WO1995008771A1

WO1995008771A1 - Immobilization of chemically cross-linked proteins on solid supports

Info

Publication number: WO1995008771A1
Application number: PCT/US1994/010032
Authority: WO
Inventors: Spencer H. Lin; Pratap Singh; Steven E. Diamond
Original assignee: Baxter Diagnostics Inc.
Priority date: 1993-09-22
Filing date: 1994-09-07
Publication date: 1995-03-30
Also published as: CA2150029A1; JPH08509064A; AU7721094A; EP0670040A1

Abstract

Methods and compositions are provided for specific binding assays in which specific binding reagents are immobilized on a solid phase. Immobilization is facilitated by coupling bifunctional reagents to specific binding assay proteins thereby chemically cross-linking the specific binding assay reagents. Such cross-linked specific binding assay reagents provide the advantage of performing assays without the use of secondary reagents. This provides for enhanced sensitivity through minimizing nonspecific background interference with the solid phase or with the target samples.

Description

IMMOBILIZATION OF CHEMICALLY CROSS-LINKED PROTEINS

ON SOLID SUPPORTS

Field of the Invention

This invention relates generally to the field of methods for immobilizing specific binding assay reagents on solid supports. In particular, this invention relates to a. method of immobilizing cross-linked reagents on solid phase supports. This invention also relates to a solid phase support having immobilized cross-linked reagents useful in diagnostic tests.

Background of the Invention

In vitrc diagnostic assays may be used to measure amounts of an analyte found in a body fluid sample or tissue sample. The analyte must be distinguished from other components found in the sample.

Analytes may be distinguished from other sample components by reacting the analyte with a specific receptor for that analyte. Assays that utilize specific receptors to distinguish and quantify analytes are often called specific binding assays.

The most common receptors are antibodies, fragments of antibodies and specific binding proteins such as Intrinsic Factor or Folate Binding Protein. Receptors are characterized by having a reversible specific binding affinity for an analyte or an analog of that analyte. The analog generally is an analyte derivative carrying a detectable marker such as an enzyme, fluorescent molecule or other known label which binds to a receptor with about the same specificity and affinity as the analyte.

In heterogeneous specific binding assays described in the technical and patent literature, the receptor or other assay reagent of the specific binding reaction is often immobilized on a solid phase.

Immobilization of these receptors is required to separate the bound components from the unbound components. The various methods by which a receptor or other reagent is immobilized on a solid phase include adsorption, absorption and covalent bonding. Procedures have been described for immobilizing an essentially soluble immunocomplex of a receptor and an antiserum to the receptor on an inert glass fiber solid phase support. These procedures are disclosed in U.S. Patent No. 4,517,288, incorporated by reference herein.

Generally, soluble immunocomplexes are prepared by combining at least two immunochemically reactive substances with one another in solution. At least one such immunochemically reactive material is selected for its immunochemical specificity for an analyte of interest. This is defined as the primary antibody. For example, if the soluble immunocomplex is to be used in an immunoassay for the detection of thyroid stimulating hormone ("TSH"), then one component of the immunocomplex is selected for its immunochemical specificity for

TSH. A typical example would be an antibody with specificity for TSH, i.e., an anti-TSH antibody.

The second component of the immunocomplex could comprise an antibody preparation directed against the anti-TSH antibody. Antiserum to anti-analyte antibodies, for example to mouse anti-TSH antibodies, can be prepared by injecting purified mouse antibody into a host animal (e.g., a goat), and thereafter harvesting the antiserum to the mouse antibody. The mouse anti-TSH antibody and the goat antiserum to mouse antibodies are thereafter worked up as standard stock solutions. Radial partition immunoassay ("RPIA") is a type of specific binding assay that typically utilizes an inert glass fiber solid phase support. The glass fiber support has low non-specific binding. In the usual RPIA procedure, a portion of a stock solution of the primary antibody is combined with the stock solution of the second component in a buffered medium. The resulting immunocomplex, in an appropriate volume of buffer, is thereafter spotted onto a delimited area of the glass fiber filter. Alternatively, the two components of the immunocomplex may be applied to the filter as separate buffered solutions and allowed to react in situ. In both instances, the point of application of the immunocomplex defines a reaction zone within the solid phase. The applied immunocomplexes become adsorbed and entrapped within the interstices of the beds of fibers within the glass fiber filter.

The method of application can include dispensing the immunocomplex solution with a manual or automated pipette, or with other automated equipment including assay analyzer instruments. Subsequent to applying the immunocomplex to the solid phase, after incubating the immunocomplex for a suitable incubation period, the solid phase is dried under controlled conditions. This process yields a stable reactive reagent which can be used in any one of a number of solid phase specific binding assay protocols.

The sample and other reactants, at least one of which is labelled, are applied to the solid phase so that they react with the immobilized receptor in the reaction zone. Separation of free analyte and/or analyte analog from bound analyte and/or analog is accomplished by application of a wash solution, with resultant chromatographic separation of the bound and unbound reagents on the solid phase support. This results in the reaction zone being essentially free of unbound materials and allows the monitoring of the amount of labelled reagent in the reaction zone.

In traditional RPIA the soluble double immunocomplex is formed by mixing the primary antibody, either monoclonal or polyclonal, with secondary polyclonal antibody as described in U.S. Patent No.

4,517,288. Many factors including temperature, salt, pH, and protein concentration influence the formation of the double immunocomplex. These conditions can be difficult to optimize in order to effectively form the double immunocomplex. In addition, this approach has inherent limitations primarily caused by the presence of the secondary antibody, for example non-specific binding. Non-specific binding is exacerbated by the presence of proteins, such as secondary antibody, that are non-specific for the analyte of interest. Thus, it is desirable to have a solid phase reagent that is substantially free of proteins non-specific for the analyte of interest, so as to minimize non-specific binding.

A further shortcoming of traditional RPIAs is that antibody fragments cannot be effectively used in these assays. A complete monomeric immunoglobulin molecule can be enzymatically digested by pepsin into an F(ab')₂ fragment, an Fc fragment and other subfragments. The F(ab')₂ fragment contains the specific antigen-binding activity of the antibody. The Fc fragment contains no antigen-binding capability but nevertheless determines important biologic characteristics of the intact molecule. One possible reason why F(ab')₂ fragments have not been effectively used in radial partition immunoassays is that the F(ab')₂ fragments may not be of sufficient size to remain imbedded in the matrix. Alternatively, such antibody fragments may be ineffective in the assay because they lack the carbohydrate portion of the Fc region. As a result, no immunocomplex is expected to form when F(ab')₂ fragments are used.

Summary of the Invention In this invention, cross-linking reagents are used to couple specific binding receptors such as antibodies, F(ab')₂ fragments or other receptor proteins to other identical or related molecules. The cross-linking agent is preferably a bifunctional reagent, preferably but not limited to bis(sulfosuccinimidyl) suberate, glutaraldehyde, or 1,4 butanediol diglycidyl ether. The specific binding receptor can be a complete antibody molecule, an operable fragment of an antibody or a non-immunological receptor protein such as folate binding protein or intrinsic factor. The cross-linked receptor protein complex can be immobilized onto a finite area of a solid phase support, such as a glass fiber matrix or polystyrene plate. The present invention comprises the method of immobilizing cross-linked receptor protein complexes on solid surfaces. The invention further comprises solid phase reagents having cross-linked protein complexes immobilized thereon, as well as specific binding assay methods utilizing such solid phase reagents.

Brief Description of the Figures Figure 1 depicts the Stratus® A, B and F rates as functions of increasing concentrations of BS³ cross-linked D70 antibodies.

Figure 2 depicts the Stratus® TSH assay using glutaraldehyde cross-linked CA2 antibodies as compared to CA2 antibodies bound by a secondary goat-anti-mouse (GAM) antibody on Stratus®. Figure 3 depicts the Stratus® TSH assay using glutaraldehyde cross-linked CA2 antibodies as compared to CA2 antibodies bound by a secondary goat-anti-mouse (GAM) antibody on Stratus® II.

Detailed Description of the Invention Applicants have discovered that chemically cross-linked protein complexes may be used for immobilizing reagents on a solid phase for use in specific-binding assays. The term "protein" as used herein is defined to include full-length proteins as well as any polypeptides, for example fragments of full-length proteins, that have utility as specific binding assay reagents. While chemically cross-linked protein complexes are useful for preparation of various solid phase reagents in immunoassays as well as other assays, the Applicants have found particularly useful application of such complexes with glass fiber filter substrates in radial partition immunoassays. Radial partition immunoassay, as disclosed in Giegel et al.,

Clin. Chem. 28:1894-98 (1982) and in U.S. Patent No. 4,517,288, is an assay procedure in which all steps are conducted directly on a solid phase. Generally, antibodies or other reagents are immobilized on a small area of glass fiber filter paper. Various calibrators containing known amounts of an analyte to be detected or various unknown samples potentially containing such analyte are then allowed to react with this immobilized receptor. Following appropriate additions of labeled analogs or other labeling reagents, excess reagents are removed from the center area of filter paper by application of a wash fluid.

In the case of an enzyme immunoassay, the wash fluid may contain the substrate for the enzyme, thus initiating the enzyme reaction simultaneously with the wash step. Preferably the action of the enzyme on the substrate generates a fluorescent signal. The enzyme activity in a part of the center area is then quantifiable by front-surface fluorometry. Depending on the assay format, i.e., direct binding assay or competitive assay, the rate of fluorescence is directly or inversely proportional to the concentration of analyte in the sample.

As described above, it is preferred that the solid phase present a relatively "inert" surface. That is, the surface should be relatively nonreactive with biological materials, particularly with respect to' nondiscriminate adsorption of proteinaceous materials. In the preferred embodiments of this invention, the physical form of the solid phase is such that the interstices or pores within the solid phase are sufficiently small so that the reaction fluids are retained and transported by capillary action. On the other hand, the solid phase pores or interstices should not be so small so as to retain undesirable components that might give rise to false positive signals.

The solid phase is advantageously composed of a mat of compressed fibers, such as glass or synthetic fibers or relatively inert cellulosic materials. The solid phase also may be constructed of other porous constituents such as sintered glass, ceramics and synthetic polymeric materials. Glass fiber filter paper is the preferred solid support of the present invention because of its inert characteristic and because of its ability to adsorb the cross-linked complexes of this invention in quantities sufficient for quantitative evaluation of retention of assay reagents. The chemically cross-linked protein complexes of this invention, once adsorbed onto a suitable solid phase, can be used in a wide variety of analytical protocols for analysis of a variety of biological materials. For example, chemically cross-linked protein complexes may be useful for immunoassay of urine, blood or blood components such as serum or plasma. Such immunoassays can be directed to the detection or quantitation of therapeutic agents, natural or synthetic steroids, hormones, enzymes, antibodies and other analytes of interest.

Therapeutic agents that can be analyzed in such protocols include without limitation digoxin, dilantin, phenobarbital, theophylline, gentamicin, quinidine, and the like. Solid phases prepared in the foregoing manner can also be used in immunoassays for the detection of steroids such as, without limitation, cortisol, aldosterone, testosterone, progesterone, and estriol or serum protein such as ferritin. Hormone levels are also capable of determination through the use of solid phase complexes of the present invention. These hormones include without limitation thyroid hormones such as tetraiodo- and triiodo-thyronine and thyroid stimulating hormone (TSH); peptide hormones such as corticotropin, gastriri, angiotensin, and proangiotensin; and polypeptide hormones such as insulin, thyrotropin, levteotropin, somatotropin and human chorionic gonadotropic hormone (HCG). Other applications of the complexes of the present invention include assay of relatively small molecules involved in metabolism, i.e., folate, to assay of polypeptide antigens and antibodies associated with infectious disease, e.g., antigens and antibodies associated with HIV, hepatitis, CMV, syphilis, Lyme disease agents, and numerous other infectious agents.

The chemically cross-linked protein complex /solid phase preparations of the present invention are applicable to a variety of specific binding assay formats. For example, various direct-binding assays may be employed with these reagents. In such assays, receptors such as antibodies or binding proteins are chemically coupled to make a cross-linked protein complex and the complex is immobilized on the solid phase. The immobilized chemically cross-linked protein complexes are contacted with a sample containing the analyte of interest. Following binding of the analyte by the immobilized complex, the solid phase is washed and then contacted with an indicator. The term indicator in the context of this invention means a labeled conjugate. The conjugate comprises an antibody, antibody fragment, binding protein or analyte depending on assay format, and the label is a fluorescent, enzymatic, colorimetric, radiometric or other labeling molecule that is associated either directly or indirectly with the conjugate. The label may be comprised of an enzymatic compound that produces fluorescence upon contact with a substrate. The extent to which the indicator is present on the solid support can be correlated with the amount of unknown analyte as disclosed, for example, in Tijssen, P., Laboratory Techniques in Biochemistry and Molecular Biology. Practice and Theory of Enzyme Immunoassay, pp. 173-219 (Chapter 10) and pp. 329-384 (Chapter 14), Elsevier Science Publishers, Amsterdam, The Netherlands, 1985. The complexes of the present invention also may be used in competitive assay formats. In such formats, the solid phase containing immobilized chemically cross-linked protein complexes with specificity for a selected analyte is contacted with a sample presumably containing such analyte and with a specific competitive reagent. The specific competitive reagent may be a labeled analog of the analyte. In this embodiment, the labeled analog competes with the sample analyte for binding to a receptor immobilized on the solid phase.

In an alternative embodiment, analyte may be coupled to a solid phase and contacted with a sample and with a specific competitive cross-linked protein reagent, for example a labeled receptor for the analyte.

In this format, sample analyte competes with solid phase analyte for binding with soluble labelled cross-linked receptor. In both embodiments, the amount of label bound to the solid phase after washing provides an indication of the levels of analyte in the sample. That is, the amount of label bound to the soluble phase is inversely proportional to the amount of analyte in the sample. Various instruments are available for applying the chemically cross-linked protein conjugates and various other binding assay reagents to a solid phase, washing, and reading the amounts of indicator bound to the solid phase. In a preferred embodiment, the solid phase comprises glass fiber filter tabs that are analyzed using the Stratus®

Immunoassay System, available from Baxter Diagnostics Inc. This instrument is a batch-processing bench-top instrument, described by Giegel et al., Clin. Chem. 28:1894-98 (1982), incorporated by reference herein. The instrument is adapted to process filter tabs in the radial partition immunoassay format, which format is also described in Giegel et al. The instrument includes fluid dispensers for sample, conjugate and substrate washes. Microprocessor-controlled stepping motors aspirate and dispense required aliquots of reagents. All timing and operational aspects of the dispensers are predetermined by a program outine within the analyzer. The instrument also includes a tab transport system, heated plate with temperature monitoring, sample and reagent fluid pumps, a read station, data processing, and means for tab disposal.

For quality control, the instrument microprocessor control program periodically verifies critical operating conditions such as reference voltages, temperatures, and dispensing settings, and flags for out-of-limit values.

The invention will be further understood with reference to the following illustrative embodiments, which are purely exemplary, and should not be taken as limiting the true scope of the present invention as described in the claims.

EXAMPLE 1

Preparation of Solid Phase Supports (Tabsl

Solid phase supports used in the present experiments comprised "tabs" as used with the Stratus® analyzer instrument or the

Stratus® II analyzer instrument, both marketed by Baxter Diagnostics Inc.

The Stratus II® instrument is identical to the Stratus® instrument except that for analysis on Stratus®, all dilutions and /or mixing of the patient sample must be carried out off-line, whereas Stratus II® has the flexibility of programming for on-line dilutions of the patient samples with one or more solutions, prior to application to the solid phase. The tabs are assembled from 1-in. (2.5 cm)-wide rolls of GF/F glass filter paper (Whatman Inc.) and snap-fit plastic tab parts, as disclosed in Giegel et al., Radial Partition Immunoassay, Clin. Chem. 28: 1894-98 (1982). Appropriate concentrations of cross-linked reagent solutions or control solutions are made up in spotting buffer. The spotting buffer composition may be varied to accommodate particular experimental or manufacturing parameters. Generally the spotting buffer may comprise, for example, an appropriate buffer including but not limited to 20mM- 200mM Tris, pH 7.0-9.0, a non-ionic surfactant such as Zonyl® FSN (E.I. DuPont DeNemours & Co., Cat. No. CH 7152S) in a concentration range of 0.1%-1.0% (weight/volume) bovine serum albumin (BSA) at 0.5%-4.0% and 0.1% sodium azide. Preferably the spotting buffer comprises 30-100 mM Tris, pH 7.0-8.5, 0.1%-0.5% Zonyl® FSN, 1.0%-3.0% BSA and 0.1% sodium azide. Most preferably the spotting buffer comprises 50mM Tris, pH 8.0, 0.1% Zonyl® FSN, 2.0% BSA and 0.1% sodium azide. Fluorinated surfactants (e.g. 3M Cat. No.'s FC 171 and FC 170C) and other appropriate surfactants known to the skilled artisan may be substituted for Zonyl® FSN.

Aliquots, typically 76 μl, of a selected solution are spotted onto the centers of blank tabs, which are then oven-dried at 80°-90°C. After cooling, the tabs may be stored at 2°-8°C until used. Spotting of the solutions on the tabs may be carried out manually with a pipetting device or may be carried out with automated manufacturing procedures. Alternatively, the tabs may be spotted and processed by the Stratus® II instrument itself, following appropriate programming of machine parameters to apply selected aliquots of stock solutions to the centers of tabs. EXAMPLE 2 Assay Procedures The radial partition assay format described in Giegel et al., Clin. Chem. 28: 1894-98 (1982) was used in all of the following experiments. Calibrator solutit s A, B, C, D, E and F were prepared in a

Tris-buffered solution (pH 7.5) including BSA, stabilizer and 0.1% sodium azide as a preservative. Calibrator solutions and the selected cross-linked reagent varied depending upon the analyte tested.

The hTSH assay is performed on the Stratus® or Stratus® II instrument by aspirating and delivering 60 μl of a selected calibrator (or sample) onto a tab that contains an optimum quantity of the cross-linked antibody complex. The Stratus® instrument substrate probe then aspirates

70 μl of the substrate wash solution containing 4-methylumbelliferryl phosphate (1.0 mM), detergents such as Brij 35 (0.1 - 1.0%) and specific alkaline phosphatase inhibitors in a diethanolamine based buffer (0.6 - 1.0

M) system at pH 9.0. The substrate wash solution is added in 20 μl and 50 μl aliquots to the tab. Depending on the assay chosen, the volumes of different reagents applied may differ. As soon as the second substrate wash aliquot is delivered, the initial fluorescence rates are read and recorded in the instrument memory.

The amount of fluorescence generated by action of, for example, alkaline phosphatase enzyme label on a methylumbelliferyl phosphate substrate is detected by the Stratus® instrument and converted to a "rate" expressed in voltage per unit time, which is presented in the Tables and Figures as mV/min ("Stratus Rates"). The Stratus rate is a measure of the intensity of the fluorescence, which is, in turn, a measure of the amount of analyte or other reactive substance bound to the reactive portion of the tab.

During a Stratus® instrument run, the fluorescence rates of individual calibrators are measured and the values directed to storage locations in a microprocessor memory. After all calibrators have been measured, the program calculates "Rodbard" parameters A, B, C and D (Davis, S.E. et al., J. Immunoassay 1: 15-25 (1980)) using a multi-pass linear regression routine that fits a mathematical relationship to the measured data points in the form shown in the following equation:

R = { ( A - D) / [ 1 + B (X/C)] + D } where R is the fluorescence rate and X is the corresponding concentration. The equation is a generalized sigmoidal curve that has been reported to give an excellent fit in various immunoassay systems. Based on the resulting A, B, C and D parameters stored in the memory, the instrument provides the concentration readout for the samples assayed.

EXAMPLE 3 Cross-linked Anti-CEA Antibodies Purified anti-carcinoembryonic antigen (CEA) monoclonal antibody (clone # CEL007, Catalog # 200088; Hybritech, Inc.) was buffer- exchanged into 0.1 M sodium phosphate (pH 7.0) and the volume reduced so that the target antibody concentration was about 6.5 +/-0.5 mg/mL. Antibody concentrations were estimated spectrophotometrically using an extinction coefficient at 280 nm of 1.48 mg(cm-im L -i ) . Bis(sulfosuccinimidyl) suberate (BS3) (Pierce Chemical Co., Cat. # 21579G) was dissolved in water to a final concentration of 20 mg/mL. The antibody solution was reacted with the BS3 solution at a molar challenge ratio of BS3:anti-CEA = 150 for two hours at room temperature (24+/-2°C) or overnight (16-24 hours) at 2-8°C. The reaction mixture was then quenched by adding 1.0 M ethanolamine hydrochloride equal to a 1/9 volume of the total mixture.

The protein component was separated from small molecular species by passage through a Sephadex G-25 column (Pharmacia, Cat # 17- 0033-02) that had been equilibrated in phosphate buffered saline (PBS) containing 0.1% NaN₃ (i.e. 0.1 g NaN₃/100 ml PBS). Alternatively, the protein component was purified by using a P-6 DG column (Bio-Rad, Cat #

150-0738). The concentration of the cross-linked antibody was determined by a BCA protein assay (Pierce, Cat. No. 23225G). Antibody solutions were adjusted to give final protein concentrations of 100 to 600 μg/mL.

After purification the cross-linked antibody was placed in

^• CEA spotting buffer (50mg sodium phosphate, 0.3 M sodium chloride, 0.1% Tween 20, 1% BSA, 2% potassium gluconate (pH 6.90)). About 76 μL of the various concentrations of protein solutions were spotted onto blank tabs and dried in an oven at about 80 °C .

A control tab was prepared using the traditional double immunocomplex assay format. The double immunocomplex was formed by mixing about 190μg/mL primary monoclonal antibody with optimized amount of secondary antibody, i.e. goat anti-mouse polyclonal (GAM) antibody, and spotted onto a glass filter tab. Calibrated standards, Cal A to Cal F, contained 0.0, 0.25, 0.75, 3.0, 12.0 and 50.0 ng/ml CEA respectively. Table 1 is a comparison of results obtained with cross-linked tabs and with the double immunocomplex control tabs.

Table 1

Tabs 100 μg/mL 400 μg/mL 600 μg/mL Control Stratus rate 2207 ^• 4859 6365 5500

(Cal E)

Extrapolation of the data shown in the table indicates that at a concentration of about 500 μg antibody/mL, the tabs utilizing cross-linked proteins were comparable to traditional tabs utilizing a double immunocomplex.

EXAMPLE 4

Cross-linked Anti-hTSH D70 Antibodies Purified anti-human thyroid stimulating hormone (hTSH) tab antibody (clone D70, manufactured by Serono) was buffer-exchanged into 0.1 M sodium phosphate (pH 7.0). The 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. The antibody solution was reacted with the BS3 solution at a molar challenge ratio of BS3:Anti-hTSH = 137. The reaction mixture was incubated overnight at 2- 8^βC. The cross-linked anti-hTSH was purified by passing the reaction mixture over a Sephadex G-25 or Bio-Rad P-6 DG column. The protein concentration was determined by BCA protein assay. The protein was then placed in TFSSB (50mM Tris, 2% BSA, 0.1% gelatin, 0.15 M NaCl, 0.1% FSN (zonyl fluoro-surfactant) (pH 8.0)). Several concentrations of the cross-linked protein complex were prepared and spotted on glass filter tabs for use in the Stratus® instrument. Calibrators A, B, C, D, E, and F used in the assay contained 0.0, 0.5, 1.5, 15.0 and 50.0 μlU/ml of hTSH. Fig. 1 depicts the Stratus® A, B and F rates as functions of increasing concentrations of cross-linked D70 antibodies. Table 2 is a comparison of TSH assays using tabs having cross- linked D70 (150 μg/mL) and tabs having the D70:GAM complex (the traditional double immunocomplex).

Table 2

A Rate B Rate F Rate B/A F/A

Cross-linked

D70 75 137 6200 1.8 83

D70 double immunocomplex 139 226 6787 1.6 49

Cal B represents the first measurable quantity of the analyte. Cal A (zero analyte) represents a measure of non-specific binding, Cal F represents the highest amount of the analyte to be detected. The ratio Cal B/Cal A is a measure of low end sensitivity and the ratio Cal F/Cal A represents overall sensitivity. The higher calibration curve ratios for the cross-linked tabs indicate an increased sensitivity for the assay using cross- linked tabs compared to the assay using the double immunocomplex tabs. Table 3 is a direct comparison of Stratus® rates and the Rodbard parameters for the TSH assay using both cross-linked D70 tabs and double immunocomplex tabs.

Table 3

Calibrator. μIU/mL Cross-linked D70 Tabs D70 double

A(0) 72 124

B(0.5) 156 237

C(1.5) 315 397

D(5.0) 883 1019

E(15.0) 2523 2686

F(50.0) 7101 6994

Rodbard parameters

A 72 124

B 1.102 0.936

C 203.00 331.92

D 36050.75 46857.50

Tab used:

Cross-linked D70 tabs: made from 150 μg/mL cross -linked protein complex

Current Tabs: TXSH-308

The data shown in Table 3 indicate the feasibility of performing a Stratus® assay using cross-linked anti-hTSH. A sensitivity run of eight replicates of A rates using cross-linked tabs revealed that the sensitivity was 0.09 μlU/mL as compared to a value of 0.1 μlU/ml for the double immuno-complex tabs. Sensitivity is defined as the concentration of TSH at which the Stratus® rate equals the average Cal A rate plu& two times the standard deviation. Notable low nonspecific binding was achieved primarily because the system used no secondary antibodies. EXAMPLE 5

Cross-linked Anti-hTSH CA2 Antibodies

A second anti-hTSH antibody, clone #CA2 (manufactured by Baxter Diagnostics Inc.) was buffer exchanged into 0.1 M sodium phosphate (pH 7.0). The volume was reduced so that the anti-hTSH concentration was about 6.0 +/- 0.5 mg/mL. Electron microscopy grade glutaraldehyde at a calculated volume of 5% was added to the antibody solution at a molar challenge ratio of glutaraldehyde:anti-hTSH = 100. The mixture was incubated at room temperature (23°C +/-2°C) for 1 hour.

The reaction was quenched by the addition of 1 M ethanolamine (pH 8) equal to a 1/9 volume of the reaction mixture and the incubation was continued at room temperature for 0.5 hours. Then the reaction mixture was purified by passing through an Ultrogel AcA-44 column (IBF Biotechnics, Cat # 230161). After protein concentration determination by

BCA, the cross-linked anti-hTSH was placed into TFSN (50mM Tris, 2% BSA, 0.1% FSN, 0.1% NaN₃, pH 8.0) containing 0.02% poly-L-lysine (Sigma, Cat # P-7890). Several concentrations of the cross-linked protein complex were prepared and spotted on glass fiber tabs for use in the Stratus® assay.

Figures 2 and 3 show the results obtained with a Stratus® TSH assay using glutaraldehyde-crosslinked CA2, compared to the traditional double immunocomplex tabs (CA2:GAM) on Stratus® and Stratus® II, respectively. Calibrators A, B, C, D, E, and F used in the assay contained 0.0, 0.25, 0.75, 3.00, 12.00 and 50.00 μlU/ml of hTSH. Raw data for the figures including the Rodbard instrument parameters are given in Table 4. Table 4

COMPARISON OF DOUBLE-IMMUNOCOMPLEX TABS WITH THOSE FROM THE CROSS-LINKED ANTIBODY TABS IN hTSH ASSAY

STRATUS® STRATUS® II

Immuno- Cross- Immuno- Cross-

CAL Complex Linked Complex Linked

(UIU/ML) (MV/MIN) (MV/MIN)

A (0.0) 125.0 104.0 130.5 109.3

B(0.25) 306.0 172.0 252.9 157.0

Q0.75) 472.0 304.0 406.2 266.2

D(3.00) 1448.0 926.0 1193.5 811.6

E(12.0) 4777.0 3314.0 4001.7 2792.0

F(50.0) 14334.0 10893.0 12800.8 9940.8

CalB/CalA 2.44 1.65 1.94 1.44

CalF/CalD 9.89 11.76 10.72 12.25

CalF/CalA 114.7 104.7 98.5 91.2

Rodbard parameters

A 125.0 104.0 103.5 109.3

B 0.886 1.040 0.927 1.083

C 224.78 130.684 240.822 104.492

D 64987.62 39637.57 64987.22 31878.42 Table 4 shows comparative performance of hTSH tabs prepared using cross-linked CA2 and tabs prepared from a double- immunocomplex on Stratus® and Stratus® II. Signal readings generated for the different calibrations using cross-linked tabs in Stratus® and Stratus® II is shown in columns 3 and 5 respectively. Corresponding data for the double-immunocomplex are shown in columns 2 and 4, respectively. Although the low end sensitivity (ratio Cal B/Cal A) for the double immunocomplex tabs is slightly better than that for the cross- linked tabs, high end sensitivity (ratio Cal F/Cal D) and overall sensitivity (ratio Cal F/Cal A) for the cross-linked tabs is better than or comparable to that of the immunocomplex tabs. These results clearly demonstrate feasibility of replacing the CA2:GAM (double-immunocomplex) tabs with those with the cross-linked CA2 tabs both on Stratus® and Stratus® II.

EXAMPLE 6

Cross-linked Anti-TAT Ffab">₂ A capture reagent consisting of an F(ab')₂ fragment of antibody against thrombin:antithrombin complex (Anti-TAT F(ab')₂) was buffer-exchanged into 0.1 M sodium phosphate (pH 7.6) and the volume was reduced so that the final F(ab')₂ concentration was about 5.00 +/- 0.25 mg/mL. A calculated volume of 100 mg/mL BS3 in water was added to the protein solution at a molar challenge ratio of BS3:F(ab')₂ = 100. The solution was incubated at room temperature (23 +/-2°C) for 2 hours. The cross-linked F(ab')₂ was purified by passing through a Sephadex G-25 column. The concentration of cross-linked F(ab')₂ was determined either by BCA protein assay or spectrophotometrically using an extinction coefficient of 1.48 mg/(mL)(cm).

An ELISA was performed using the cross-linked F(ab')₂. The plates were first coated with the cross-linked F(ab')₂ overnight at 2-8°C. TAT calibrators (0-50 nM) were added and the plates were incubated at ambient temperature for 0.5 hour. Anti-TAT antibody-HRP (horseradish peroxidase) conjugate was added and incubated at ambient temperature for 15 minutes followed by the addition of the substrate. The incubation then was continued for additional 15 minutes at ambient temperature. The plates were washed twice with a wash buffer before each new reagent was added. The plates were monitored at 405 nm using a plate reader in order to determine the colorimetric readout.

Table 5 is a comparison of ELISA performance between cross- linked F(ab')₂ plates and non-cross-linked F(ab')₂ plates. Two monoclonal antibody-HRP conjugates made from clones 4C72 and 5B46 (both manufactured by Baxter Diagnostics, Inc.) were used in the cross-linked F(ab')₂ assays.

Table 5

F(ab")2ρlates Crosslinked F(ab">2

Plates

Conjugate F Cal A Cal F/A F Cal A Cal F/A

I 2.295 0.151 15.2 2.491 0.128 19.5 π 1.942 0.139 13.8 2.238 0.109 20.5 m 1.942 0.119 16.3 2.017 0.093 21.7

IV 1.310 0.092 14.2 1.391 0.074 18.8

V 1.606 0.123 13.1 1.744 0.084 20.8

Where, I: 5.5 μg/mL 4C72 cσ nj. + 5.5 μg/mL 5B46 conj II: 2.5 μg/mL 4C72 conj. + 5.5 μg/mL 5B46 conj.

HI: 1.0 μg/mL 4C72 conj. + 5.5 μg/mL 5B46 conj. IV: 5.5 μg/mL 4C72 conj. alone V: 5.5 μg/mL 5B46 conj. alone

This table shows the signal generated in the presence of calibrators F (50 nM) and A (0.0 nM) as measured by absorption at 405 nm in the ELISA assays using untreated F(ab')₂ and cross-linked F(ab')₂- For all the combinations of the two conjugates tested, overall sensitivity (ratio Cal F/Cal A) of the plates prepared from the cross-linked antibody was higher than those prepared from the untreated antibody. This increase in sensitivity for the cross-linked antibody plates was not only due to decrease in non-specific binding (lower signal for Cal A) but also due to higher response for the Cal F resulting from more efficient immobilization of the cross-linked antibody on the plates.

The foregoing detailed description has been provided for a better understanding of the invention only and no unnecessary limitation should be understood therefrom as some modifications will be apparent to those skilled in the art without deviating from the spirit and scope of the appended claims.

Claims

We claim:

1. A method for immobilizing reagents onto a solid phase support, comprising the steps of: reacting a protein composition with a cross-linking agent to form a cross-linked protein complex, said protein complex comprising a specific binding receptor and being substantially free of non-specific proteins; and applying said cross-linked protein complex to a solid support under conditions effective for immobilizing said cross-linked protein complex on said solid support.

2. The method of claim 1 wherein said cross-linking agent is a bifunctional reagent.

3. The method of claim 2 wherein said bifunctional reagent is bis(sulfosuccinimidyl) suberate.

4. The metho. ,..i claim 2 wherein said 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. The method of claim 6 wherein said specific binding protein is intrinsic factor.

8. The method of claim 6 wherein said antibody is anti- carcinoembryonic antigen monoclonal antibody.

9. The method of claim 6 wherein said antibody is anti-human thyroid-stimulating hormone monoclonal antibody.

10. The method of claim 6 wherein said antibody fragment is anti-TAT F(ab')₂.

11. The method of claim 1 wherein said solid support is a polystyrene plate.

12. The method of claim 1 wherein said solid support is a glass fiber filter.

13. A specific binding assay solid phase reagent, comprising a cross-linked protein complex cross-linked by means of a bifunctional agent, said complex comprising at least two specific binding receptors and a solid support having said cross-linked protein complex immobilized thereon, said solid support being substantially free of non-specific proteins.

14. The specific binding assay solid phase reagent of claim 13, wherein said specific binding receptors are selected from the group consisting of antibodies, antibody fragments and specific binding proteins.

15. The specific binding assay solid phase reagent of claim 13, wherein said specific binding receptor is anti-carcinoembryonic antigen monoclonal antibody.

16. The specific binding assay solid phase reagent of claim 13, wherein said specific binding receptor is anti-human thyroid-stimulating hormone monoclonal antibody.

17. The specific binding assay solid phase reagent of claim 13, wherein said specific binding receptor protein is anti-TAT F(ab')₂-

18. The specific binding assay solid phase reagent of claim 13, wherein said bifunctional reagent is bis(sulfosuccinimidyl) suberate.

19. The specific binding assay solid phase reagent of claim 13, wherein said bifunctional reagent is glutaraldehyde.

20. The specific binding assay solid phase reagent of claim 13, wherein said bifunctional reagent is 1,4 butanediol diglycidyl ether.

21. The specific binding assay solid phase reagent of claim 13, wherein said solid support is a polystyrene plate.

22.. The specific binding assay solid phase reagent of claim 13, wherein said solid support is a glass fiber filter.

23. A method for conducting a specific binding assay to determine the concentration or presence of an analyte in a sample, comprising: (a) applying said sample under binding conditions to the specific binding assay solid phase reagent of claim 13 or 14; and (b) measuring the specific binding reactivity of said analyte with said solid phase reagent to detect or quantify said analyte in said sample.

24. The method of claim 23, wherein said measuring comprises: (a) applying a selected amount of indicator under binding conditions to said solid phase reagent; (b) determining the amount of said indicator bound to said solid phase; and (c) correlating said amount of said indicator with the concentration or presence of said analyte in said sample.

25. The method of claim 24 wherein said sample and said indicator are applied simultaneously to said solid phase.

26. The method of claim 23, wherein said measuring comprises: (a) applying a labeled specific competitive reagent under binding conditions to said solid phase; (b) determining the amount of label bound to said solid phase; and (c) correlating said amount of said label with the concentration or presence of said analyte in said sample.

27. The method of claim 26 wherein said specific competitive reagent is a labeled analogue of said analyte.

28. The method of claim 27 wherein said specific competitive reagent is a labeled receptor for said analyte.