CN102103143A - Colloidal gold test strip for double-index detection and preparation method thereof - Google Patents
Colloidal gold test strip for double-index detection and preparation method thereof Download PDFInfo
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- CN102103143A CN102103143A CN2011100448777A CN201110044877A CN102103143A CN 102103143 A CN102103143 A CN 102103143A CN 2011100448777 A CN2011100448777 A CN 2011100448777A CN 201110044877 A CN201110044877 A CN 201110044877A CN 102103143 A CN102103143 A CN 102103143A
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Abstract
The invention belongs to the field of clinic medical tests, and in particular relates to a colloidal gold test strip for double-index detection and a preparation method thereof. The colloidal gold test strip for the double-index detection comprises a test strip substrate, a sample pad, a gold labeled antibody-coated polyester film, a coating film and water absorbent paper, wherein the sample pad, the gold labeled antibody-coated polyester film, the coating film and the water absorbent paper are mutually lapped and adhered on the test strip substrate in turn; the coating film is provided with a rabbit anti-mice IgG antibody-coated control line and two detection lines which are parallel to the control line; the two detection lines are used for coating antibodies which are specifically combined with one of antigens to be detected respectively; and two gold labeled antibodies are available and are the colloidal gold labeled antibodies which can be specifically combined with one of the antigens to be detected respectively. The test strip can simultaneously detect two indexes, has the advantages of accurate result and high specificity, and is easy and convenient to operate.
Description
Technical field
The invention belongs to the clinical medical inspection field, be specifically related to a kind of pair of index and detect with colloidal gold strip and preparation method thereof.
Background technology
For satisfying the fast detecting requirement of biological medicine detection range, adopt the test strip and the kit widespread use of Preparation of Colloidal Gold.But, because a reagent strip can only detect an index, and in the practice, often needing to detect simultaneously a plurality of indexs, the kit and the reagent strip that adopt a plurality of individual events to detect are operated just show loaded down with trivial details time-consuming respectively.
In order to address the above problem the Chinese patent (patent No.: 01242327.0) disclose a kind of multi-joint kit, can place many reagent strips, be used for detecting simultaneously several indexs.But this kit structure is complicated, also easy contaminated environment and both hands during toward the well application of sample.And also there are not at present a kind of method or means that can detect N-akrencephalon natriuretic peptide precursor and cardiac muscle troponin I simultaneously.
Summary of the invention
The objective of the invention is to above-mentioned deficiency, provide a kind of pair of index to detect and use colloidal gold strip, can solve the problem that can not in a test strips, detect two indexs simultaneously that prior art exists at prior art.
Another object of the present invention provides the preparation method of this test strips.
Purpose of the present invention can be by being achieved by the following technical solutions:
A kind of pair of index detects uses colloidal gold strip, comprise the test strips end liner, and the sample pad, the polyester film that is coated with golden labeling antibody, coated film and the thieving paper that overlap stickup on the test strips end liner in turn mutually, coated film is provided with bag by the control line of rabbit anti-mouse igg antibody, coated film also is provided with two detection lines parallel with described control line, respectively the bag by can with the antibody of one of antigen to be checked specific bond; Described golden labeling antibody has two kinds, be respectively colloid gold label can with the antibody of one of antigen to be checked specific bond.
The antibody of one of described and antigen to be checked specific bond be can with monoclonal antibody, polyclonal antibody, antibody fragment or the chimeric antibody of one of antigen to be checked specific bond.
A bag is by N-akrencephalon natriuretic peptide precursor polyclonal antibody in described two detection lines, and another bag is by the cardiac muscle troponin I monoclonal antibody.
The N-akrencephalon natriuretic peptide precursor monoclonal antibody that described golden labeling antibody is a colloid gold label and the cardiac muscle troponin I monoclonal antibody of colloid gold label.
A kind of pair of index detects the preparation method who uses colloidal gold strip, comprises the steps:
1) preparation coated film: with wrap be cushioned liquid dilute respectively can with the antibody and the rabbit anti-mouse igg antibody of one of antigen to be checked specific bond, and three kinds of antibody after will diluting are sprayed on respectively on the middle part of nitrocellulose filter abreast, and three kinds of antibody infiltrate the control line that forms two detection lines and rabbit anti-mouse igg antibody sandwich behind the nitrocellulose filter respectively;
2) polyester film of preparation coated with gold labeling antibody: prepare respectively two kinds of colloid gold labels can with the antibody of one of antigen to be checked specific bond, utilize this two kinds of mixtures of antibodies to apply polyester films;
3) assembling test strips: on the test strips end liner, overlap mutually in turn and paste sample pad, the polyester film that is coated with golden labeling antibody, coated film and thieving paper.
The described preparation coated film of step 1): be cushioned liquid dilution N-akrencephalon natriuretic peptide precursor polyclonal antibody, cardiac muscle troponin I monoclonal antibody and rabbit anti-mouse igg antibody with bag, and three kinds of antibody after will diluting are sprayed on respectively on the middle part of nitrocellulose filter abreast, three kinds of antibody infiltrate form respectively behind the nitrocellulose filters bag by the detection line of N-akrencephalon natriuretic peptide precursor polyclonal antibody, bag by the control line of the detection line of cardiac muscle troponin I monoclonal antibody and rabbit anti-mouse igg antibody.
Step 2) method of the polyester film of described preparation coated with gold labeling antibody is: collaurum is adjusted under the alkali condition, add streptavidin solution, behind the capping, add the antibody response that is connected to biotin, the centrifugal supernatant that goes, precipitation are preserved liquid with golden labeling antibody and are recovered volume.The described antibody that is connected to biotin preferably is connected to the N-akrencephalon natriuretic peptide precursor monoclonal antibody of biotin or is connected to the monoclonal antibody of the cardiac muscle troponin I of biotin.
Of the present invention pair of index detects colloidal gold strip and detects two kinds of application in the determined antigen at the same time.
The of the present invention pair of index detects colloidal gold strip and detects application in N-akrencephalon natriuretic peptide precursor and the cardiac muscle troponin I at the same time.
This test strips can be used for detecting the fluid sample of cardiac muscle troponin I and N-akrencephalon natriuretic peptide precursor, during operation, with whole blood or blood plasma, serum is added on the sample pad of this test strips by certain minim, when the result who occurs is when an aubergine band respectively occurring on the N-of test strips akrencephalon natriuretic peptide precursor detection line, cardiac muscle troponin I detection line and control line position, it is positive showing as the both.A red stripes respectively appears in cardiac muscle troponin I detection line and control line, illustrates that cardiac muscle troponin I is positive; A red stripes respectively appears in N-akrencephalon natriuretic peptide precursor detection line and control line, illustrates that N-akrencephalon natriuretic peptide precursor is positive; When only an aubergine band occurring on control line 8 positions of test strips, negative result; When the aubergine band not occurring on controlled 8 positions of test strips is null result.
Beneficial effect:
The present invention has adopted most popular colloidal gold technique in the modern POCT detection, by the control line of the detection line, the detection line that is coated with the cardiac muscle troponin I monoclonal antibody and the rabbit anti-mouse igg antibody that are coated with N-akrencephalon natriuretic peptide precursor polyclonal antibody is set on the test strips simultaneously, can two indexs of synchronous detection (determined antigen or albumen), and two kinds of antigen can the phase mutual interference, the result is accurate, fast, easy and simple to handle, can be widely used in myocardial damage, detect in the time of myocardial infarction and heart failure and use.
Description of drawings
The cross-sectional configuration synoptic diagram of Fig. 1 colloidal gold strip of the present invention;
Wherein: 1. end liner; 2. sample pad; 3. polyester film; 4. coated film; 5. thieving paper; 6. bag is by the detection line of N-akrencephalon natriuretic peptide precursor polyclonal antibody; 7. bag is wrapped by the control line of rabbit anti-mouse igg antibody by the detection line 8. of cardiac muscle troponin I monoclonal antibody.
Embodiment:
The present invention is further detailed explanation below in conjunction with the drawings and specific embodiments.
Embodiment 1
As shown in Figure 1, a kind of colloidal gold strip, comprise test strips end liner 1, and mutually the overlap joint sample pad 2 of pasting, polyester film 3, coated film 4 and the thieving paper 5 of coated with gold labeling antibody in turn on the test strips end liner, coated film 4 middle parts have a bag by the detection line 6 of goat-anti N-akrencephalon natriuretic peptide precursor polyclonal antibody, bag by the detection line 7 of cardiac muscle troponin I monoclonal antibody 4C2 and a bag by the control line 8 of rabbit anti-mouse igg antibody.Above antibody except N-akrencephalon natriuretic peptide precursor polyclonal antibody available from the strong glad Bioisystech Co., Ltd in Nanjing, other all is available from Fitzgerald company.
Have in the polyester film of coated with gold labeling antibody by the N-akrencephalon natriuretic peptide precursor monoclonal antibody M907241 of colloid gold particle mark with by the monoclonal antibody M155 of the cardiac muscle troponin I of colloid gold particle mark.
The concrete preparation method of colloidal gold strip is as follows:
1) preparation of coated film 4:
A) bag is cushioned the preparation of liquid: with 0.025M, the PBS of PH7.4 uses the 0.22u membrane filtration, place 4 ℃ standby, the term of validity 7 days.
B) preparation of confining liquid: will contain 1%BSA, 1% sucrose, 0.025M, the PBS of PH7.5 uses the 0.22U membrane filtration, place 4 ℃ standby, the term of validity is 3 days.
C) preparation of N-akrencephalon natriuretic peptide precursor polyclonal antibody detection line 6: the concentration that goat-anti N-akrencephalon natriuretic peptide precursor polyclonal antibody is pressed 2mg/ml, peristaltic pump is awarded liquid measure 0.4ml/min, line speed 50m/20min, 20 ℃ of forced air dryings are 12 hours in drying box.
D) preparation of cardiac muscle troponin I monoclonal antibody detection line 7: the concentration that cardiac muscle troponin I monoclonal antibody 4C2 is pressed 4.5mg/ml, peristaltic pump is awarded liquid measure 0.4ml/min, line speed 50m/20min, line on the nitrocellulose membrane middle part, this line is parallel with detection line 6, the two lines 4mm of being separated by, careful evenly 20 ℃ of forced air dryings 12 hours in drying box.
E) preparation of control line 8: with the concentration that rabbit anti-mouse igg antibody is pressed 8mg/ml, peristaltic pump is awarded liquid measure 0.4ml/min, line speed 50m/20min, in nitrocellulose membrane top line, this line is parallel with detection line 7, the two lines 4mm of being separated by, careful even, put into 20 ℃ of forced air dryings of drying box 12 hours.
F) nitrocellulose filter that will contain detection line 6, detection line 7 and controlled 8 with above-mentioned confining liquid is in 37 ℃ of sealings 60 minutes, take out rearmounted 37 ℃ down oven dry handled two hours, envelope is standby.
2) preparation of the polyester film 3 of coated with gold labeling antibody:
A) preparation of aqueous solution of chloraurate: with the dissolving of the tri-distilled water of 10g gold chloride 1000ml, be made into 1% aqueous solution, place 4 ℃ standby, the term of validity 3 months.
B) preparation of trisodium citrate: dissolve trisodium citrate with tri-distilled water, be made into 1% aqueous solution, with 0.22U membrane filtration mistake, place 4 ℃ standby, the term of validity 7 days.
C) preparation of 1% wet chemical: 1g sal tartari is dissolved with tri-distilled water with 100ml, with 0.22U membrane filtration mistake, place 4 ℃ standby, the term of validity is 7 days.
D) golden labeling antibody is preserved the preparation of liquid: with the sucrose of 15g, the Sodium azide of 20ul, in the 1%BSA dissolving of the PH=7.4 of 100ml, with 0.22U membrane filtration mistake, place 4 ℃ standby, the term of validity is 7 days.
E) preparation of the particle of collaurum: 1% gold chloride is diluted to 0.01% with tri-distilled water, place 95 ℃ of reactions 10 minutes, add the 1ml trisodium citrate, continue reaction 15 minutes, treat the colloidal gold solution color by indigo plant after purple stain is red, it is standby that cooling back adds the 2ml1% solution of potassium carbonate.Outward appearance should be pure, and is bright, do not have precipitation and floating thing.
F) preparation of golden labeling antibody: regulate collaurum pH value to 7.5 with 1% solution of potassium carbonate, amount according to 10ug streptavidin solution/ml collaurum adds the abundant mixing of streptavidin solution, place 25 ℃ of water-baths to add 5%BSA after 30 minutes again, seal after 20 minutes, the centrifuging and taking precipitation, recovering its final concentration with BSA is that 1%, 4 ℃ of preservation is standby.The amount of pressing 50ug monoclonal antibody-biotin/ml collaurum adds N-akrencephalon natriuretic peptide precursor monoclonal antibody M907241-biotin in the collaurum, 37 ℃ of stirring reactions 40 minutes, add 5%BSA, sealing was stirred 20 minutes, centrifugal 20 minutes of 6000r/min, abandon supernatant, precipitation is preserved liquid with golden labeling antibody and is recovered volume, 4 ℃ of preservations.The monoclonal antibody M155 of gold mark cardiac muscle troponin I prepares as stated above.Two kinds of golden labeling antibodies were mixed 4 ℃ of preservations by 1: 1.
G) collaurum that mixes is layered on the polyester film uniformly, discharge rate is the 1.0ul/cm*3 road, drying, and envelope, standby.
3) processing of sample pad 2
Sample pad is soaked a few hours with 100mM PBS damping fluid, after the taking-up drying.
4) assembling of colloidal gold strip:
End liner 1, sample pad 2 and thieving paper 5 are the general parts in this area.Above-mentioned sample pad 2, the polyester film 3 that is coated with golden labeling antibody, coated film 4 and thieving paper 5 are overlapped stickup in turn mutually obtain test paper plate, the test strips that at last test paper plate is cut into different in width gets final product.
Embodiment 2
The test of N-akrencephalon natriuretic peptide precursor (NT-proBNP) blood sample:
Measure the serum specimen of the N-akrencephalon natriuretic peptide precursor that contains different definite values through the supporting detection kit of the present invention of Luo Shi E170 Chemiluminescence Apparatus, the difference of the degree that relatively develops the color and reference serum the results are shown in Table 1.
Table 1
The test of cTnI blood sample:
Measure the serum specimen of the cardiac muscle troponin I (cTnI) that contains different definite values through the supporting detection kit of the present invention of Luo Shi E170 Chemiluminescence Apparatus, the difference of the degree that relatively develops the color and reference serum the results are shown in Table 2.
Table 2
CTnI and the test of NT-proBNP blood sample:
Measure the NT-proBNP that contains different definite values and the serum specimen of cTnI simultaneously through the supporting detection kit of the present invention of Luo Shi E170 Chemiluminescence Apparatus, the difference of the degree that relatively develops the color and reference serum the results are shown in Table 3.
Table 3
The blood sample test of no cTnI and NT-proBNP:
Through the different definite value serum specimens that supporting cTnI detection kit of Luo Shi E170 Chemiluminescence Apparatus and NT-proBNP detection kit are measured simultaneously, the difference of the degree that relatively develops the color and reference serum the results are shown in Table 4.
Table 4
By result 4 visible detection numerical value cTnI<1.0ng/ml as a result, NT-proBNP<300pg/ml is shown as negative findings.
Positive:
Article two, or three aubergine bands occur, one is positioned at Quality Control district (C), if another is positioned at NT-proBNP test section (T), can judge that the NT-proBNP index is positive; Equally, if another is positioned at cTnI test section (T), can judge that the cTnI index is positive; If be positioned at NT-proBNP test section (T) and red stripes all appears in cTnI test section (T), can judge that NT-proBNP and cTnI index are all positive.
Negative:
Only an aubergine band appears in Quality Control district (C), (T) no aubergine band occurs or aubergine band of Quality Control district (C) appearance in the test section, colour developing of (T) NT-proBNP detection line and colour developing degree<"+" in the test section, show and do not contain cTnI in the sample, but or cTnI content be lower than sensing range; NT-proBNP content is lower than 300pg/ml.
The concentration of NT-proBNP or cTnI is recorded by Luo Shi E170 Chemiluminescence Apparatus in table 1~table 4, the result that the colour developing degree records for test strips of the present invention, "+" expression positive findings, the order of severity that the quantitaes colour developing degree of "+" is promptly positive, many more representative colour developings are dark more to be that positive severity is high more, and it is negative findings that "-" expression does not develop the color.
In above embodiment, various processes of Xiang Ximiaoshuing and method are not conventional methods as known in the art.
The above only is preferred embodiment of the present invention, is not to be the restriction of the present invention being made other form, and any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the equivalent embodiment of equivalent variations.But every technical solution of the present invention content that do not break away to any simple modification, equivalent variations and remodeling that above embodiment did, still belongs to the protection domain of technical solution of the present invention according to technical spirit of the present invention.
Claims (10)
1. two indexs detect and use colloidal gold strip, comprise the test strips end liner, and the sample pad, the polyester film that is coated with golden labeling antibody, coated film and the thieving paper that overlap stickup on the test strips end liner in turn mutually, coated film is provided with a bag by the control line of rabbit anti-mouse igg antibody, it is characterized in that coated film also is provided with two detection lines parallel with described control line, respectively the bag by can with the antibody of one of antigen to be checked specific bond; Described golden labeling antibody has two kinds, be respectively colloid gold label can with the antibody of one of antigen to be checked specific bond.
2. the according to claim 1 pair of index detects colloidal gold strip, the antibody that it is characterized in that one of described and antigen to be checked specific bond be can with monoclonal antibody, polyclonal antibody, antibody fragment or the chimeric antibody of one of antigen to be checked specific bond.
3. according to claim 1 pair of index detects colloidal gold strip, it is characterized in that a bag is by N-akrencephalon natriuretic peptide precursor polyclonal antibody in described two detection lines, and another bag is by the cardiac muscle troponin I monoclonal antibody.
4. the according to claim 3 pair of index detects colloidal gold strip, it is characterized in that the N-akrencephalon natriuretic peptide precursor monoclonal antibody that described golden labeling antibody is a colloid gold label and the cardiac muscle troponin I monoclonal antibody of colloid gold label.
5. the described a kind of pair of index of claim 1 detects the preparation method who uses colloidal gold strip, it is characterized in that comprising the steps:
1) preparation coated film: with wrap be cushioned liquid dilute respectively can with the antibody and the rabbit anti-mouse igg antibody of one of antigen to be checked specific bond, and three kinds of antibody after will diluting are sprayed on respectively on the middle part of nitrocellulose filter abreast, and three kinds of antibody infiltrate the control line that forms two detection lines and rabbit anti-mouse igg antibody sandwich behind the nitrocellulose filter respectively;
2) polyester film of preparation coated with gold labeling antibody: prepare respectively two kinds of colloid gold labels can with the antibody of one of antigen to be checked specific bond, utilize these two kinds golden labeling antibody potpourris to apply polyester films;
3) assembling test strips: on the test strips end liner, overlap mutually in turn and paste sample pad, the polyester film that is coated with golden labeling antibody, coated film and thieving paper.
6. a kind of pair of index according to claim 5 detects the preparation method with colloidal gold strip, it is characterized in that the described preparation coated film of step 1): be cushioned liquid dilution N-akrencephalon natriuretic peptide precursor polyclonal antibody with bag, cardiac muscle troponin I monoclonal antibody and rabbit anti-mouse igg antibody, and three kinds of antibody after will diluting are sprayed on respectively on the middle part of nitrocellulose filter abreast, and three kinds of antibody infiltrate the cellulose nitrate thickness and form bag respectively by the detection line of N-akrencephalon natriuretic peptide precursor polyclonal antibody, bag is by the control line of the detection line of cardiac muscle troponin I monoclonal antibody and rabbit anti-mouse igg antibody.
7. a kind of pair of index according to claim 5 detects the preparation method with colloidal gold strip, it is characterized in that step 2) method of the polyester film of described preparation coated with gold labeling antibody is: collaurum is adjusted under the alkali condition, add streptavidin solution, behind the capping, add the antibody response that is connected to biotin, the centrifugal supernatant that goes, precipitation are preserved liquid with golden labeling antibody and are recovered volume.
8. a kind of pair of index according to claim 7 detects the preparation method with colloidal gold strip, it is characterized in that the described antibody that is connected to biotin is the monoclonal antibody that is connected to the N-akrencephalon natriuretic peptide precursor monoclonal antibody of biotin or is connected to the cardiac muscle troponin I of biotin.
9. described pair of index of claim 1 detects colloidal gold strip and detects two kinds of application in the determined antigen at the same time.
10. described pair of index detection of claim 1 colloidal gold strip detects the application in N-akrencephalon natriuretic peptide precursor and the cardiac muscle troponin I at the same time.
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