in the polymerlsation of the polymerisate of the outer layer are monomers which are mentioned earlier.
They are either used all before the polymerisation or are used in part before the polymerisation, whereby the remain-ing part is added to the reaction medium in the course of the polymerisation in successive fractions or continuously, or all is added in the course of the polymerisation in successive fractions or continuously.
As copolymerisable ethylenic monocarboxylic acids or polycarboxylic acids there may be named acrylic acid, methacrylic acid, maleic acid, fumaric acid, crotonic acid, sorbic acid, cinnamic acid, itaconic acid and aconitic acid, such acids being used in an amount between 0.5 wt.%
and lS wt.%, preferably between 0.5 wt.% and 10 wt.%, based on the monomer(s).
.
...: :
~ ~1 3~
- 14 -The copolymerisable unsaturated alkali organo-sulphonates are, for example, sodium vinylsulphonate, sodium methallylsulphonate, sodium 2-sulphoethylacrylate, sodium 2-sulphoethylmetacrylate and 2-acrylamido-2-methylpropane-sulphonate. They are used in an amount between 0.1 wt.and 3 wt.% based on the monomer(s).
The copolymerisable ethylenic monocarboxylic acids or polycarboxylic acids and the copolymerisable unsaturated alkali organosulphonates can be used individually or in combination in the given amounts.
The manufacture of the core polymerisate Ls carried out in emulsion according to any classical process in the presence of an initiator and an emulsifier.
...
As the initiator there is preferably used an alkali persulphate, a water-soluble diazo derivative or a Redox system based on hydrogen peroxide, an organic peroxide or hydroperoxide in an amount in the order of 0.01 wt.% to 5 wt.%, preferably 0.03 wt.% to 3 wt.%, based on the mono-mer(s).
The emulsifier used can be anion-active and/or non--ionic. The emulsifier is a classical emulsifier used in emulsion polymerisation.
As anion-active emulsifiers there may be named salts of fatty acids; alkali alkylsulphates, alkali alkyl-,; . .
;
The copolymerisable ethylenic monocarboxylic acids or polycarboxylic acids and the copolymerisable unsaturated alkali organosulphonates can be used individually or in combination in the given amounts.
The manufacture of the core polymerisate Ls carried out in emulsion according to any classical process in the presence of an initiator and an emulsifier.
...
As the initiator there is preferably used an alkali persulphate, a water-soluble diazo derivative or a Redox system based on hydrogen peroxide, an organic peroxide or hydroperoxide in an amount in the order of 0.01 wt.% to 5 wt.%, preferably 0.03 wt.% to 3 wt.%, based on the mono-mer(s).
The emulsifier used can be anion-active and/or non--ionic. The emulsifier is a classical emulsifier used in emulsion polymerisation.
As anion-active emulsifiers there may be named salts of fatty acids; alkali alkylsulphates, alkali alkyl-,; . .
;
- 15 -sulphonates, alkali alkylarylsulphonate, alkali alkylsulpho-succinates, alkali alkylphosphates; sulphosuccinic acid alkyl ester; sulphonates of alkylphenol polyglycol ethers;
salts of esters of alkylsulphopolycarboxylic acids; con-densation products of fatty acids with oxyalkanesulphonicacids and aminoalkanesulphonic acids; sulphated derivatives of polyglycol ethers; sulphated esters of fatty acids and polyglycols; alkanolamides of sulphated fatty acids.
As non-ionic emulsifiers there can be mentioned fatty acid esters of polyalcohols, alkanolamides of fatty acids, polyethyleneoxides, copolyethyleneoxide/propyleneoxide, oxyethylated aLkylphenols.
The amount of emulsifier(s) which is used lies in the order of 0.01 wt.% to 5 wt.% based on the monomer(s) and its introduction is carried out either all together before the polymerisation or in part before the polymerisation, whereby the remaining part is added to the reaction medium in the course of the polymerisation in successive fractions or continuously, or all together in the course of the polymerisation in successive fractions or continuously.
me amount of water which is used in the polymer-isation of the core polymerisate must be such that the concentration of the monomer(s) preferably does not exceed 60 wt.%.
Although it is not absolutely necessary, it is pos-sible to add to the reaction medium compounds which are
salts of esters of alkylsulphopolycarboxylic acids; con-densation products of fatty acids with oxyalkanesulphonicacids and aminoalkanesulphonic acids; sulphated derivatives of polyglycol ethers; sulphated esters of fatty acids and polyglycols; alkanolamides of sulphated fatty acids.
As non-ionic emulsifiers there can be mentioned fatty acid esters of polyalcohols, alkanolamides of fatty acids, polyethyleneoxides, copolyethyleneoxide/propyleneoxide, oxyethylated aLkylphenols.
The amount of emulsifier(s) which is used lies in the order of 0.01 wt.% to 5 wt.% based on the monomer(s) and its introduction is carried out either all together before the polymerisation or in part before the polymerisation, whereby the remaining part is added to the reaction medium in the course of the polymerisation in successive fractions or continuously, or all together in the course of the polymerisation in successive fractions or continuously.
me amount of water which is used in the polymer-isation of the core polymerisate must be such that the concentration of the monomer(s) preferably does not exceed 60 wt.%.
Although it is not absolutely necessary, it is pos-sible to add to the reaction medium compounds which are
- 16 _ ~ ~i3~3 either capable of modifying the ionic strength of the medium and accordingly the particle distribution such as mineral acids or electrolytes in an amount up to 3 wt.%
based on the monomers or which are capable of modifying the pH value of the medium such as, for example, buffers, acids or bases. However, it has been established in certain cases that for the promotion of the copolymerisation it is preferred for the medium to be neutral or acid.
The temperature at which the polymerisation is carried out is a function of the initiator used and of the polymerisate to be manufactured and generally lies between -5C and +gOC.
The lattices obtained have polymerisate particles with a diameter between 0.03 ~m and 5 ~m, preferably between 0.05 ~m and 1 ~m. These particles are generally not calibrated, but it is possible to obtain them calibrated when the known calibration process is used for the emulsion polymerisation, such as the controlled addition of the emulsifier and/or of the monomer(s) and especially inoculation. In the latter case, the emulsifier can be contained in the inoculation material.
The particles are formed from homopolymerisates or copolymerisates having a surface of carboxyl and/or sul-phonate functions. The existence of these functions can be confirmed by conductometric titration.
- ,~
based on the monomers or which are capable of modifying the pH value of the medium such as, for example, buffers, acids or bases. However, it has been established in certain cases that for the promotion of the copolymerisation it is preferred for the medium to be neutral or acid.
The temperature at which the polymerisation is carried out is a function of the initiator used and of the polymerisate to be manufactured and generally lies between -5C and +gOC.
The lattices obtained have polymerisate particles with a diameter between 0.03 ~m and 5 ~m, preferably between 0.05 ~m and 1 ~m. These particles are generally not calibrated, but it is possible to obtain them calibrated when the known calibration process is used for the emulsion polymerisation, such as the controlled addition of the emulsifier and/or of the monomer(s) and especially inoculation. In the latter case, the emulsifier can be contained in the inoculation material.
The particles are formed from homopolymerisates or copolymerisates having a surface of carboxyl and/or sul-phonate functions. The existence of these functions can be confirmed by conductometric titration.
- ,~
- 17 - ~ ~ 3~53 The manufacture of the polymerisate of the outer layer is carried out in aqueous emulsion in the presence of the core polymerisate, a chain-propagating agent, an initiator and, if desired, an emulsifier.
The amount of the core polymerisate used lies between 30 wt.% and 99.5 wt.~ and preferably amounts to 60 wt.% to 99 wt.% based on the sum of core polymerisate and monomer to be polymerised or monomers to be polymerised.
The chain-propagating agent of the aminophenyldi-sulphide or aminophenylmercaptan type is especially o,o'--dithiobisaniline, p,p'-dithiobisaniline, 2-mercapto-aniline, 3-mercaptoaniline or 4-mercaptoaniline. This agent is in general used in solution in the monomer(s) in an amount between 0.1 wt.% and 10 wt.%, preferably between 0.5 wt.% and 5 wt.% based on the monomer(s).
The initiators required for the polymerisation of the monomer(s) of the outer layer are diazo initiators, azo-nitriles such as azo bis-isobutyronitrile or sulphonated azonitriles as are described in French Patent No. 1 233 5a2.
Of these there can be mentioned azobis-(isobutyronitrile sodium sulphonate), azobis-(~-methylbutyronitrile sodium sulphonate), azobis-(a-methyl-~-ethoxycarbonylbutyronitrile sodium sulphonate); carboxylated azonitriles such as 4,4'--azobis-(4-cyanopentanoic acid) and their salts, azobis--alkylamidinium salts such as a,a'-azobis-isobutyramidinium :, ., ~ . . ,:
. :, : .
- ' . " ~' ~ . . ,:~ ' ,.:
:: - :.: :. :
, .. .
3~a5i3
The amount of the core polymerisate used lies between 30 wt.% and 99.5 wt.~ and preferably amounts to 60 wt.% to 99 wt.% based on the sum of core polymerisate and monomer to be polymerised or monomers to be polymerised.
The chain-propagating agent of the aminophenyldi-sulphide or aminophenylmercaptan type is especially o,o'--dithiobisaniline, p,p'-dithiobisaniline, 2-mercapto-aniline, 3-mercaptoaniline or 4-mercaptoaniline. This agent is in general used in solution in the monomer(s) in an amount between 0.1 wt.% and 10 wt.%, preferably between 0.5 wt.% and 5 wt.% based on the monomer(s).
The initiators required for the polymerisation of the monomer(s) of the outer layer are diazo initiators, azo-nitriles such as azo bis-isobutyronitrile or sulphonated azonitriles as are described in French Patent No. 1 233 5a2.
Of these there can be mentioned azobis-(isobutyronitrile sodium sulphonate), azobis-(~-methylbutyronitrile sodium sulphonate), azobis-(a-methyl-~-ethoxycarbonylbutyronitrile sodium sulphonate); carboxylated azonitriles such as 4,4'--azobis-(4-cyanopentanoic acid) and their salts, azobis--alkylamidinium salts such as a,a'-azobis-isobutyramidinium :, ., ~ . . ,:
. :, : .
- ' . " ~' ~ . . ,:~ ' ,.:
:: - :.: :. :
, .. .
3~a5i3
- 18 -chloride and azobis-N,N'-dimethyleneisobutyramidinium chloride.
The initiator, which is used in an amount of 0.01 wt~ to 3 wt.%, preferably 0.1 wt.% to 2 wt.~, based on the monomer(s), is used all together or in part before the polymerisation, whereby the other part is added to the reaction medium in the course of the polymeris~tion in successive fractions or continuously, especially when the life of the initiator is short at the polymerisation temperature. The initiator can also be added continuously to the reaction mixture all together in the course of the polymerisation.
The emulsifier, which is optionally used, is chosen from the anion-active and/or non-ionic emulsifiers of the kind mentioned earlier in connection with the manufacture of the core polymerisate. The emulsifier can be similar or different to the emulsifier used for the manufacture of the core polymerisate. It ls used in an amount up to 10 wt.% based on the monomer(s) and its introduction can be carried out, depending on the desired average diameter of the latex particles, either all together before the poly-merisation or in part before the polymerisation, whereby the remaining part is added in the course of the poly-merisation in the successive fractions or continuously, or it can be carried out all together in the course of the polymerisation in successive fractions or continuously.
The initiator, which is used in an amount of 0.01 wt~ to 3 wt.%, preferably 0.1 wt.% to 2 wt.~, based on the monomer(s), is used all together or in part before the polymerisation, whereby the other part is added to the reaction medium in the course of the polymeris~tion in successive fractions or continuously, especially when the life of the initiator is short at the polymerisation temperature. The initiator can also be added continuously to the reaction mixture all together in the course of the polymerisation.
The emulsifier, which is optionally used, is chosen from the anion-active and/or non-ionic emulsifiers of the kind mentioned earlier in connection with the manufacture of the core polymerisate. The emulsifier can be similar or different to the emulsifier used for the manufacture of the core polymerisate. It ls used in an amount up to 10 wt.% based on the monomer(s) and its introduction can be carried out, depending on the desired average diameter of the latex particles, either all together before the poly-merisation or in part before the polymerisation, whereby the remaining part is added in the course of the poly-merisation in the successive fractions or continuously, or it can be carried out all together in the course of the polymerisation in successive fractions or continuously.
- 19 ~ 3~
The amount of water which is used in the poly-merisation of the outer layer must be such that the concen-tration of core polymerisate and monomer to be polymerised or monomers to be polymerised prefarably does not exceed S 60 wt.%, preferably 45 wt.%.
The temperature at which the polymerisation is carried out is a function of the chosen initiator and generally lies between 5C and 100C, preferably between 40C and 90C.
The lattices obtained have polymerisation particles whose diameter lies between 0~03 ~m and 5 ~m, preferably between 0.05 ~m and 1 ~m. Since the amount of the outer layer is not very large, it does not modify the size of the p~rticles of the core polymerisate in an appreciable manner.
The particles can be calibrated or not, but for certain uses it is preferred on grounds of reproducibility that they are calibrated, i.e. that they have a narrow particle size distribution.
The lattices are mechanical and are stable upon storase as well as towards electrolytes, i.e. they do not floc.~ out when there are added to them mineral salts such as, for exa~ple, the chlorides, nitrates, borates and phosphates of sodium, calcium, magnesium and potassium.
B
, ~.;~ .
.
.. . .. . .
3 ~
The amount of water which is used in the poly-merisation of the outer layer must be such that the concen-tration of core polymerisate and monomer to be polymerised or monomers to be polymerised prefarably does not exceed S 60 wt.%, preferably 45 wt.%.
The temperature at which the polymerisation is carried out is a function of the chosen initiator and generally lies between 5C and 100C, preferably between 40C and 90C.
The lattices obtained have polymerisation particles whose diameter lies between 0~03 ~m and 5 ~m, preferably between 0.05 ~m and 1 ~m. Since the amount of the outer layer is not very large, it does not modify the size of the p~rticles of the core polymerisate in an appreciable manner.
The particles can be calibrated or not, but for certain uses it is preferred on grounds of reproducibility that they are calibrated, i.e. that they have a narrow particle size distribution.
The lattices are mechanical and are stable upon storase as well as towards electrolytes, i.e. they do not floc.~ out when there are added to them mineral salts such as, for exa~ple, the chlorides, nitrates, borates and phosphates of sodium, calcium, magnesium and potassium.
B
, ~.;~ .
.
.. . .. . .
3 ~
- 20 -The particles are formed from polymerisates and have a surface with carboxyl and/or sulphonate functions as well as groups of the formula -S~
N~2 Although the outer layer of the core polymerisate is polymerised, the carboxyl and/or sulphonate functions remain accessible, as can be shown by conductometric titration, and the groups of the formula ~ -- S~
are available for further reactions.
The immunologically active materials (antigen or antibody) can be bound physically and/or chemically to the latex polymers used in accordance with the invention.
In one embodiment, the reagent provided by the present lS invention is manufactured by forming an azo bond between the latex and the immunologically active material. For this purpose, the primary aromatic amino groups of the latex are converted into a diazonium salt by treatment of the latex in aqueous acidic solution with a nitrite.
N~2 Although the outer layer of the core polymerisate is polymerised, the carboxyl and/or sulphonate functions remain accessible, as can be shown by conductometric titration, and the groups of the formula ~ -- S~
are available for further reactions.
The immunologically active materials (antigen or antibody) can be bound physically and/or chemically to the latex polymers used in accordance with the invention.
In one embodiment, the reagent provided by the present lS invention is manufactured by forming an azo bond between the latex and the immunologically active material. For this purpose, the primary aromatic amino groups of the latex are converted into a diazonium salt by treatment of the latex in aqueous acidic solution with a nitrite.
- 21 ~ 3~3 An inorganic acid such as hydrochloric acid, sulphuric acid or perchloric acid can be used, for example, as the acid. Sodium nitrite or potassium nitrite is preferably used as the nitrite. The treatment is preferably carried out at 0-5C because of the instability of the diazonium salt.
The immunologically active material is subsequently reacted with the diazotised carrier in a~ueous medium, preferably at a temperature between 0C and room tem-perature.
In a further embodiment of the process provided bythe present-invention, the immunologically active material can be bound to the latex with the aid of a polyfunctional compound via an intermediate stage.
Suitable polyfunctional compounds are those which react with the aromatic amino groups of the latex polymers or which enter into a substitution reaction on the aromatic ring of the latex polymers and simultaneously react with functional groups of the immunologically active materials (e.g. amino, mercapto, carboxyl and hydroxyl groups) or enter into a substitution reaction on the aromatic ring of the immunologically active materials.
Examples of such polyfunctional compounds are com-pounds containing azo, isocyano, isothiocyano or aldehyde , ., :
..: .
. .
3~
The immunologically active material is subsequently reacted with the diazotised carrier in a~ueous medium, preferably at a temperature between 0C and room tem-perature.
In a further embodiment of the process provided bythe present-invention, the immunologically active material can be bound to the latex with the aid of a polyfunctional compound via an intermediate stage.
Suitable polyfunctional compounds are those which react with the aromatic amino groups of the latex polymers or which enter into a substitution reaction on the aromatic ring of the latex polymers and simultaneously react with functional groups of the immunologically active materials (e.g. amino, mercapto, carboxyl and hydroxyl groups) or enter into a substitution reaction on the aromatic ring of the immunologically active materials.
Examples of such polyfunctional compounds are com-pounds containing azo, isocyano, isothiocyano or aldehyde , ., :
..: .
. .
3~
- 22 -groups such as, for example, bis-diazobenzidine, bis-diazo-benzidine-disulphonic acid, bis-diazo-p-phenyldiamine, phenyldiisocyanate, toluenediisocyanate and glutardialde-hyde.
When the reaction is carried out in the presence of a bifunctional compound, the immunologically active material is reacted with the carrier in aqueous medium, preferably at room temperature (20C to 25C). The temperature can, however, also lie between 0C and 40C.
The amount of bifunctional compound used depends on the number of amino groups on the latex. Preferably, there is used a ten-fold to hundred-fold molar excess of the bifunctional compound based on the number of the amino groups on the latex used.
In both embodiments for the manufacture of the reagent provided by the present invention the pH value of the reaction medium is important. The pH value must be such that a protein reaction partner is not denatured. As a rule, the pH value lies between 5 and 9. This pH value is maintained using suitable customary buffer systems such as phosphate buffer and the like.
The end product is a water-insoluble material, which is suspended in an aqueous buffer solution of pH value 5.0 to 9, whereby the pH value of the solution is dependant on ' ~
When the reaction is carried out in the presence of a bifunctional compound, the immunologically active material is reacted with the carrier in aqueous medium, preferably at room temperature (20C to 25C). The temperature can, however, also lie between 0C and 40C.
The amount of bifunctional compound used depends on the number of amino groups on the latex. Preferably, there is used a ten-fold to hundred-fold molar excess of the bifunctional compound based on the number of the amino groups on the latex used.
In both embodiments for the manufacture of the reagent provided by the present invention the pH value of the reaction medium is important. The pH value must be such that a protein reaction partner is not denatured. As a rule, the pH value lies between 5 and 9. This pH value is maintained using suitable customary buffer systems such as phosphate buffer and the like.
The end product is a water-insoluble material, which is suspended in an aqueous buffer solution of pH value 5.0 to 9, whereby the pH value of the solution is dependant on ' ~
- 23 -the individually used system and on the requirements on the stability of the immunologically active materials. The specific weight of the product corresponds to about that of water (0.97-1.02), whereby a stable suspension of the product is achieved. The products can be isolated, for example by centrifugation, in the form of a white or yellowish precipitate.
The amount of immunologically active material which is bound to the immunologically inert latex polymer-carrier amounts as a rule to 0.01 wt.% to 15.0 wt.%. However, each individual immunologically active material is used in an amount which is most convenient in a particular diag-nostic test.-- On this basis, each immunologically active material is combined with the carrier in a ratio which best corresponds to the respective specific req,uirements. The present invention therefore includes the use of such an amount of immunologically active material in combination with an immunologically inert latex polymer-carrier which is suitable to yield a reagent which is useful for such diagnostic purpose.
After its manufacture, the product can be used in specific diagnostic tests which are based on immunological principles.
In accordance with the present invention, the deter-mination of the immunologically active substance can be ,!
'' ' ' ` ' ' " ~' ..
~' ." ' ' ' " " ' '` " . . .
. . ` ' . '.'. " ' ' ` . ' `` :' ~53~
The amount of immunologically active material which is bound to the immunologically inert latex polymer-carrier amounts as a rule to 0.01 wt.% to 15.0 wt.%. However, each individual immunologically active material is used in an amount which is most convenient in a particular diag-nostic test.-- On this basis, each immunologically active material is combined with the carrier in a ratio which best corresponds to the respective specific req,uirements. The present invention therefore includes the use of such an amount of immunologically active material in combination with an immunologically inert latex polymer-carrier which is suitable to yield a reagent which is useful for such diagnostic purpose.
After its manufacture, the product can be used in specific diagnostic tests which are based on immunological principles.
In accordance with the present invention, the deter-mination of the immunologically active substance can be ,!
'' ' ' ` ' ' " ~' ..
~' ." ' ' ' " " ' '` " . . .
. . ` ' . '.'. " ' ' ` . ' `` :' ~53~
- 24 -carried ou~ not only in a direct test method but also in an indirect (inhibition) test method.
In the direct test method for the determination of an immunologically active substance, the analytical sample and the carrier particles sensitised with the corresponding immunological reaction partner are mixed and the occurrence of an agglutination is observed. The test is positive when an agglutination is established.
In the case of the indirect (inhibition) test method for the determination of an immunologically active sub-stance, the analytical sample is mixed with a determined amount of the corresponding immunological reaction partner (e.g. antiserum~ and latex particles which are sensitised with the immunologically active substance and the occur-rence of an agglutination is observed. The test is positive when no agglutination is established.
The reagents used in such immunological test methods can advantageously be packed for commercial purposes in a diagnostic test kit.
In the case of a direct test, the reagent kit for the determination of an immunologically active substance contains in a container an aqueous suspension of latex particles sensitised with the corresponding immunological reaction partner.
- .
.: . ,. : ~ ::
In the direct test method for the determination of an immunologically active substance, the analytical sample and the carrier particles sensitised with the corresponding immunological reaction partner are mixed and the occurrence of an agglutination is observed. The test is positive when an agglutination is established.
In the case of the indirect (inhibition) test method for the determination of an immunologically active sub-stance, the analytical sample is mixed with a determined amount of the corresponding immunological reaction partner (e.g. antiserum~ and latex particles which are sensitised with the immunologically active substance and the occur-rence of an agglutination is observed. The test is positive when no agglutination is established.
The reagents used in such immunological test methods can advantageously be packed for commercial purposes in a diagnostic test kit.
In the case of a direct test, the reagent kit for the determination of an immunologically active substance contains in a container an aqueous suspension of latex particles sensitised with the corresponding immunological reaction partner.
- .
.: . ,. : ~ ::
- 25 -In the case of an indirect test, the reagent kit for the determination of an immunologically active substance contains in a first container a solution of the cor-responding immunological reaction partner (e.g. antiserum) and in a second container an aqueous suspension of latex particles sensitised with the immunologically active material.
In both cases, the aqueous suspension of the immuno-logically active materials bound to latex or the immuno-logical reaction partners bound to latex can be present inany concentration. However, a concentration of 0.5 wt.%
to 5 wt.% is preferred.
: ,. ~: :,
In both cases, the aqueous suspension of the immuno-logically active materials bound to latex or the immuno-logical reaction partners bound to latex can be present inany concentration. However, a concentration of 0.5 wt.%
to 5 wt.% is preferred.
: ,. ~: :,
26 ~ 3~
The following Examples illustrate the present invention:
Example 1 . .
A latex of core polymerisate is manufactured in a 25 litre autoclave, there being used:
4800 g of deionised water, 50 g of potassium persulphate, 50 g of sodium pyrophosphate, 10 g of sodium lauryl sulphate, 50 g of sodium methallyl sulphonate, 100 g of acrylic acid, 100 g of itaconic acid, 2135 g of styrene and 2865 g of butadiene.
The polymerisation is carried out at 75C under a nitrogen atmosphere, the monomers being introduced con-tinuously over a period of 7 hours and the reaction being continued for 8 hours.
After cooling down, there is obtained a latex of pH
2.5, whose concentration of polymerisate particles amounts to 51 wt.%.
By electron microscopy it is established that the particles have an average diameter of 0.145 ~m and 90~ of the particles have a diameter between 0.14 and 0.15 ~m.
.., , . .~ . .. .
, : . . ..
The following Examples illustrate the present invention:
Example 1 . .
A latex of core polymerisate is manufactured in a 25 litre autoclave, there being used:
4800 g of deionised water, 50 g of potassium persulphate, 50 g of sodium pyrophosphate, 10 g of sodium lauryl sulphate, 50 g of sodium methallyl sulphonate, 100 g of acrylic acid, 100 g of itaconic acid, 2135 g of styrene and 2865 g of butadiene.
The polymerisation is carried out at 75C under a nitrogen atmosphere, the monomers being introduced con-tinuously over a period of 7 hours and the reaction being continued for 8 hours.
After cooling down, there is obtained a latex of pH
2.5, whose concentration of polymerisate particles amounts to 51 wt.%.
By electron microscopy it is established that the particles have an average diameter of 0.145 ~m and 90~ of the particles have a diameter between 0.14 and 0.15 ~m.
.., , . .~ . .. .
, : . . ..
- 27 -The composition of the polymerisate is essentially similar to that of the monomers used. The particles carry on their surface carboxyl and sulphonate functions which are determined by conductometric titration.
406 g of the foregoing latex and 1541 g of deionised water are introduced into a reactor. The mixture is heated ~o 70C while stirring and this temperature is maintained during the entire duration of the reaction.
As soon as the mixture has attained 70C, it is held ; 10 under a nitrogen atmosphere and there are simultaneously added thereto in 3 hours with constant velocity 1.25 g of ` sodium dihexylsulphosuccinate in 150 g of water, 0.20 g of a,a'-azobis-isobutyramidinium chloride in 210 g of water and 18 g of styrene containing 0.45 g of p,p'-dithiobis-aniline.
The polymerisation is then continued for 5 hours.
The mixture is thereafter cooled down.
Properties of the resulting latex:
_ _ pEI: 3.1;
concentration of polymerisation particles: 9.3 wt.%;
electrolyte stability: 5;
average diameter of the particles: 0.15 ~m, 90%
having a diameter between 0.145 ~m and 0.155 ~m.
:~ .
The particles carry on their surface carboxyl and sulphonate functions, which are confirmed by conductometric titration, and groups of the formula - --S~
1 ml of the 10~ latex obtained is added to 20 ml of water and the mixture is centrifuged for 1.5 hours at 35,000 g. The supernatant is decanted off, the residue is taken up in 20 ml of water and again centrifuged for 1.5 hours at 35,000 g. This operation is repeated twice and the thus-obtained latex is denoted in the following Examples as "washed latex".
5 mg of human immunoglobulin G (Cohn fraction II) is denatured by heating to 60C for 3 hours in 1 ml of 0.1-M
glycine/hydrochloric acid buffer pH 4.
1 ml of washed latex is added to 5 ml of 0.05 M
hydrochloric acid and the mixture is cooled down to 0C.
To this mixture is added 0.1 ml of a 0.01-M sodium nitrite solution and the resulting mixture is stirred at 0C for 15 minutes. The diazotised latex is centrifuged at 5C for 1.5 hours at 35,000 g and the supernatant is decanted off.
The residue is taken up in 5 ml of ice-cold 0.1-M glycine/
hydrochloric acid buffer pH 4.0 and added to 1 ml of 0.5%
denatured human immunoglobulin G in the same buffer, stirred .. . .. .
' '; ' ' , . . : . .
- 29 - ~ ~315~
for 1 hour in an ice-bath and subsequently left to stand at 10C overnight. The latex is centrifuged for 1.5 hours at 35,000 g, the supernatant is decanted off and the residue is washed twice with in each case 25 ml of 0.1-M glycine/
sodium hydroxide buffer pH 8.2 by centrifugation and suspension of the residue. Sufficient buffer is now added to the latex to give a solution containing 30 mg/ml.
Agglutination test:
For the test tube agglutination test the following buffer is used: 7.5 g of glycine, 6.0 g of calcium chloride, 3 g of bovine albumin and 1 g of sodium azide dissolved in 1 litre of water. The pH value is ad]usted to 8.2 wi-th sodium hydroxide. For the detection of the rheumatoid factors in serum, 20 ~1 of latex is diluted with 3 ml of buffer in a small test tube and 25 ~1 of the serum to be investigated are added. After intermixing, the test tubes are held at 37C during 2 hours in a heat-block. A
positive serum agglutinates under these conditions while a negative control system shows no agglutination.
Example 2 1 ml of washed latex is manufactured and diazotised as described in Example 1. To the diazotised latex residue are added 5 ml of ice-cold 0.1-M glycine/sodium hydroxide buffer pH 6.0, 5 ml of goat anti-human albumin immunoglobulin G dissolved in 1 ml of foregoing buffer are . : , : ~; :
_ 30 _ 1 1~ 3~
added and, after stirring for 1 hour at 10C, the mixture is left to stand overnight. The latex is centrifuged at 35,000 g for 1.5 hours, the supernatant is discarded and the sediment is washed twice with in each case 25 ml of 0.1-M glycine/sodium hydroxide pH 8.2. After washing, the latex is mixed with sufficient buffer to give a 3% solution.
Agglutination test:
For the test tube agglutination test the following buffer is used: 7.5 g of glycine, 6.0 g of calcium chloride, 3 g of bovine albumin and 1.0 g of sodium azide dissolved in 1 litre of water. The pH is adjusted to 6.0 with hydrochloric acid. A concentration series of human albumin in 3 ml of buffer is set up in small test tubes, in each case 2 ~1 of latex are added and, after intermixing, held for 2 hours at 37C in a heat-block.
¦ 20 ¦ 10 ¦1 1 1 1 05 ~ l ~ g human albumin/ml buffer = agglutinates in 2 hours - = does not agglutinate in 2 hours From this Table it is evident that 0.05 ~g of human albumin/ml can be determined with the latex reagent of this Example.
,~ , :. .... . ~
.. , ~. . . .
11`~ 33L~i~
Example 3 1 ml of the washed latex of Example 1 is added to 5 ml of 0.1-M phosphate buffer pH 5.0, 5 mg of sheep anti--human IgG immunoglobulin G in 1 ml of buffer are added and the mixture is stirred well. Subsequently, 0.1 ml of a 0.01-M-p-phenyldiisothiocyanate solution in dimethyl-formamide is added, the mixture is stirred for 1 hour and left to stand at room temperature overnight. The latex is centrifuged for 1.5 hours at 35,000 g, the supernatant is discarded and the residue is washed twice with in each case 25 ml of 0.1-M glycine/sodium hydroxide buffer pH 8.2.
For the agglutination test the latex is used in a con-centration of 30 mg/ml. -~-Agglutination test:
For the test tube agglutination test there is used a 0.1-M phosphate buffer pH 6.0 with 0.1% bovine albumin.
A concentration series of human IgG in 3 ml of buffer is prepared. To each test tube are added 20 ~1 of latex reagent, the test tube contents are intermixed and incubated for 2 hours at 37C in a heat-block.
¦loo ¦ 10 ¦1 ¦0-1 10-o5 ¦O ¦ ~g human IgG/ml buffer I + I+ 1+1+ 1- T-l + = agglutinates in 2 hours - = does not agglutinate in 2 hours : : :. ,. .:., .:, .: ~
- 32 - 1~3~-3 The Table shows that 0.1 ~g/ml of human IgG can be determined with the latex reagent of this Example.
Example 4 36.8 mg of benzidine are dissolved in 0.5 ml of 2-N
sodium chloride and diluted with 7.5 ml of water. The mixture is cooled in an ice-bath and added dropwise while stirring to 27.2 mg of sodium nitrite in 2 ml of water.
The thus-obtained bisdiazotised benzidine can be stored at -20C for weeks.
101 ml of the washed latex of Example 1 is taken up in - 5 ml of 0.1-M phosphate buffer pH 7.0 and 5 mg of human immunoglobulin G in 1 ml of the foregoing buffer are added.
The suspension is cooled down to 0C and, while stirring, 0.01 ml of a 0.02-M bisdiazotised benzidine solution is added. The mixture is then left to stand at 10C over-night. The latex is centrifuged at 30,000 g for 1.5 hours, the supernatant is discarded and the sediment is washed twice with in each case 25 ml of 0.1-M glycine/
sodium hydroxide pH 8.2. After washing, the latex ls mixed with sufficient buffer to give a 3% solution.
Agglutination test:
.
For the determination of IgG in an inhibition test, a 0.1-M phosphate buffer pH 6.0 is used. In each case 3 ml of a 11500 diluted sheep anti-human IgG serum and increasing ':
' , . ~ ,.. .
'` . ' ''~''''", : ' ~ ' -3~3 amounts of human IgG are added to small test tubes. After incubation for 15 minutes at 37C, there is added to each test tube 20 ~l of latex reagent and the mixture is incubated for 3 hours at 37C.
¦ ~ l ¦0;0l 1 lO 1 50 ¦l 1~5 ¦ ~g human IgG/3 ml + = agglutinates in 3 hours - = does not agglutinate in 3 hours 0.16 ~g of human IgG/ml can be determined with the latex reagent of this Example.
~' ' ,, :' .::
:. .~. ~ : : :
406 g of the foregoing latex and 1541 g of deionised water are introduced into a reactor. The mixture is heated ~o 70C while stirring and this temperature is maintained during the entire duration of the reaction.
As soon as the mixture has attained 70C, it is held ; 10 under a nitrogen atmosphere and there are simultaneously added thereto in 3 hours with constant velocity 1.25 g of ` sodium dihexylsulphosuccinate in 150 g of water, 0.20 g of a,a'-azobis-isobutyramidinium chloride in 210 g of water and 18 g of styrene containing 0.45 g of p,p'-dithiobis-aniline.
The polymerisation is then continued for 5 hours.
The mixture is thereafter cooled down.
Properties of the resulting latex:
_ _ pEI: 3.1;
concentration of polymerisation particles: 9.3 wt.%;
electrolyte stability: 5;
average diameter of the particles: 0.15 ~m, 90%
having a diameter between 0.145 ~m and 0.155 ~m.
:~ .
The particles carry on their surface carboxyl and sulphonate functions, which are confirmed by conductometric titration, and groups of the formula - --S~
1 ml of the 10~ latex obtained is added to 20 ml of water and the mixture is centrifuged for 1.5 hours at 35,000 g. The supernatant is decanted off, the residue is taken up in 20 ml of water and again centrifuged for 1.5 hours at 35,000 g. This operation is repeated twice and the thus-obtained latex is denoted in the following Examples as "washed latex".
5 mg of human immunoglobulin G (Cohn fraction II) is denatured by heating to 60C for 3 hours in 1 ml of 0.1-M
glycine/hydrochloric acid buffer pH 4.
1 ml of washed latex is added to 5 ml of 0.05 M
hydrochloric acid and the mixture is cooled down to 0C.
To this mixture is added 0.1 ml of a 0.01-M sodium nitrite solution and the resulting mixture is stirred at 0C for 15 minutes. The diazotised latex is centrifuged at 5C for 1.5 hours at 35,000 g and the supernatant is decanted off.
The residue is taken up in 5 ml of ice-cold 0.1-M glycine/
hydrochloric acid buffer pH 4.0 and added to 1 ml of 0.5%
denatured human immunoglobulin G in the same buffer, stirred .. . .. .
' '; ' ' , . . : . .
- 29 - ~ ~315~
for 1 hour in an ice-bath and subsequently left to stand at 10C overnight. The latex is centrifuged for 1.5 hours at 35,000 g, the supernatant is decanted off and the residue is washed twice with in each case 25 ml of 0.1-M glycine/
sodium hydroxide buffer pH 8.2 by centrifugation and suspension of the residue. Sufficient buffer is now added to the latex to give a solution containing 30 mg/ml.
Agglutination test:
For the test tube agglutination test the following buffer is used: 7.5 g of glycine, 6.0 g of calcium chloride, 3 g of bovine albumin and 1 g of sodium azide dissolved in 1 litre of water. The pH value is ad]usted to 8.2 wi-th sodium hydroxide. For the detection of the rheumatoid factors in serum, 20 ~1 of latex is diluted with 3 ml of buffer in a small test tube and 25 ~1 of the serum to be investigated are added. After intermixing, the test tubes are held at 37C during 2 hours in a heat-block. A
positive serum agglutinates under these conditions while a negative control system shows no agglutination.
Example 2 1 ml of washed latex is manufactured and diazotised as described in Example 1. To the diazotised latex residue are added 5 ml of ice-cold 0.1-M glycine/sodium hydroxide buffer pH 6.0, 5 ml of goat anti-human albumin immunoglobulin G dissolved in 1 ml of foregoing buffer are . : , : ~; :
_ 30 _ 1 1~ 3~
added and, after stirring for 1 hour at 10C, the mixture is left to stand overnight. The latex is centrifuged at 35,000 g for 1.5 hours, the supernatant is discarded and the sediment is washed twice with in each case 25 ml of 0.1-M glycine/sodium hydroxide pH 8.2. After washing, the latex is mixed with sufficient buffer to give a 3% solution.
Agglutination test:
For the test tube agglutination test the following buffer is used: 7.5 g of glycine, 6.0 g of calcium chloride, 3 g of bovine albumin and 1.0 g of sodium azide dissolved in 1 litre of water. The pH is adjusted to 6.0 with hydrochloric acid. A concentration series of human albumin in 3 ml of buffer is set up in small test tubes, in each case 2 ~1 of latex are added and, after intermixing, held for 2 hours at 37C in a heat-block.
¦ 20 ¦ 10 ¦1 1 1 1 05 ~ l ~ g human albumin/ml buffer = agglutinates in 2 hours - = does not agglutinate in 2 hours From this Table it is evident that 0.05 ~g of human albumin/ml can be determined with the latex reagent of this Example.
,~ , :. .... . ~
.. , ~. . . .
11`~ 33L~i~
Example 3 1 ml of the washed latex of Example 1 is added to 5 ml of 0.1-M phosphate buffer pH 5.0, 5 mg of sheep anti--human IgG immunoglobulin G in 1 ml of buffer are added and the mixture is stirred well. Subsequently, 0.1 ml of a 0.01-M-p-phenyldiisothiocyanate solution in dimethyl-formamide is added, the mixture is stirred for 1 hour and left to stand at room temperature overnight. The latex is centrifuged for 1.5 hours at 35,000 g, the supernatant is discarded and the residue is washed twice with in each case 25 ml of 0.1-M glycine/sodium hydroxide buffer pH 8.2.
For the agglutination test the latex is used in a con-centration of 30 mg/ml. -~-Agglutination test:
For the test tube agglutination test there is used a 0.1-M phosphate buffer pH 6.0 with 0.1% bovine albumin.
A concentration series of human IgG in 3 ml of buffer is prepared. To each test tube are added 20 ~1 of latex reagent, the test tube contents are intermixed and incubated for 2 hours at 37C in a heat-block.
¦loo ¦ 10 ¦1 ¦0-1 10-o5 ¦O ¦ ~g human IgG/ml buffer I + I+ 1+1+ 1- T-l + = agglutinates in 2 hours - = does not agglutinate in 2 hours : : :. ,. .:., .:, .: ~
- 32 - 1~3~-3 The Table shows that 0.1 ~g/ml of human IgG can be determined with the latex reagent of this Example.
Example 4 36.8 mg of benzidine are dissolved in 0.5 ml of 2-N
sodium chloride and diluted with 7.5 ml of water. The mixture is cooled in an ice-bath and added dropwise while stirring to 27.2 mg of sodium nitrite in 2 ml of water.
The thus-obtained bisdiazotised benzidine can be stored at -20C for weeks.
101 ml of the washed latex of Example 1 is taken up in - 5 ml of 0.1-M phosphate buffer pH 7.0 and 5 mg of human immunoglobulin G in 1 ml of the foregoing buffer are added.
The suspension is cooled down to 0C and, while stirring, 0.01 ml of a 0.02-M bisdiazotised benzidine solution is added. The mixture is then left to stand at 10C over-night. The latex is centrifuged at 30,000 g for 1.5 hours, the supernatant is discarded and the sediment is washed twice with in each case 25 ml of 0.1-M glycine/
sodium hydroxide pH 8.2. After washing, the latex ls mixed with sufficient buffer to give a 3% solution.
Agglutination test:
.
For the determination of IgG in an inhibition test, a 0.1-M phosphate buffer pH 6.0 is used. In each case 3 ml of a 11500 diluted sheep anti-human IgG serum and increasing ':
' , . ~ ,.. .
'` . ' ''~''''", : ' ~ ' -3~3 amounts of human IgG are added to small test tubes. After incubation for 15 minutes at 37C, there is added to each test tube 20 ~l of latex reagent and the mixture is incubated for 3 hours at 37C.
¦ ~ l ¦0;0l 1 lO 1 50 ¦l 1~5 ¦ ~g human IgG/3 ml + = agglutinates in 3 hours - = does not agglutinate in 3 hours 0.16 ~g of human IgG/ml can be determined with the latex reagent of this Example.
~' ' ,, :' .::
:. .~. ~ : : :
Claims (14)
1) A water-insoluble reagent in the form of an aqueous suspension having a pH of 5 to 9, for an immunological determination, having a specific weight corresponding to about that of water, and being in the form of discrete latex particles, to which is bound an immunologically active material, wherein the latex consists of a dispersion of particles of vinyl polymerisates, which carry as the end-groups groups of the formula , whereby the particles are formed from a core of vinyl and/or diene polymerisate, which carries carboxyl and/or sulphonate functions, and of an outer layer of vinyl polymerisate, which carries as the end-groups groups of the formula , and have an average diameter between 0.03 µm and 5 µm.
2) A reagent according to claim 1, wherein the vinyl polymerisates are either homopolymerisates of monomers which are selected from styrene and its derivatives, vinyl chloride, vinylidene chloride, vinyl acetate, acryl derivatives or copolymerisates of these monomers with one another and/or with modified vinyl comonomers which con-stitute up to 5 wt.% of the copolymerisate.
3) A reagent according to claim 1, wherein the diene polymerisates are homopolymerisates of butadiene and its derivatives or copolymerisates of these monomers with one another and/or with vinyl monomers and/or modified vinyl comonomers which constitute up to 5 wt.% of the copolymerisate.
4) A reagent according to claim 2, wherein the diene polymerisates are homopolymerisates of butadiene and its derivatives or copolymerisates of these monomers with one another and/or with vinyl monomers and/or modified vinyl comonomers which constitute up to 5 wt.% of the copolymerisate.
5) A reagent according to any one of claims 1 to 3 inclusive, wherein the vinyl polymerisates which form the outer layer are homopolymerisates of styrene and its derivatives, of alkyl acrylates and alkyl methacrylates (the alkyl groups containing 1 to 10 carbon atoms), of acrylonitrile or methacrylonitrile, as well as copolymerisates of these monomers with one another and/or with modified acting vinyl comonomers which constitute up to 5 wt.% of the copolymerisates.
6) A reagent according to any one of claims 1 to 3 inclusive, wherein the core polymerisate comprises 30 wt.%
to 99.5 wt.% of the particle and the polymerisate of the outer layer constitutes 70 wt.% to 0.5 wt.% of the particle.
to 99.5 wt.% of the particle and the polymerisate of the outer layer constitutes 70 wt.% to 0.5 wt.% of the particle.
7) A reagent according to any one of claims 1 to 3 inclusive, wherein the amount of immunologically active material amounts to 0.01 wt.% to 15.0 wt.%.
8) A reagent according to any one of claims 1 to 3 inclusive, wherein the immunologically active material is bound covalently to the discrete particles of the latex polymers.
9) A reagent according to any one of claims 1 to 3 inclusive, wherein the immunologically active material is human immunoglobulin G.
10) A reagent according to any one of claims 1 to 3 inclusive, wherein the immunologically active material is goat anti-human albumin immunoglobulin G.
11) A reagent according to any one of claims 1 to 3 inclusive, wherein the immunologically active material is sheep anti-human IgG immunoglobulin G.
12) A process for the manufacture of a reagent as set forth in any one of claims 1 to 3 inclusive, which process comprises reacting the latex, after diazotisation or in the presence of suitable bifunctional compounds, with the immunologically active material.
13) A method for the determination of an immumologically active substance in a sample, which method comprises mixing the sample and particles of a latex defined in any one of claims 1 to 3 inclusive sensitised with the corresponding immunological reaction partner and observing the occurrence of an agglutination.
14) A method for the determination of an immunologically active substance in a sample, which method comprises mixing the sample with a determined amount of the corresponding immunological reaction partner and with particles of a latex defined in the claims 1 to 3 inclusive sensitised with the immunologically active substance and observing the occurrence of an agglutination.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CH954277A CH628738A5 (en) | 1977-08-03 | 1977-08-03 | IMMUNOLOGICAL DIAGNOSTIC REAGENT. |
| CH9542/77 | 1977-08-03 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1103153A true CA1103153A (en) | 1981-06-16 |
Family
ID=4353079
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA307,224A Expired CA1103153A (en) | 1977-08-03 | 1978-07-12 | Vinyl polymerisate latex particles bonded to immunological material |
Country Status (12)
| Country | Link |
|---|---|
| US (1) | US4226747A (en) |
| EP (1) | EP0000772A1 (en) |
| JP (1) | JPS5428816A (en) |
| AU (1) | AU528150B2 (en) |
| CA (1) | CA1103153A (en) |
| CH (1) | CH628738A5 (en) |
| DE (1) | DE2833886A1 (en) |
| DK (1) | DK343678A (en) |
| FR (1) | FR2399665A1 (en) |
| GB (1) | GB2001996B (en) |
| NL (1) | NL7808143A (en) |
| SE (1) | SE7808349L (en) |
Families Citing this family (26)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CH628738A5 (en) * | 1977-08-03 | 1982-03-15 | Hoffmann La Roche | IMMUNOLOGICAL DIAGNOSTIC REAGENT. |
| GB2027031A (en) * | 1978-08-02 | 1980-02-13 | Hoffmann La Roche | Diagnostic Reagent Comprising a Proteinaceous Material Bonded to a Latex |
| DE2918342A1 (en) * | 1979-05-07 | 1980-11-20 | Behringwerke Ag | LATEX REAGENT |
| DE3048883A1 (en) * | 1980-12-23 | 1982-07-15 | Boehringer Mannheim Gmbh, 6800 Mannheim | Hydrophilic latex particles prodn. by emulsion polymerisation - without added surfactant, useful as carriers for biological or immunological cpds. |
| JPS57168163A (en) * | 1981-04-10 | 1982-10-16 | Japan Synthetic Rubber Co Ltd | Carier for immunoserological inspection reagent |
| DE3116995A1 (en) * | 1981-04-29 | 1982-11-25 | Röhm GmbH, 6100 Darmstadt | LATEX FOR IMMOBILIZING BIOLOGICALLY EFFECTIVE SUBSTANCES |
| JPS585658A (en) * | 1981-07-02 | 1983-01-13 | Japan Synthetic Rubber Co Ltd | Carrier for immunity serological inspecting reagent |
| US4792527A (en) * | 1981-07-17 | 1988-12-20 | Toray Industries, Inc. | Method of assaying biologically active substances and labelling agents therefor |
| DE3130924A1 (en) * | 1981-08-05 | 1983-02-17 | Röhm GmbH, 6100 Darmstadt | SURFACE-BASED SYSTEMS FOR FIXING SUBSTRATES CONTAINING NUCLEOPHILE GROUPS |
| US4401765A (en) * | 1981-09-01 | 1983-08-30 | E. I. Du Pont De Nemours And Company | Covalently bonded high refractive index particle reagents and their use in light scattering immunoassays |
| JPS5847258A (en) * | 1981-09-01 | 1983-03-18 | イ−・アイ・デユポン・ド・ネモア−ス・アンド・コンパニ− | Granular reagent for light-scattering immunoassay |
| US4480042A (en) * | 1981-10-28 | 1984-10-30 | E. I. Du Pont De Nemours And Company | Covalently bonded high refractive index particle reagents and their use in light scattering immunoassays |
| JPS5897656A (en) * | 1981-12-07 | 1983-06-10 | Sekisui Chem Co Ltd | Latex for diagnosis reagent |
| US4713350A (en) * | 1983-10-24 | 1987-12-15 | Technicon Instruments Corporation | Hydrophilic assay reagent containing one member of specific binding pair |
| US4654325A (en) * | 1984-05-24 | 1987-03-31 | Selenke William M | Medicament for reducing nephrotoxicity caused by positively charged agents such as aminoglycosides and methods of use thereof |
| JPH0692970B2 (en) * | 1986-02-28 | 1994-11-16 | 日本合成ゴム株式会社 | Method for producing carrier particles for diagnostic agent |
| FR2595826B1 (en) * | 1986-03-13 | 1990-09-07 | Lurhuma Zirimwabagabo | IMMUNOASSAY PRODUCT, PREPARATION METHOD THEREOF, USE THEREOF, IMMUNOGENIC COMPLEX COMPRISING THE SAME, AND USE OF THE COMPLEX |
| US5166077A (en) * | 1987-04-30 | 1992-11-24 | Nitto Denko Corporation | Latex for immobilization of physiologically active substances for immuno nephelometry |
| WO1992005440A1 (en) * | 1990-09-26 | 1992-04-02 | Akers Research Corporation | Improved ligand assay |
| US5231035A (en) * | 1991-01-11 | 1993-07-27 | Akers Research Corporation | Latex agglutination assay |
| US5973029A (en) * | 1993-10-12 | 1999-10-26 | The Sherwin-Williams Company | Corrosion-resistant waterborne paints |
| US5432210A (en) * | 1993-11-22 | 1995-07-11 | Rohm And Haas Company | Polymer particles and method for preparing by polymerization of encapsulated monomers |
| US5981296A (en) * | 1997-02-18 | 1999-11-09 | Dade Behring Inc. | Stabilization of particle reagents |
| IT1291164B1 (en) † | 1997-03-04 | 1998-12-29 | Coral Spa | UNIVERSAL DUCT FOR THE CONVEYANCE OF HARMFUL SMOKES OR GAS FROM A WORKING PLACE. |
| DE602006012489D1 (en) * | 2005-07-01 | 2010-04-08 | Roche Diagnostics Gmbh | CARBOXYLATED LATEX PARTICLES |
| RU2444541C2 (en) | 2006-08-24 | 2012-03-10 | Маллард Крик Полимерс, Инк. | Cationic latex as carrier for bioactive ingredients and methods of obtaining and using said latex |
Family Cites Families (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1237268A (en) * | 1967-11-01 | 1971-06-30 | Miles Lab | Proteinaceous reagent and process for producing same |
| GB1384399A (en) * | 1971-02-01 | 1975-02-19 | Hoffmann La Roche | Automated method of obtaining serological data |
| US3857931A (en) * | 1971-02-01 | 1974-12-31 | Hoffmann La Roche | Latex polymer reagents for diagnostic tests |
| US4118349A (en) * | 1971-05-12 | 1978-10-03 | Behringwerke Aktiengesellschaft | Process for the manufacture of polystyrene latex compounds |
| US3896217A (en) * | 1973-03-19 | 1975-07-22 | Summa Corp | Method and apparatus for radioimmunoassay with regeneration of immunoadsorbent |
| FR2321519A1 (en) * | 1975-08-22 | 1977-03-18 | Rhone Poulenc Ind | STYRENIC POLYMER LATEX |
| FR2331567A1 (en) * | 1975-11-13 | 1977-06-10 | Inst Nat Sante Rech Med | Fixation of organic cpds. contg. sugar residues - by oxidn. and coupling to a support contg. amino gps. |
| US4140662A (en) * | 1977-03-25 | 1979-02-20 | Ortho Diagnostics, Inc. | Attachment of proteins to inert particles |
| US4134872A (en) * | 1977-05-20 | 1979-01-16 | The Dow Chemical Company | Heterogeneous polymer particles comprising an interpolymer domain of a monovinylidene aromatic monomer, an open chain aliphatic conjugated diene and a monoethylenically unsaturated acid |
| CH628738A5 (en) * | 1977-08-03 | 1982-03-15 | Hoffmann La Roche | IMMUNOLOGICAL DIAGNOSTIC REAGENT. |
| US4156669A (en) * | 1978-04-24 | 1979-05-29 | The Dow Chemical Company | Latexes of encapsulated vinylidene chloride copolymer particles |
-
1977
- 1977-08-03 CH CH954277A patent/CH628738A5/en not_active IP Right Cessation
-
1978
- 1978-07-12 CA CA307,224A patent/CA1103153A/en not_active Expired
- 1978-07-27 AU AU38407/78A patent/AU528150B2/en not_active Expired
- 1978-07-31 US US05/929,410 patent/US4226747A/en not_active Expired - Lifetime
- 1978-08-01 FR FR7822676A patent/FR2399665A1/en active Granted
- 1978-08-02 DK DK343678A patent/DK343678A/en not_active Application Discontinuation
- 1978-08-02 JP JP9373978A patent/JPS5428816A/en active Pending
- 1978-08-02 EP EP78100581A patent/EP0000772A1/en not_active Withdrawn
- 1978-08-02 GB GB7831927A patent/GB2001996B/en not_active Expired
- 1978-08-02 NL NL787808143A patent/NL7808143A/en not_active Application Discontinuation
- 1978-08-02 DE DE19782833886 patent/DE2833886A1/en not_active Withdrawn
- 1978-08-02 SE SE7808349A patent/SE7808349L/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| DK343678A (en) | 1979-02-04 |
| DE2833886A1 (en) | 1979-02-22 |
| GB2001996A (en) | 1979-02-14 |
| FR2399665A1 (en) | 1979-03-02 |
| JPS5428816A (en) | 1979-03-03 |
| EP0000772A1 (en) | 1979-02-21 |
| CH628738A5 (en) | 1982-03-15 |
| SE7808349L (en) | 1979-02-04 |
| GB2001996B (en) | 1982-03-24 |
| NL7808143A (en) | 1979-02-06 |
| AU528150B2 (en) | 1983-04-14 |
| US4226747A (en) | 1980-10-07 |
| AU3840778A (en) | 1980-01-31 |
| FR2399665B1 (en) | 1982-11-05 |
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| MKEX | Expiry |