JPH08509064A - Immobilization of chemically crosslinked proteins on a solid support - Google Patents

Abstract

  (57) [Summary] Methods and compositions for specific binding assays are provided in which the specific binding reagent is one that is bound to a solid support. Immobilization is streamlined by binding a bifunctional reagent to a specific binding assay protein, thereby chemically cross-linking the specific binding reagent. Such cross-linked specific binding reagents offer the advantage of performing the assay without the use of secondary reagents. This provides improved sensitivity through minimizing non-specific background interference with the solid support or target sample.

Description

Detailed Description of the Invention Immobilization of chemically crosslinked proteins on a solid supportField of the invention   The present invention generally involves a method of immobilizing a specific binding assay reagent on a solid support. About the field. In particular, the invention relates to a method of immobilizing cross-linked reagents on a solid support. To do. The present invention also provides a solid phase support having immobilized cross-linking reagents useful in diagnostic tests. Regarding holding body.Background of the invention   To determine the amount of analyte found in a liquid or tissue sample, In vitro diagnostic assays can be used. Specimen found in sample Must be distinguished from other ingredients. Specimens should be tested from other sample components. Can be distinguished by reacting analytes with specific receptors for the body . Assays that utilize specific receptors to distinguish and quantify analytes are often It is often called a specific binding assay.   The most common receptors are antibodies, fragments of antibodies and intrinsic factors or folate binding. It is a specific binding protein such as a protein. Receptor is a specimen or an analogue of that specimen It is characterized by having a reversible specific binding affinity for the body. Affinity The body generally uses the enzyme, firefly, which binds to the receptor with about the same specificity and affinity as the analyte. An analyte attractant carrying a detectable marker such as a photomolecule or other known label Conductor.   In the heterogeneous specific binding assay described in the technology and patent literature In some cases, receptors for specific binding reactions or other assay reagents are often immobilized on a solid phase. Be converted. Immobilization of these receptors separates bound from unbound components. It is necessary to Various methods of immobilizing receptors or other reagents to a solid phase include Includes adsorption, absorption and covalent bonding. Receptor and antiserum fruit for the receptor Procedures for immobilizing qualitatively soluble immune complexes to inert glass fiber solid phase supports Has been described. These operations are incorporated herein by reference, US Pat. No. 4,51 No. 7,288.   Generally, a soluble immune complex dissolves at least two immunochemically reactive substances. It is made by binding to each other in liquid. Such immunochemically reactive At least one of the materials is selected for immunochemical specificity for the analyte of interest Be done. For example, if soluble immune complexes are used for the detection of thyroid stimulating hormone (TSH) A component of the immune complex, if it should be used in an immunoassay for Is selected for its immunochemical specificity for TSH. A typical example is T It will be an antibody with specificity for SH, ie an anti-TSH antibody.   The second component of the immune complex consists of an antibody preparation directed against the anti-TSH antibody. Could be Antisera against anti-specimen antibodies, such as mouse anti-TSH antibody, The purified mouse antibody is injected into a host animal (eg, goat) and then paired with the mouse antibody. It can be prepared by harvesting antisera that produce Mouse anti-TSH antibody And goat antisera to mouse antibodies are then processed into standard Hituk's solution.   Radial Heartion Immunoassay (RPIA) is an inert glass fiber solid phase A type of specific binding assay that typically utilizes a support. It's Ip. Glass fiber supports have low nonspecific binding. Normal RPIA operation In the procedure, a portion of the stock solution of the primary antibody is used as a buffer for the second component in the buffered medium. Combined with the stock solution. The resulting immune complex in the appropriate amount of buffer is then glass. Spotted on a limited area of fiber filter. Instead of the immune complex Allow components to be applied as separate buffer solutions to the filter and react in situ can do. In both cases, the application point of the immune complex shaped the reaction zone in the solid phase. To achieve. The applied immune complex is adsorbed, and the fibers in the glass fiber filter are adsorbed. Captured in the void of the fiber bed.   The method of application is to enter the immune complex solution or with an automatic pipette, or Dispensing by other automated equipment, including deposition machines. Suitable immune complex to solid phase After incubating the immune complex for an appropriate incubation time, the solid phase Are dried under controlled conditions. This process involves a number of solid-phase specific binding assays. Obtain a stable reaction reagent that can be used in any of the protocols.   Samples and / or other reagents labeled on at least one of the Applied to the solid phase to react with the immobilized receptor therein. Bound analyte and / or Separation of free analyte and / or analyte analog from the analog is achieved by binding on a solid support. By the application of a wash solution, which results in a chromatographic separation of unbound and Achieved. This ensures that the reaction zone is substantially free of unbound material, and Allows monitoring of the amount of labeling reagent in the reaction zone.   In traditional RPIA, as described in US Pat. No. 4,517,288, Monoclonal or polyclonal primary antibody versus secondary polyclonal antibody Soluble double immunity by mixing with An epidemiological complex is generated. Many factors including temperature, salt, pH and protein concentration Influence the formation of double immune complexes. These conditions make the double immune complex efficient Can be difficult to optimize for formation. In addition, this approach Antibodies, for example, have inherent limitations primarily caused by the presence of non-specificity. Non-specific binding is a tampering with a secondary antibody, such as a secondary antibody, that is non-specific for the analyte of interest. Exacerbated by the presence of ku. This is of interest to minimize non-specific binding. It is desirable to have a solid-phase reagent that is substantially free of non-specific proteins for certain analytes Be done.   Another drawback of traditional RPIA is that it effectively uses antibody fragments in these assays. It cannot be used. Complete monomeric immunoglobulin molecules are produced by pepsin. F (ab ')₂Fragments, Fc fragments, and other subflags Can be enzymatically digested into menthol. F (ab ')₂Fragment is an antibody The specific antigen binding activity of The Fc fragment has no antigen binding ability, Nevertheless, it determines important biological characteristics of intact molecules. F (ab ' )₂Was not used effectively in radial partition immunoassays One possible reason is that F (ab ')₂The fragment is embedded in the matrix It is not large enough to be kept in storage. Instead, such an antibody fragment Fragments are not present in the assay because they do not have the carbohydrate portion of the Fc region. And may not be effective. As a result, F (ab ')₂Use fragments It is not expected that immune complexes will form whenSummary of the invention   In the present invention, the cross-linking reagent is an antibody, F (ab ')₂Fragmen Or other specific binding protein, such as Used to link to a molecule. The crosslinker is preferably a bifunctional reagent, preferably Is bis (sulfosuccinimidyl) suberate, glutaraldehyde, or 1 , 4-butanediol diglycidyl ether, but not limited to Yes. The specific receptor is the complete antibody molecule, a usable fragment of the antibody, or Can be a non-immunological receptor such as a folate binding protein or an intrinsic factor It The cross-linked receptor protein complex is a glass fiber matrix or poly It can be immobilized on a limited area on a solid support such as a styrene plate. Wear.   The present invention provides a method of immobilizing a crosslinked receptor protein complex on a solid surface. Become. The invention further has a cross-linked protein complex immobilized thereon. Includes solid phase reagents, and specific binding assay methods using such solid phase reagents. I'm out.Brief description of the drawings   Figure 1 shows BS³Stratus as a function of increasing concentration of cross-linked D70 antibody The A, B and F rates are illustrated.   Figure 2. Binding by secondary goat anti-mouse (GAM) antibody on Stratus Stratu with glutaraldehyde cross-linked CA2 compared to other CA2 antibodies s illustrates the TSH assay.   Figure 3. Binding by secondary goat anti-mouse (GAM) antibody on Stratus II. Strat using glutaraldehyde cross-linked CA2 as compared to CA2 antibody Figure 3 illustrates the us TSH assay.Detailed Description of the Invention   Applicants have used chemically cross-linked protein complexes for specific binding assays. It has been discovered that it can be used for immunoreagents on a solid phase for use in b. here The term "protein" used in is not only full-length protein, but also specific binding. Any polypeptide having utility as an assay reagent, eg, full length tamper It is defined to include a fragment of A chemically crosslinked protein complex Useful for the production of various solid phase reagents in immunoassays and other assays However, Applicants have found that glass fiber filters in radial partition immunoassays -We have found a particularly useful application of such complexes for substrates.   Giegel et al. ,Clin. Chem.28: 1894-98 ( 1982) and U.S. Pat. No. 4,517,288. Sion immunoassay is an assay procedure in which all steps are performed on a solid phase. Is. In general, antibodies or other reagents should be immobilized on a small area on glass fiber filter paper. Is standardized. Various calibration standards containing known amounts of analyte to be detected or Various unknown samples potentially containing such analytes then react with this immobilized receptor. Allowed to respond. After proper addition of labeled analogs or other labeling reagents Excess reagent is removed from the central area of the filter paper by the application of wash solution.   In the case of enzyme immunoassays, the wash solution may contain a substrate for the enzyme. Therefore, it can be said that the enzyme reaction is started at the same time as the washing step. Preferably The action of the enzyme on the substrate produces a fluorescent signal. The enzymatic activity of a part of the central area is It can be quantified by front surface fluorometry. Assay format That is, depending on whether the direct binding assay is a competitive assay, the fluorescence rate depends on the analyte in the sample. It is directly or inversely proportional to the concentration.   As mentioned above, the solid phase preferably provides a relatively inert surface. sand In other words, the surface is relatively Must be non-reactive. In a preferred embodiment of the invention, solid phase physics The pores in the solid phase have sufficiently small gaps or pores that the reaction solution is It is like being held and transported by use.   The solid phase is advantageously glass or synthetic fiber or a relatively inert cellulosic material. Composed of a mat of compressed fibers. Solid phase is sintered glass, ceramic Cux and other porous components such as synthetic polymeric materials. Moth Lath fiber filter paper has a quantitative evaluation for its properties and for assay reagent retention. Due to its ability to adsorb the crosslinked complex of the invention in a sufficient amount to It is a solid support.   The chemically cross-linked protein complex of the present invention, once adsorbed to a suitable solid phase , Used in a wide range of analytical protocols for the analysis of various biological materials Can be For example, chemically cross-linked protein complexes can bind urine, blood, Can be useful in immunoassays of blood components such as serum, plasma. like that Immunoassays include therapeutic agents, natural or synthetic steroids, hormones, enzymes, antibiotics. It can be directed to the detection or quantification of substances or other analytes of interest.   The therapeutic agents that can be analyzed in such protocols are limited. But not digoxin, dilantin, phenobarbital, theophylline, Includes gentamicin, quinidine, etc. Since The solid phase prepared in the above manner also includes, but is not limited to, cortisol. , Aldosterone, testosterone, progesterone, and estriol For the detection of steroids such as, and serum proteins such as ferritin It can be used for Hormone levels can also be determined by using the solid phase complex of the present invention. Can be measured by. These hormones include, but are not limited to, tetrayo Doe and triiodothyronine and thyroid stimulating hormone (TSH), cortico Like tropine, gastrin, angiotensin, and proangiotensin Peptide hormones, and insulin, thyrotropin, levuteotropin, Polypep such as somatotropin and human gonadotropin hormone (HCG) Contains chid hormone. Other uses of the conjugates of the invention include the relatively small components involved in metabolism. Offspring such as folate assays, polypeptide antigens and antibodies associated with infectious diseases such as It is responsible for HIV, hepatitis, CMV, syphilis, Lyme disease and many other infectious agents. Including sei.   The chemically cross-linked protein complex / solid phase preparations of the present invention can be used with various specific binding agents. Applicable to essay format. For example, various direct binding assays Reagents may be used. In such assays, the antibody or binding protein Such receptors are chemically linked to form cross-linked protein complexes, which The complex is immobilized on the solid phase. Immobilized chemically cross-linked protein complex Are contacted with a sample containing the analyte of interest.   After binding the analyte with the immobilized complex, the solid phase is washed and then the indicator is Contacted. The term "indicator" in the context of the present invention refers to a labeled conjugate. Means the body. The zygote is an assay Depending on the mat, contains antibodies, antibody fragments, binding proteins or analytes, labeled Is fluorescence, enzyme, color, radiometry or other directly or indirectly associated with the conjugate Is a labeled molecule. The label consists of an enzymatic compound that fluoresces when in contact with the substrate. May be. The degree of indicator present on the solid phase can be determined, for example, by Tijssen, P .; , L Laboratory Technologies in Biochemistry   and Molecular Biology, Practice and Theory of Enzyme Immunoassay, pp. 173- 219 (Chapter 10), pp. 329-384 (Chapter 1 4), Elsevier Science Publishers, The Netherlands, Correlates with the amount of unknown analyte, as disclosed in Amsterdam (1985) .   The complexes of the invention can also be used in competitive assay formats . In such a format, with specificity for the selected analyte, Solid phases containing immobilized chemically cross-linked protein complexes have been used in such assays. Contacted with a sample suspected of containing the body, and with a specific competitive reagent It The specificity competing reagent may be a labeled analyte analog. In this example, The labeled analog competes with the sample analyte for the receptor immobilized on the solid phase.   In an alternative embodiment, the analyte is linked to a solid phase, with the sample, and with specificity competition. Can be contacted with a cross-linking protein reagent, such as a labeled receptor for the analyte It In this format, the sample analyte is bound to the soluble labeled cross-linking receptor. And compete with the solid phase sample. Both In one embodiment, the amount of label bound to the solid phase after washing depends on the level of analyte in the sample. Provide instructions. That is, the amount of label bound to the soluble phase depends on the analyte in the sample. Inversely proportional to the amount of.   Various instruments have been used to test chemically cross-linked protein conjugates and other binding assay assays. For applying the drug to the solid phase, washing, and reading the amount of indicator bound to the solid phase It is available. In a preferred embodiment, the solid phase is Baxter, Diagnostic Stratus Immunoasset from Chicks, Inc. B) consists of glass fiber filter tabs analyzed using the system. This equipment Are hereby incorporated by reference. Giegel et al. , Clin. C hem. 28: 1894-98 (1982) batch processing pliers. It is a top device. This instrument is a radial partition immunoassay format. It is adapted to handle filter tabs in This format is also G iegel et al. It is described in. This equipment is used for samples, joints and And a liquid distributor for the substrate wash. Microprocessor control system A popping motor aspirates and dispenses the appropriate amount of reagent. All distributor timings The working and working planes are predetermined by the program routine in the analyzer. ing. This equipment also has a tub transport system, a heating plate equipped with a temperature monitor. , Sample and reagent fluid pumps, read stations, data processing, and It includes tab processing means.   For quality control, the instrument microprocessor control program should Degree, and important work like flags for distribution settings and outliers. Periodically verify business conditions.   The present invention will be further understood with reference to the following illustrative embodiments. They are It is purely illustrative and limits the true scope of the invention as claimed. Do not understand.                                 Example 1                       Preparation of solid support (table) Including "tab". Both devices are compatible with Baxter Diagnostics Inc. By All patient sample dilutions and / or mixing is performed offline. A process for online dilution of patient samples with one or more prior solutions. St except with the flexibility of programming   Tabs are Giegel et al., Radial Partition Immunoassay, Clin. Chem. 28:18 94-98 (1982), GF / F glass fiber paper (Whatman I nc.) 1-inch (2.5 cm) wide roll and snap-fit plastic tag It can be assembled from parts. The appropriate concentration of crosslinker solution or control solution is Made in spot buffer. The composition of this spot buffer depends on the specific experimental and Can be varied to suit manufacturing parameters. Generally, this spot Buffer is, for example, 20 mM to 200 mM Tris, pH 7.0 to 9.0, 0.1% to 1 Zo in the concentration range of 0.0% (weight / weight) Nonionic surfactants such as 0.5% to 4.0% bovine serum albumin (BSA), and 0.1% sodium azide, but is not limited thereto. Preferably, the spot buffer is at a concentration of 30-100 mM. Contains 3.0% BSA and 0.1% sodium azide. Most preferably, the spot Buffer is 50 mM Tris, pH 8.0, 0.1% Mu. Fluorinated surfactants (eg 3M Catalog No. FC 171 and FC 170C) And other suitable surfactants known to one of ordinary skill in the art   Spot aliquots, typically 76 μL, of the selected solution in the center of the blank tab Which is then oven dried at 80-90 ° C overnight. After cooling, tabs 2-8 Store at ℃ until use. Use a pipette device to spot the solution on the tab. Manually or using an automated manufacturing procedure. As an alternative The tab is an instrument for applying a selected portion of stock solution in the center of the tab. It may be spotted and processed by itself.                                 Example 2                              Assay procedure   In all the experiments below, Giegel et al., Clin. Chem. 28: 1894-98 (1982 The radial partition assay format described in (1) was used. Calibration solutions A, B, C, D, E and F were treated with BSA, stabilizer and 0.1% sodium azide. Prepared in squirrel buffer (pH 7.5). The calibration solution and the crosslinker selected were analyzed It was changed according to the object. Aspirate, 60 μL of the selected calibration standard (or sample) and By releasing onto the tab containing the antibody conjugate prepared. Stratus equipment Substrate probe of 4-methylumbelliferyl phosphate (1.0 mM) (0.1-1.0%) and specific alkaline phosphatas, such as Brij 35 Enzyme inhibitor in a buffer solution (0.6-1.0M) based on diethanolamine at pH 9.0 Aspirate 70 μL of substrate wash containing. Add 20 μL and 50 μL of this substrate wash solution to the tub. Add in aliquots of μL. Depending on the assay chosen, the amount of various reagents added will vary. It As soon as a partial volume of the second substrate wash solution is released, the first fluorescence rate is read and And is recorded in the memory of the device.   For example, alkaline phosphine to methylumbelliferyl phosphate substrate The amount of fluorescence generated by the action of the enzyme tase is Stratus. Is converted to. They are given in the tables and figures as mV / min (“Stratus rate”) ing. This Stratus rate is a measure of the intensity of fluorescence, which in turn It is a measure of the amount of analyte or other reactive material bound to the responsive moiety.   During operation of the Stratus instrument, the fluorescence rates of individual calibration standards are measured and The value of is directed to a memory location in the microprocessor's memory. All calibration standards Is measured, the program measures in the form shown in the equation below. Uses a multi-pass linear regression routine that fits a mathematical relationship to the data points "Rodbard" parameters, A, B, C and D (Davis, S.E. et al., J. Im. munoassay 1: 15-25 (1980)).           R = {(A-D) / [1 + B (X / C)] + D} Where R is the fluorescence index and X is the corresponding concentration. This equation is for various im Non-assay system reported to give excellent fit and generalized Create a sigmoid curve. The obtained A, B, C, stored in memory And D, the instrument will give a concentration reading for the assayed sample. give.                                 Example 3                       Cross-linked anti-CEA antibody   Purified anti-carcinoembryonic antigen (CEA) monoclonal antibody (clone #CEL 007, Catalog # 200088, Hybridtech, Inc.) with 0.1M sodium phosphate. Buffer exchange into a pH 7.0 buffer and target antibody concentration of approximately 6.5 ± 0.5 mg / mL The liquid volume was reduced so that The antibody concentration is 1.48 extinction coefficient at 280 nm. mg (cm^-1L^-1) Was used for the spectrophotometric estimation. Suberic acid bis (sulfuric acid Hosuccinimidyl) [bis (sulfosuccinimidyl) suberate] (BS³) (Pierc e Chemical Co., Catalog # 21579G) in water to a final concentration of 20 mg / mL. It was This antibody solution was added to the molar challenge ratio BS.³: Anti-CEA = 150 for 2 hours at room temperature (24 BS at ± 2 ℃³Allow to react with solution or let sit at 2-8 ° C overnight (16-24 hours). I responded. The reaction mixture is then added to an amount of 1/9, which is equal to 1/9 of the total mixture The reaction was quenched by adding M ethanolamine hydrochloride.   The protein component is 0.1% sodium azide (ie 0.1 g NaN₃/ 100 mL   PBS containing phosphate buffered saline (PBS) ) Sephadex G-25 column (Pharmacia, Catalog # 17-0033-) It was separated from the smaller molecular species by passing through 02). Alternatively, tan The protein component was purified using a P-6 DG column (Bio-Rad, Catalog # 150-0738). Made The concentration of cross-linked antibody is determined by the BCA protein assay (Pierce, Cat. Log # 23225G). Antibody solution should be 100-600 μg / mL final Adjusted to protein concentration.   After purification, the cross-linked antibody was transferred to CEA spot buffer (50 mg sodium phosphate). Um, 0.3M sodium chloride, 0.1% Tween 20, 1% BSA, 2% gluco It was placed in potassium phosphate (pH 6.90)). Protein of various concentrations of about 76 μL The quality solution was spotted on a blank tub and dried in an oven at about 80 ° C.   Control tabs were prepared using the traditional dual immune complex assay format. Double immunity Conjugates contain approximately 190 μg / mL primary monoclonal antibody in an optimal amount of secondary antibody, Formed by mixing with goat anti-mouse polyclonal (GAM) antibody, Then spotted onto a glass fiber tab. Calibration standards CalA to CalF Contained 0.0, 0.25, 0.75, 3.0, 12.0 and 50.0 ng / mL CEA, respectively. . Table 1 shows the results obtained for the cross-linking tab and for the dual immune complex control tab. Is a comparison.   The extrapolation of the data presented in the table is for a concentration of about 500 μg antibody / mL. In, tabs with cross-linked proteins are used in transduction using double immune complexes. It shows that it is comparable to the traditional tab.                                 Example 4                  Cross-linked anti-hTSH D70 antibody   Purified anti-human thyroid stimulating hormone (hTSH) tab antibody (clone D70: Buffer manufactured by Serono) into 0.1M sodium phosphate, pH 7.0 did. The liquid volume was reduced so that the antibody concentration was about 5.5 ± 0.5 mg / mL. BS³ Was dissolved in water to a final concentration of 20 mg / mL. BS this antibody solution³Solution and Molar challenge ratio BS3: Anti-h-TSH = 137. This reaction mixture Incubated overnight at 2-8 ° C. Cross-linked anti-h-TSH was added to the reaction mixture. The product was purified by passing through a Sephadex G-25 or Bio-Rad P-6 DG column. Ta Protein concentration was measured by BCA protein assay. This protein Then TFSSB (50 mM Tris, 2% BSA, 0.1% gelatin, 0 .15m sodium chloride, 0.1% FSN (Zonyl fluorinated surfactant) (pH8.0 )) Put it inside.   Preparation of cross-linked protein complexes at several concentrations, and Stratus The calibration standards A, B, D, C, E and F used in the above were 0.0, 0.5, 1.5, 15.0 and 150μ. It contained U / mL hTSH. Figure 1 shows Stratu Draw as. Table 2 shows tabs with D70 (150 μg / mL) cross-linked and D70 : TSH assess with tab with GAM complex (traditional double immune complex) It is a comparison of b.   CalB represents the first measurable amount of the analyte. CalA (zero analyte ) Represents non-specific binding and CalF represents the highest concentration of analytical control to be detected. Ratio C alB / CalA is a measure of the edge on the low sensitivity side, and the ratio CalF / CalA is the whole. Represents the target sensitivity. Higher calibration curve ratios for cross-linked tabs indicate double immunoconjugate Increased sensitivity of assay with cross-linked tabs compared to assay with body tabs It shows that it is doing.   Table 3 uses both cross-linked D70 tabs and double immunocomplex tabs It is a direct comparison of data. Tabs used: Cross-linked D70 tab: made from 150 μg / mL cross-linked protein complex It was Current tab: TXSH-308   The data presented in Table 3 shows that Stratu with cross-linked anti-hTSH was used. Sensitivity measurements of 8 replicates for A rate showed that the sensitivity was for the double immune complex tab. Of 0.09 μIU / mL, which is comparable to the value of 0.1 μIU / mL I made it Sensitivity is the average CalA rate + To be done. Remarkably non-specific, mainly because the system does not use secondary antibodies A markedly low level of physical association was achieved.   Table 4 shows the hTSH tabs produced with cross-linked CA2 and the two. The signal readings obtained for various calibration standards with cross-linked tabs are The columns and the fifth column are shown respectively. Corresponding data on dual immune complexes Are shown in the second and fourth columns, respectively. About double immune complex tab The insensitive end of the (CalB / CalA ratio) is that of the crosslinked tab. Slightly better than the edge on the sensitive side for the cross-linked tab (ratio CalF / CalD) and overall sensitivity (ratio CalF / CalA) : Clearly shows that the GAM (double immunocomplex) tab can be replaced.                                 Example 6                Cross-linked anti-TAT F (ab ')₂   Thrombin: Antibodies to antithrombin complex (anti-TAT F (ab '))₂) F (ab ')₂The capture reagent consisting of a fragment was added to 0.1 M sodium phosphate ( buffer exchange into pH 7.6) and final F (ab ')₂Concentration is about 5.00 ± 0.25m The liquid volume was reduced to g / mL. Calculated amount of 100 mg / mL B in water S³The mole challenge ratio BS³: F (ab ')²= 100, add to the protein solution It was This solution was incubated at room temperature (23 ± 2 ° C) for 2 hours. Cross-linked F (Ab ')₂Was purified by passing through a Sephadex G-25 column. Cross-linked F (ab ')₂Of BCA protein assay or 1.48 mg / It was measured spectrophotometrically using an extinction coefficient of (mL) (cm).   ELISA is a cross-linked F (ab ')₂Was carried out. Plate first , The crosslinked F (ab ')₂Coating overnight at 2-8 ° C. TAT calibration Standards (0-50 nM) are added and the plates are incubated at room temperature for 0.5 hours It was Add anti-TAT antibody-HRP (horseradish peroxidase) conjugate and let Incubate for 15 minutes, then add substrate. Then at room temperature for another 15 minutes Incubated. Plates should be washed twice with wash buffer before adding each new reagent. Washed. Read the plate using a plate reader to measure the colorimetric reading. Monitored at 405 nm.   Table 5 shows crosslinked F (ab ').₂Plate and non-crosslinked F (ab ')₂Plate and Is a comparison of the ELISA performance during the period. Clone 4C72 and 5B46 (both Also manufactured by Baxter Diagnostics, Inc.) The clonal antibody-HRP conjugate was added to the crosslinked F (ab ')₂Used in assay did.   This table shows unprocessed F (ab ')₂And crosslinked F (ab ')₂EL using As measured by absorbance at 405 nm in an ISA assay, Shows the signal generated in the presence of calibration standards F (50 nM) and A (0.0 nM) . Tested these two For all combinations of conjugates, plates made of cross-linked antibody Overall sensitivity (ratio CalF / CalA) made from untreated antibody It was very expensive. This increased sensitivity for cross-linked antibody plates is due to non-specific binding. Cross-linking on the plate, not only due to the loss of signal (decrease in CalA signal) Also due to the high response for CalF resulting from the more efficient immobilization of the antibody It was a thing.   The above detailed description has been presented only for better understanding of the invention, Modifications will be apparent to those skilled in the art without departing from the spirit and scope of the appended claims. So don't read unnecessary limits from it.

─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Diamond, Stephen, Yi             United States 46530 Gray, Indiana             Ranger, Quail Valley Drive             51868

Claims (1)

[Claims] 1. A method for immobilizing a reagent on a solid support, the method comprising reacting a protein composition with a cross-linking agent to form a cross-linked protein complex, the protein complex comprising a specific binding receptor. And substantially free of non-specific proteins, and immobilizing the crosslinked protein complex on a solid phase support and the crosslinked protein complex on the solid phase support. Applying under conditions effective to oxidize. 2. The method of claim 1, wherein the cross-linking agent is a bifunctional reagent. 3. The method of claim 2, wherein the bifunctional reagent is bis (sulfosuccinimidyl) suberate. 4. The method of claim 2, wherein the bifunctional reagent is glutaraldehyde. 5. The method of claim 2 wherein said bifunctional reagent is 1,4-butanediol diglycidyl ether. 6. The method of claim 1, wherein said protein is selected from the group consisting of antibodies, antibody fragments and specific binding proteins. 7. 7. The method of claim 6, wherein the specific binding protein is intrinsic factor. 8. The method of claim 6, wherein said antibody is an anti-carcinoembryonic antigen monoclonal antibody. 9. 7. The method of claim 6, wherein the antibody is an anti-human thyroid stimulating hormone monoclonal antibody. Ten. 7. The method of claim 6, wherein the antibody fragment is anti-TAT F (ab ') ₂ . 11. The method of claim 1, wherein the solid support is a polystyrene plate. 12. The method of claim 1, wherein the solid support is a glass fiber filter. 13. A specific binding assay solid phase reagent comprising a cross-linked protein complex cross-linked by a bifunctional reagent, said complex having at least two specific binding receptors and said cross-linked protein complex immobilized thereon. A specific binding assay solid-phase reagent comprising a solid-phase support that has been derivatized, and the solid-phase support is substantially free of non-specific proteins. 14. 14. The specific binding assay solid phase reagent of claim 13, wherein the specific binding receptor is selected from the group consisting of antibodies, antibody fragments and specific binding proteins. 15. 14. The specific binding assay solid phase reagent of claim 13, wherein the specific binding receptor is an anti-carcinoembryonic antigen monoclonal antibody. 16． 14. The specific binding assay solid phase reagent of claim 13, wherein the specific binding receptor is an anti-human thyroid stimulating hormone monoclonal antibody. 17. 14. The specific binding assay solid phase reagent of claim 13, wherein the specific binding receptor protein is anti-TAT F (ab ') ₂ . 18. 14. The specific binding assay solid phase reagent of claim 13, wherein the bifunctional reagent is bis (sulfosuccinimidyl) suberate. 19. 14. The specific binding assay solid phase reagent of claim 13, wherein the bifunctional reagent is glutaraldehyde. 20. 14. The specific binding assay solid phase reagent of claim 13, wherein the bifunctional reagent is 1,4-butanediol ether. twenty one. 14. The specific binding assay solid phase reagent of claim 13, wherein the solid phase support is a polystyrene plate. twenty two. 14. The specific binding assay solid phase reagent of claim 13, wherein the solid phase support is a glass fiber filter. twenty three. A method for performing a specific binding assay to determine the concentration or presence of an analyte in a sample, comprising: (a) subjecting the sample to binding conditions under specific binding conditions. Applying to an assay solid phase reagent, and (b) measuring the specific binding reactivity of the analyte with the solid phase reagent to detect or quantify the analyte in the sample. Method. twenty four. The measurement comprises: (a) applying a selected amount of the indicator to the solid support under binding conditions, (b) measuring the amount of the indicator bound to the solid support, and (c) 24. The method of claim 23, comprising correlating the amount of the indicator with the concentration or presence of the analyte in the sample. twenty five. 25. The method of claim 24, wherein the sample and the indicator are applied simultaneously to the solid phase. 26. The measurement comprises: (a) applying a labeled specific competitive reagent to the solid phase under binding conditions, (b) measuring the amount of label bound to the solid phase, and (c) the label. 24. The method of claim 23, comprising correlating the amount with the concentration or presence of the analyte in the sample. 27. 27. The method of claim 26, wherein the specific competitive reagent is a labeled analog of the analyte. 28. 28. The method of claim 27, wherein the specific competitive reagent is a labeled receptor of the analyte.