WO1991013358A1

WO1991013358A1 - Stereochemically controlled immobilization of active molecules

Info

Publication number: WO1991013358A1
Application number: PCT/US1991/001094
Authority: WO
Inventors: Willfried Schramm
Original assignee: Bioquant, Inc.
Priority date: 1990-02-23
Filing date: 1991-02-19
Publication date: 1991-09-05
Also published as: AU7457791A

Abstract

A method of immobilizing a protein on the surface of a support matrix includes the steps of binding the protein to the surface of the support matrix, each of the protein molecules including an active domain. A second molecule is bound to the matrix about the proteins. A dense lattice is formed of the second molecule about the protein to maintain the active domain of the protein exposed away from the surface of the support matrix. A matrix made in accordance with the above method is also disclosed. The matrix includes a support surface and a dense lattice bound to the support surface. The dense lattice includes primary molecules, such as proteins, bound to the support surface. The primary molecules include active domains. Secondary molecules are bound to the support surface about the primary molecules for maintaining the active domains of the primary molecules exposed away from the support surfaces.

Description

STEREOCHEMICALLY CONTROLLED IMMOBILIZATION OP ACTIVE MOLECULES

TECHNICAL FIELD

The present invention relates to matrices of the type having reactive molecules bound thereto and methods of immobilizing the reactive molecules on the surface of the matrix. More specifically, the present invention relates to the immobilization of proteins having active sites thereon onto support matrices.

BACKGROUND ART

Proteins have been immobilized to solid matrices for a number of different applications. Enzymes bound to gels can be used for bioprocessing (U.S. Patent 4,572,897, S. A oz et al; U.S. Patent 4,246,350, E.D. Hier and J.P. Oriel). Immobilized binding proteins such as Protein A or Protein G have been applied in chromatographic columns to purify antibodies (U.S. Patent 4,801,.687, T.T. Ngo) . For analytical processes such as immunoassays, antibodies have been immobilized on many different surfaces. For most applications, at least two criteria are important in the process of immobilizing proteins on a solid matrix: a) the protein should be permanently bound to the surface, and b) the active center of the protein should remain intact.

While prior art teaches different methods for immobilization of proteins, these methods do not allow for controlled binding of the proteins so that the active center is freely accessible. The most commonly used method is binding of proteins via the -amino groups of lysine residues. This frequently results in loss of specific binding activity of the bound molecule due to multi-site attachment and multiple orientations of the bound species (Schneider et al 1982) .

Proteins bind readily to surfaces by physical adsorption (Cantarero et al 1980; Morrissey and Han 1976) . Although this method of immobilization is simple to accomplish, the disadvantage is that substantial amounts can be released during incubation with medium (Zollinger et al 1976; Lehtonen and Viljanen 1980).

To assure permanent binding, proteins are usually chemically bound to modified or non-modified solid matrices (U.S. Patent 4,363,634, R.F. Schall, Jr.; U.S. Patent 4,419,444, G.A. Quash; U.S. Patent 4,562,157; C.R. Lowe et al; U.S. Patent 4,582,875, T.T. Ngo) . Cross-linking of active protein molecules to inactive protein species for the purpose of forming polymers, films or foams with or without a solid support has also been described (U.S. Patent 4,464,468 S. Avrameas et al) . However, this prior art does not teach methods for the immobilization of proteins such that the active center remains intact. Prior art teaches the immobilization of protein via a second binding protein: either via Protein A or via Protein G, both of which bind specifically to the Fc region of many IgGs (Schramm et al 1987) or via the Fab fragment of an antibody that also binds specifically the Fc region of the primary IgG (Sankolli et al 1987) . Although this enhances substantially the reproducibility of immobilizing IgGs to surfaces, it does not guarantee ends-on immobilization. Ends-on immobilization refers to immobilization with the Fab region exposed, as discussed below. Another disadvantage of this method is that the interaction of the IgG and the second binding protein is reversible; i.e. if the sample to be analyzed contains IgG itself (e.g. blood serum samples) , the specific antibody might be dissociated from Protein A or another such binding protein, hence, potentially providing false analytical results.

Other prior art describes the chemical immobilization of IgGs to modified surfaces.

Introducing to the solid matrix an abundance of a ino groups, the carbohydrate residues on the IgG can be used to chemically bind the protein to the surface (U.S. Patent 4,419,444 G.A. Quash). Although this method precludes potential dissociation of the IgG, it does not prevent side-on immobilization of the antibody of the surface; that is, binding of both the Fc region and part of the Fab region, as discussed in detail below. Immobilized immunoglobulins are often used in analytical systems (solid-phase immunoassays) . Since IgG can very selectively detect antigens in extremely small concentrations, a small amount of IgG immobilized on the surface of the solid matrix provides a more sensitive assay. However, the larger the surface available for the IgG molecule, the easier it is to assume a thermodynamically favorable position by matching its lipophilic/hydrophilic domains with that of the surface. Consequently, the active site of the protein might bind to the solid matrix. With regard to IgGs, this means that fewer idiotypic sites are accessible.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided a method of immobilizing a molecule on a surface of a support matrix. The method includes the steps of binding primary molecules to the surface of a support matrix, each of the primary molecules including an active domain. Secondary molecules are bound to the matrix about the primary molecules. A dense lattice of the secondary molecules is formed about the primary molecules to maintain the active domain of the primary molecules exposed away from the surface of the support matrix.

The present invention further provides a matrix of the type having reactive molecules bound thereto. The matrix includes a support surface and a dense lattice bound to the support surface. The dense lattice includes the primary molecules bound to the support surface, the primary molecules including active domains. The dense lattice further includes secondary molecular means bound to the support surface about the primary molecules for maintaining the active domains of the primary molecules exposed away from the support surface.

FIGURES IN THE DRAWINGS

Other advantages of the present invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein:

Figure 1 is a schematic structure of an immunoglobulin G (IgG) . The protein consists of two identical heavy chains and two identical light chains. Part of the heavy-light chain combination forms the idiotypic (antigen binding) site. The IgG is shown to be bivalent;

Figure 2 is a schematic representation of the immobilization of IgG in different configurations;

Figure 3 is a schematic representation of the chemical binding of IgG to an activated surface; Figure 4 is a schematic representation of the immobilization of IgG in the presence of a secondary molecule to a polymer on a solid matrix; Figure 5 is a bar graph showing uniform binding of 125i-labeled derivative of the progesterone (counts per minute, cpm) to a monoclonal antibody immobilized in the presence of avidin, a bar represents the average of 8 wells of one strip, the standard deviations being indicated by the symbols, the total variation of binding between 96 wells being 3%;

Figure 6 is a bar graph showing the same amount of monoclonal antibody as shown in Figure 1 being immobilized to microwells in the absence of avidin, higher well-to-well and strip-to-strip variation being observed, the average amount of antibody per well being lower, the total variation of binding between 96 wells being 9%;

Figure 7 is a graph showing low variability in a dose response curve for testosterone in a solid-phase assay where the antibody was chemically bound to the modified surface of microwells in the presence of an excess of Fc fragments of a nonspecific antibody as a secondary molecule to support the orientation of the antibody molecules, the bars indicating the standard deviation of triplicates; Figure 8 is a graph showing the high variation in a dose response curve for testosterone in a solid-phase assay on microwells, the wells being prepared as described in Figure 7 with the exception that the antibody was immobilized in the absence of Fc fragments; and

Figure 9 is a graph showing the immobilization of different concentrations of antibody to LH in the presence (A) and in the absence (B) of fluorescein on derivatized surfaces of glass tubes, the higher binding of the antibody in the presence of fluorescein being attributed to better accessibility of the idiotypic sites of the IgG molecules.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is provided a matrix of the type having reactive molecules bound thereto and a method of immobilizing the molecules on the surface of the support matrix. Generally, the matrix made in accordance with the present invention includes a support surface. Examples of support surfaces are silanized glass, plastic materials, and other support surfaces well known in the art. A dense lattice is bound to the support surface. The dense lattice includes primary molecules bound to the support surface, the primary molecules including active domains. The primary molecules can be proteinacious, such as antibodies, enzymes, as well as nonproteinacious materials having active sites, such as carbohydrates or other molecules. Secondary molecules, as described below in greater detail, are bound to the support surface about the primary molecules for maintaining the active domains of the primary molecules exposed away from the support surface.

The importance of maintaining the active domains or active centers of a protein or other molecules after immobilization can be demonstrated by utilizing an immunoglobulin as an example of a primary molecule. Immunoglobulins (antibodies) are proteins that specifically bind their counterparts, the antigens. Immunoglobulins are used widely in analytical procedures such as immunoassays for the quantitative or qualitative detection of antigens, the antigens being the analytfe_. of the assay. Many of these procedures use immobilized immunoglobulins. A subclass of these proteins, im unoglobulin G (IgG) , consist of two identical heavy chains and two identical light chains. The structure of these IgGs is often symbolized by a shape resembling the letter "Y^w as shown in Figure 1. To clarify the present invention, some features of an IgG molecule are described. Two major regions of the protein structure have been defined as the Fab region 10 and the Fc region 12. While the Fc region is not involved in antigen binding, the Fab region contains a very distinct sequence of amino acids that is unique for each population of antibodies, referred to as an idiotypic site. For antibodies that recognize an antigen, the idiotypic site binds specifically to the antigen, this site being referred to as the antigen site. Since IgG contains two identical idiotypic sites, the binding protein is considered bivalent. On what is referred to as the heavy chain in the Fc region is a sequence of carbohydrate groups 16.

The immobilization of IgGs on solid matrices can occur in different configurations. An example of several of these configurations is shown in Figure 2. Several IgG molecules 18,20,22,24 are shown bound to a support surface 26 of a matrix 28. Most desirable is the "ends-on" immobilization of the molecule labeled 18 via the end of the Fc region that does not participate in antigen binding. In this configuration, the idiotypic sight is directed towards the reaction solution, the idiotypic site being the active domain of the molecule.

The least desirable configuration is a potential "top-on" immobilization, as shown by molecule 24 because the idiotypic site is not accessible for antigen binding. In other words, the active domain of the molecule 24 is bound to the matrix 28 as opposed to being exposed.

Most often, the protein will bind to the surface 26 as shown by molecules 20,22 with either the lipophilic domains or hydrophilic domains (depending on the physical properties of the surface 26) of the molecules 20,22 in a "side on" configuration. Thus, antigens, in particular large antigens or antigen-enzyme conjugates frequently used as signal generators in immunoassays, have only limited access to the active sites of the binding protein.

As discussed in the Background Art section, IgGs have been chemically immobilized to modified surfaces. Although these methods proclude potential dissociation of the IgG, the methods do not prevent side-on immobilization of the antibody to the surface as shown schematically in Figure 3. In Figure 3, an abundance of a ino groups 30 are disposed about a modified surface 32 of a matrix 34. Preferably, the carbohydrate residues 16 of the IgG molecules schematically illustrated are used to chemically bind the protein to the amino groups 30 on the surface 32 of the matrix 34. This still tends to result in many side-on immobilized antibodies bound to the surface 32 of the matrix 34. The present invention provides for the co- immobilization of two molecules to form a lattice on the surface of the solid matrix. The primary molecule, such as an enzyme or a binding protein such as IgG, is bound to the support surface. The secondary molecule serves as a filler to maintain a lattice that keeps the active domain of the primary protein exposed for its specific reaction.

One embodiment of this invention describes the chemical binding of an IgG (antibody) to different surfaces, although those skilled in the arts can apply similar methods to other active proteins. The surfaces used for immobilization might contain functional groups that are suitable for chemical reactions with protein (chemical binding) . On the other hand, other forces (e.g., lipophilic or electrostatic interactions ) can be used to immobilize the primary protein and the secondary molecule (physical binding) . Alternatively, combinations of chemical and physical binding for the primary protein and the secondary molecule might be adequate for many applications.

Surfaces can be activated to introduce functional groups for chemical binding. For example, glass has been silanized with -aminopropyl silanes to introduce an abundance of amino groups on the surface for further reaction. (Weetall and Filbert 1974) . The introduction of other functional groups (thio, carboxyl, aldehyde, oxirane, etc.) and variations of the method have been described (Japan Kokai 78 29,921, A. Kosaka; Fr. Demande 2,435,715, J. Kikutake et al; U.S. Patent 4,273,865, O. von Stetten et al; U.S. Patent 4,118,536, J. L. Beardsley et al; U.S. Patent 4,210,722, S.F. Silver; U.S. Patent 4,794,090, M.E. Parham et al; U.S. Patent 4,757,014, C.E. Hendrickson et al; U.S. Patent 4,654,299, D. Lentfer) .

Another method for introducing functional groups is to coat the surfaces either with a suitable polymer (e.g. , poly-lysine) or with protein molecules which are subsequently chemically cross-linked. Although this provides a coat that is physically adsorbed, there is an excess of attachment points between the polymer and the solid matrix so that the binding energy between the two entities essentially approaches a chemical bond.

If an IgG is chemically bound to the modified surface of the solid matrix by its carbohydrate groups, the chemical reaction will not alter the idiotypic binding site. However, as shown in Figure 3, the antibody binding site may still be sterically hindered. Therefore, a preferred embodiment of the present invention includes a chemically modified oligosaccharide at the IgG so that a functional group is created which extends from the body of the protein molecule thereby introducing a spacer link. To preserve the antibody binding site on the IgG, the carbohydrate residue on the Fc region is also, utilized for the introduction of the spacer molecule (Figure 4) .

In Figure 4, a matrix 36 is shown having a chemically modified surface, such as having amino groups 38 extending therefrom. Secondary molecules are schematically shown at 40 forming a lattice with primary molecules in the form of IgG molecules 42. Carbohydrate sites 44 of the molecules 42 are chemically modified so that functional groups 46 are created which extend from the body of the protein molecule to be bound at 48 to the amino groups 38 on the surface of the matrix 36.

Alternatively, the surface of the solid matrix can be modified such that active functional groups extend far enough from the surface to form a suitable linker arm for unimpared immobilization of the primary protein. However, an extension between the solid matrix and the primary molecule by means of a linker arm might not be required for many applications.

The secondary molecule is co-immobilized with the primary protein. The objective is to obtain a dense packing of a monomolecular layer of two molecules so that the primary protein cannot collapse towards the surface. This can be achieved by spatially filling the spaces between the primary protein with other molecules, or by changing the surface charge with secondary molecules so that electrostatic interaction between the primary protein and the surface is reduced. A combination of both methods is possible.

For example, if an immunoglobulin is immobilized in the presence of an Fc fragment from another IgG, the fragments occupy the spaces between the immunoglobulin molecules. Chemical binding of an IgG to amino groups from poly-lysine in the presence of excess fluorescein is based on another mechanism. The two acidic groups of fluorescein can easily interact with excess amino groups from poly-lysine on the surface of the solid matrix. This changes the surface charge on the polymer and, therefore, the electrostatic environment of the immobilized IgG. A combination of the two above mentioned effects constitutes the co-immobilization of avidin and immunoglobulin. Avidin fills the available spaces between the IgG molecules and also creates a modified electrostatic environment due to its high content of carbonhydrate residues and its high isoelectric point (pl=10.5) . For some applications, such as analytical procedures (e.g. , immunoassays) , the concentration of the primary protein must be controlled. For example, for antibodies with a high affinity constant to the antigen and for highly sensitive assays, the amount of antibody forming a monomolecular layer of dense packing on the surface of the solid matrix would be too high. Thus, although a monomolecular layer of dense packing of molecules is desired, only a fraction is an IgG and the remaining component constitutes the secondary molecule (Figure 4) . This can be achieved by reacting an appropriate ratio of primary protein and secondary molecule.

Some primary proteins have the tendency to lay flat on the surface, depending on the lipophilicity of protein and surface. A secondary molecule can prevent the primary proteins from laying flat.

The sequence of binding the primary protein and molecule to the surface can vary from application to application. If the primary protein is bound first, it is erected from its potential side-on configuration as the secondary molecule diffuses to the surface. A functional group with an extended link to the primary protein might facilitate its erection.

In some embodiments, the active group on the surface might also be present in the primary protein (e.g., if it is an amino group). Therefore, the secondary molecule could bind to the primary protein instead of to the modified surface. However, by introducing an excess of activated groups to the surface (e.g., with amino-silanized glass, or coating with poly-lysine of plastic materials) , the probability of encountering an activated group for reaction on the surface far exceeds the probability of the secondary molecule meeting a similar group on the primary protein.

EXAMPLES

Example 1:

This example describes the co- immobilization of a monoclonal antibody and avidin to microplate wells which were coated with poly-lysine. The antibody binds specifically the steroid hormone progesterone.

Preparation of derivatized IgG. Immunoglobulins were precipitated by adding an equal volume of 90% saturated ammonium sulphate, pH 7.4, at 4°C to the serum (for polyclonal antibodies) or to peritoneal exudates from mice inoculated with hybridoma (monoclonal antibodies) . Recovered immunoglobulin was dissolved in phosphate buffer and precipitated twice again. The protein was further purified on DEAE-Sephadex gel. The gel was equilibrated to Tris buffer (10 mmol/L, pH 8.0). To a column containing 20 mL of gel-bed volumne, 1 L of a solution of 5 mg/mL partially purified immunoglobulin was applied. The protein was eluted with phosphate buffer containing different concentrations of sodium chloride: 50,100, 200, and 300 mmol/L. The IgG fraction eluted with the buffer containing 200 mmol/L. The protein concentration was determined by UV-spectrophotometry at 280 nm. The immunoglobulin was dissolved in sodium acetate buffer (5 mg/mL, 32.2 nmol in 4 mL) and reacted with 0.65 mg of NaI0.(3 umol) in 1 mL of sodium acetate buffer, pH 4.5 for 2 hours at room temperature. The oxidized IgG was separated by gel chromatography on P-30 (equilibrated in sodium acetate buffer; 6 L of gel-bed volume; 1 mL reaction mixture applied per column) . The fractions 4 and 5 were collected (0.5 mL fraction) and all fractions containing derivatized IgG were combined. An amount of 94.1 g of N-aminocaproyl-galactopyranose (0.3 mol) was dissolved in 1 mL of sodium acetate buffer (10 molar excess based on IgG) . The IgG (fractions 4 + 5 from chromatography) was added to this solution and kept at 4"C overnight. The reaction mixture was dialyzed against phosphate buffer (0.05 mol/L, pH 7.4) . The protein concentration was adjusted to 1 mg/mL (determined by UV-spectrophotometry at 280 nm) .

Immobilization of IgG. Polystyrene microwells consisting of 12 strips with 8 brake-apart wells (Costar, Cambridge, MA) were filled with 200 uL of a solution of 1 mg/L of poly-lysine in carbonate buffer (0.05 mol/L sodium carbonate, pH 9.6) and incubated overnight at room temperature in a closed container. The plates were emptied and washed three times with 300 uL of de-ionized water. A solution containing 0.7 nmol/L of the derivatized antibody, 3.5 nmol/L avidin, and 0.3 mmol/L of sodium periodate in phosphate buffer (0.05 mol/L of phosphate, pH 7.4) was distributed in a volume of 200 uL into the poly- lysine treated wells and the plates were incubated at 4"C in closed containers in the dark for 24 h.

Thereafter, the incubation solution was discarded, the wells washed three times with 300 uL of de- ionized water, air dried, and stored in a closed container with silica gel as desiccant at 4"C. Immobilization of antibody in the presence of avidin increases the uniformity of the immunoglobulin coat. This is expressed in a small well-to-variation of antigen binding (Figure 5) . In comparison, if the same amount of immunoglobulin is bound to the wells in the absence of a secondary molecule, a higher variation of binding between the wells and between the strips of on plate of microwells is observed (Figure 6) . In this case, the "edge effect", i.e., significantly different binding around the periphery of one plate (Burt et al, 1979; Kricka et al, 1980) becomes clearly prominent. Example 2 :

Polyclonal antibodies to the steroid hormone testosterone were co-immobilized on polystyrene microwells with the Fc fragment obtained from an antibody that does not specifically bind a steroid. The microwells were coated with cross- linked BSA that contained amino-groups with an extended linker arm for coupling.

Preparation of IgG. Immunoglobulin was precipitated from a sheep anti-testosterone antiserum with ammonium sulphate as described in Example 1. The IgG fraction was dialyzed against phosphate buffer and used without further purification.

Preparation of Fc fragments. Fc fragments were obtained from a monoclonal mouse antibody with no specificity to testosterone by proteolytic papain digestion generally known to those skilled in the arts (Mage, 1980) .

Immobilization of IgG. Polystyrene microwells were filled with a solution of 2 ug/mL of bovine serum albumin (BSA) in 0.05 mol/L of phosphate buffer (pH 7.4) and 7.5 uL/mL of glutardialdehyde. This forms a monomolecular layer of crosslinked protein on the surface of the microwells. The wells were incubated for 4 hours at room temperature and washed three times with de-ionized water, filled with 200 uL of a solution of 0.01 mmol/L of sper idine trihydrochloride in phosphate buffer and incubated for 2 hours at room temperature. This deactivates excess aldehyde groups and introduces an excess of amino groups with an extended linker arm. A solution containing 0.6 nmol/L of IgG, 5.4 nmol/L of Fc fragment, and 0.3 mmol/L of sodium periodate in phosphate buffer was distributed in a volume of 200 uL into the BSA-treated wells and the plates were incubated at 4^βC in closed containers in the dark for 24 hours. Thereafter, the incubation solution was discarded, the wells washed three times with 300 uL of de-ionized water, air dried, and stored at 4^βC in a closed container with silica gel as desiccant. Dose-response curves with wells prepared as described above yielded triplicates with a low variability at different concentrations of testosterone (Figure 7) . If the Fc fragment was omitted during the immobilization reaction, the variation of triplicates in a dose-response curve was significantly higher (Figure 8) . E ample 3 :

A monoclonal antibody to the prostanoid thromboxane B, was co-immobilized with a monoclonal antibody that does not bind specifically a prostaglandin on polystyrene wells. The wells were coated with poly-lysine.

Preparation of IgG. Immunoglobulin was purified by affinity chromatography on immobilized Protein A (Goding, 1978) . Immobilization of IgG. Polystyrene microwells were coated with poly-lysine as described in Example 1. A solution containing 3.3 nmol/L of a monoclonal antibody to thromboxane B_, 13.3 nmol/L of the non-specific antibody, and 0.3 mmol/L of sodium periodate in phosphate buffer (0.05 mol/L of phosphate, pH 7.4) was distributed in a volume of 200 uL into the poly-lysine treated wells and the plates were incubated at 4^βC in closed containers in the dark for 24 hours. Thereafter, the incubation solution was discarded, the wells washed three times with 300 uL of de-ionized water, air dried, and stored in a closed container with silica gel as desiccant at 4"C. Example 4 :

A monoclonal antibody to luteinizing hormone (LH) was co-immobilized with fluorescein on glass test tubes. The test tubes were derivatized with -aminopropyl silane (Weetall, 1976) . Preparation of derivatized IgG. Immunoglobulin was used after ammonium sulphate precipitation. N-aminocaproyl-galatopyranosylamine was bound to the antibody as described in Example 1 to attach a linker arm to the molecule.

Immobilization of IgG. The surface of borosilicate test tubes (12 x 75 mm) was derivatized with -amino propyl tri ethoxysilane under acid catalysis. The test tubes were washed with de- ionized water and baked for 4 hours at 120"C. A solution containing 3.2 nmol/L of a monoclonal antibody to LH, 8 umol/L of the sodium salt of fluorescein, and 0.3 mmol/L of sodium periodate in phosphate buffer (0.05 mol/L of phosphate, pH 7.4) was distributed in a volume of 500 uL into the tubes and incubated at 4°C in closed containers in the dark for 24 hours. Thereafter, the incubation solution was discarded, the tubes washed three times with l mL of de-ionized water, air dried, and stored in a closed container with silica gel as desiccant at 4^βC. The above Examples illustrate the effect of the present invention which provides an increased concentration of active domains per unit volume of matrix having primary molecules bound thereto (comparing Figures 5 and 6 and also comparing Figures 7 and 8) . Further, the Examples illustrate the higher binding of antibody in the presence of fluorescein (Figure 9) which is attributed to better accessibility of the idiotypic sites of the IgG molecules on matrices made in accordance with the present invention. Therefore, the present invention provides a matrix and a process of immobilizing proteins on a solid matrix wherein the protein is permanently bound to the surface and the active center or domain of the protein remains intact and exposed.

The invention has been described in an illustrative anner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims wherein reference numerals are merely for convenience and are not to be in any way limiting, the invention may be practiced otherwise than as specifically described.

Claims

What is claimed is:

1. A method of immobilizing a molecule on a surface of a support matrix, said method including the steps of: binding primary molecules to the surface of the support matrix, each of the primary molecules including an active domain; binding secondary molecules to the matrix about the primary molecules; and forming a dense lattice of the secondary molecules about the primary molecules to maintain the active domain of the primary molecules exposed away from the surface of the support matrix.

2. A method as set forth in claim 1 wherein said forming step is further defined as forming a monolayer of the primary and secondary molecules in the lattice, the secondary molecules preventing the collapsing of the primary molecules towards the support matrix.

3. A method as set forth in claim 1 wherein said first binding step is further defined as immobilizing proteinacious molecules to the surface of the support matrix, each of the proteinacious molecules including the active domain.

4. A method as set forth in claim 1 wherein said forming step is further defined as sterically interacting the secondary molecules with the primary molecules and preventing the primary molecules from collapsing towards the support matrix.

5. A method as set forth in claim 1 further including the step of reducing the electrostatic interacting of the bound primary molecules with the surface of the support matrix to prevent the primary molecules from collapsing towards the support matrix.

6. A method as set forth in claim 5 wherein said reducing step is further defined as changing the surface of the support matrix with the bound secondary molecules and electrostatically interacting the secondary molecules with the primary molecules.

7. A method as set forth in claim 6 wherein the matrix possesses lipophilic or hydrophilic properties, further including the step of modifying the lipophilic or hydrophilic matrix to potentiating said electrostatic interacting step.

8. A method as set forth in claim 1 further including the step of adding functional groups to the support matrix.

9. A method as set forth in claim 1 wherein said first binding step is further defined as chemically reacting the primary molecules with the surface of the matrix.

10. A method as set forth in claim 7 further including the step of chemically binding a functional group of the primary molecule to position the molecule at a preferred distance from the support matrix.

11. A method as set forth in claim 1 wherein said first binding step is further defined as physically reacting the primary molecules with the surface of the matrix.

12. A method as set forth in claim 1 wherein said second binding step chemically binding a functional group of the secondary molecule to position the molecule at a preferred distance from the support matrix.

13. A method as set forth in claim 12 further including the step of chemically binding a functional group of the secondary molecules including adding functional groups to the support matrix.

14. A method as set forth in claim 1 wherein said second binding step is further defined as physically reacting the secondary molecules with the surface of the matrix.

15. A method as set forth in claim 1 further including the step of chemically interacting the primary and secondary molecules.

16. A method as set forth in claim 1 further including the step of physically interacting the primary and secondary molecules.

17. A method as set forth in claim 1 further including the step of binding spacer molecules to the matrix to distance the primary and/or secondary molecules from the support matrix at predetermined distance.

18. A matrix of the type having reactive molecules bound thereto, said matrix comprising: a support surface and a dense lattice bound to said support surface, said dense lattice including primary molecules bound to said support surface, said primary molecules including active domains and secondary molecules bound to said support surface about said primary molecules for maintaining the active domains of the primary molecules exposed away from said support surface.

19. A matrix as set forth in claim 18 wherein said lattice consists of a monolayer of said primary molecules and said secondary molecular means.

20. A matrix as set forth in claim 18 wherein said primary molecules are proteinacious.

21. A matrix as set forth in claim 20 wherein said primary molecules are antibodies.

22. A matrix as set forth in claim 18 wherein said secondary molecular means physically interacts with said primary molecules to sterically prevent said primary molecules from collapsing towards said support matrix.

23. A matrix as set forth in claim 18 wherein said secondary molecular means includes electrostatic means for reducing the electrostatic interaction of said primary molecular bound to said support surface with said support surface.

24. A matrix as set forth in claim 18 wherein said primary molecules is chemically bound to said support surface.

25. A matrix as set forth in claim 24 wherein said primary molecule is IgG antibody, said antibody including carbohydrate groups chemically bound to said support surface.

26. A matrix as set forth in claim 25 wherein said antibody includes a spacer molecule chemically bound to said carbohydrate groups and to said support surface.

27. A matrix as set forth in claim 24 wherein said antibody includes an Fc region, said matrix including a spacer molecules bound between said Fc region and said support surface.