Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54306—Solid-phase reaction mechanisms

Definitions

• This invention is directed to a method and a composition of matter. More specifically, this invention concerns itself with a multi-test solid phase or support (i.e., a porous medium) used to conduct a variety of immunoassays.
• Description of the Prior Art The various techniques which have been previously described in the technical and patent literature relating to immunoassay, in general, involve four basic processes for the isolation/detection of a reaction product (generally containing the analyte of interest) .
• a common form of immunoassay involves the immunochemical interaction of an analyte of interest with a ligand (antigen or antibody) thereby resulting in the formation of an immunoprecipitate; the presence of the precipitate being indicative of the presence of the analyte.
• homogeneous immunoassay (semi-quantitative and quantitative) are generally referred to as a homogeneous immunoassay; and, the solid phase immunoassays, which include the classical heterogenous systems (i.e. U.S. Patent No. 3,654,090), the multiple zone solid phase systems (i.e. U.S. Patent No. 4,258,001) and the solid phase radial partition immunoassay system (i.e. U.S. Patent No. 4,517,208) .
• the entire assay is conducted within a single, essentially fluid phase.
• the reactants, sample and indicator are contained within the same liquid fraction and the monitoring of the amount of indicator is performed without any separation between the immunochemically bound materials and those which remain free in the fluid.
• one component of the immunochemical -reaction is often immobilized on a solid phase or support.
• the need for such immobilization is the requirement, in such system, to separate the immunochemically bound constituents (presumably bound to the solid phase) from the unbound constituents.
• the demands of this system traditionally require that the bound materials (solid phase) and the unbound materials (liquid phase) be separated into two (2) mutually exclusive fractions.
• the immobilization of one of the immunochemical reactants on the solid phase in this system provides the most practical means for achieving such separation.
• the multiple zone solid phase system described in the above referenced patent involves immunochemical interactions which are similar to the classical heterogenous assay described previously; however, the separation of the immobilized constituents from the more mobile constituents is effected by transfer or elution thereof from one contiguous zone (i.e. contiguous laminae) to another.
• the fourth immunoassay system which has most recently evolved, is a hybrid of both classical heterogeneous and the homogeneous immunoassay systems and yet distinctive from the multiple zone system discussed hereinabove.
• This hybrid more descriptively characterized as "solid phase, radial partition immunoassay", initially involves the immobilization, within a solid phase, of an im unoreagent (i.e., an antibody). Subsequent thereto, an aliquot of patient's sample is applied to the portion of the solid phase containing the immobilized immunoreagent.
• a second immunoreagent (generally a ligand conjugated to an indicator) is applied to the solid phase in a similar fashion, and allowed to interact with either the immunoreagent, which has been immobilized within the solid phase, or with the analyte from the patient's sample, depending upon the nature of the analytical protocol (i.e., competitive or sandwich assay) .
• the manner in which the various materials are applied to the solid phase containing the immobilized immunoreagent defines the reaction zone within the solid phase.
• a wash solution may be dispensed onto this reaction zone, whereby unbound materials (i.e., materials which have not immunochemically reacted directly or indirectly with the immobilized reagent) are eluted from the reaction zone in a radial pattern, thereby producing an area within the reaction zone which is essentially free of unbound materials.
• unbound materials i.e., materials which have not immunochemically reacted directly or indirectly with the immobilized reagent
• an immunoreagent can be immobilized upon the surface of a solid phase
• any one or a combination of mechanisms including adsorption, absorption or covalent bonding, see U.S. Patent No. 4,168,146 (to Grubb) .
• Grubb uses a variety of reagents, including cyanogen bromide and glutaraldehyde to covalently bond antibody to a variety of different substrates (i.e. cellulose containing materials, cellulose acetate film or a microporous plastic sheet containing silica) .
• substrates i.e. cellulose containing materials, cellulose acetate film or a microporous plastic sheet containing silica
• the amount of soluble immunocomplex adsorbed on to the porous medium was based upon what was empirically determined to be effective to selectively bind and retain an analyte of interest to the immunoreagent within a defined area of the porous medium in the context of a particular immunoassay. Accordingly, the prescription of an effective amount can vary from immunoassay to immunoassay, and be further dependent upon the physical geometry of the solid phase and the manner in which the assay results are monitored.
• a shortcoming of this method is that different soluble immunocomplexes adsorbed on to the porous medium are needed for each different type of immunoassay; as such a need exists to develop a multi- test immunoreactant that can be used in a variety of immunoassays.
• this invention provides an immunochemical reagent made of secondary antibodies absorbed on to an inert porous medium. More specifically, this invention provides an immunochemical reagent made of anti-serum to a specific species adsorbed on to an inert porous medium.
• the anti-serum may be for example, goat anti-mouse, goat anti-rabbit, goat anti- sheep, sheep anti-goat, rabbit anti-goat, donkey anti- goat or more generally, a second species anti first species.
• anti-sera may be referred to herein as secondary antibody.
• the secondary antibody may be purified or unpurified.
• combinations of the various specific animal species may also be applied to a solid phase.
• this invention provides a method for conducting a solid phase immunoassay of a fluid sample for an unknown amount of analyte, the method involving: a method for conducting a solid phase immunoassay of a fluid sample for an unknown amount of analyte, the solid phase consisting essentially of secondary antibodies immobilized on a solid support, the method involving: combining under binding conditions, a fluid sample containing the analyte and the primary antibodies to form a fluid mixture of analyte-primary antibody complexes and primary uncomplexed antibodies; applying the fluid mixture under binding conditions to the solid support to bind analyte-primary antibody complexes and uncomplexed primary antibody; applying an indicator to the solid phase under binding conditions to bind with bound uncomplexed antibody or to bind with bound analyte; observing the extent to which the indicator is present on the solid support; and correlating the extent to which the indicator is present of the solid support with the amount of unknown analyte in the sample.
• the indicator may also be added to the fluid sample; this can be done by either adding the components at the same time or allowing the analyte and antibody to briefly incubate before the addition of the indicator. Binding conditions are further delineated in P. Tijssen, Laboratory Techniques in Biochemistry. Molecular Biology, Practice and Theory of Enzyme Immunoassay 123-145. (4th ed. 1987) (hereby incorporated by reference) .
• the term indicator in the context of this invention means a labeled conjugate.
• the conjugate is antibody or an analyte depending on the assay format.
• the label is a fluorescent, enzymatic, colorometric or radio etric compound 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. P. Tijssen, Laboratory Techniques in Biochemistry. Molecular Biology, Practice and Theory of Enzyme Immunoassay 173-217 and 368-376. (4th ed. 1987) (hereby incorporated by reference) .
• This invention also provides a method for conducting a solid phase immunoassay of a fluid sample for an unknown amount of analyte, the solid phase consisting essentially of secondary antibodies immobilized within a finite zone of the interstices of a solid, inert, porous support, the method involving: combining under binding conditions a fluid sample containing the analyte and primary antibodies to form a fluid mixture of analyte-primary antibody complexes and uncomplexed primary antibodies; applying the fluid mixture under binding conditions to the center of the finite zone to bind analyte-primary antibody complexes and uncomplexed antibody; applying an indicator to the solid support under binding conditions to bind with uncomplexed antibody; observing the extent to which the indicator is present within a delimited area of the reaction zone; and correlating the extent to which the indicator is present in the delimited area with the amount of unknown analyte in the sample.
• this invention provides a method for conducting a solid phase immunoassay of a fluid sample for an unknown amount of analyte, the method involving: a method for conducting a solid phase immunoassay of a fluid sample for an unknown amount of analyte, the solid phase consisting essentially of secondary antibodies immobilized on a solid support, the method involving: applying under binding conditions primary antibody to the solid support; applying under binding conditions the fluid sample to the solid support; applying an indicator to the solid phase under binding conditions to bind with bound uncomplexed antibody; observing the extent to which the indicator is present on the solid support; and correlating the extent to which the indicator is present of the solid support with the amount of unknown analyte in the sample.
• This invention also provides a method for conducting a solid phase immunoassay of a fluid sample for an unknown amount of analyte, the solid phase consisting essentially of secondary antibodies immobilized within a inite zone of the interstices of a solid, inert, porous support, the method involving: applying under binding conditions primary antibody to the center of the finite zone of the solid support; applying an indicator to the solid support under binding conditions to bind with uncomplexed antibodies, applying under binding conditions the fluid sample to the center of the finite zone of the solid support; observing the extent to which the indicator is present within a delimited area of the reaction zone; and correlating the extent to which the indicator is present in the delimited area with the amount of unknown analyte in the sample.
• Fig.l shows response of a calibrator versus percent (%) goat anti-mouse immunoglobulin adsorbed on an inert porous matrix.
• Fig.2 shows response versus percent (%) goat anti- rabbit in normal rabbit serum.
• Fig.3 shows titration of rabbit anti- ⁇ tradiol
• Pig.4 shows comparison of STRATUS* (Baxter Diagnostics Inc.) e ⁇ tradiol versus multi- test calibration-curves.
• Fig.5 shows a comparison of response versus concentration of estradiol in terms of STRATUS ⁇ (Baxter Diagnostics Inc.) performance versus the multi-test method.
• the multi-test immunoreagents contemplated by this invention are porous inert medium having adsorbed thereto species specific antiserum or antiserum from a homologous species. These reagents do not contain specific antibody and are capable of binding any primary antibody. In particular, goat anti-rabbit and goat anti-mouse antiserum function well as the antiserum adsorbed to the porous inert medium.
• Antisera is prepared by injecting the immunoglobulin from a first species into a second species. The second species develops antiserum against the immunoglobulin of the first species, or more generally a second species anti first species.
• anti-sera may be referred to herein as secondary antibody.
• the secondary antibody may be purified or unpurified.
• a primary antibody is an antibody which is specific for an analyte, for example anti-hCG antibody is specific for hCG.
• Primary antibodies may be developed from a variety of species using standard techniques, however, the species most commonly used are rabbits and mice. Any primary antibody developed in a species will be recognized by antiserum against that species. For example, a primary antibody developed in a mouse will be recognized by antisera such as goat anti-mouse or rabbit anti-mouse. Since rabbits and mice are the most common species used to develop primary antibodies and most common antisera is second species anti-mouse or anti-rabbit.
• homologous species Because of the homology between certain strains of primary antibody, a secondary antibody developed against a species may also react with other closely related species. For example, goat anti-mouse will often react with primary antibody prepared from a rat. For purposes of this invention these species are referred to herein as homologous species.
• Primary antibody as used herein means an antibody specific for an analyte that immunologically reacts with the appropriate antisera and includes primary antibody from the specific species and any homologous species. The benefit of this is that it provides a wider selection of antibodies.
• the solid phase which is compatible with the foregoing objectives of this invention may be "inert". More specifically, the characterization of a solid phase as “inert” (in the context of this invention) is intended as referring to the relative nonreactive character of the surface of the solid phase with respect to its ability to indiscriminately adsorb proteinaceous materials. As noted earlier herein, in the Background section of this disclosure, glass and other materials similar in chemical composition, are generally regarded as relatively inert in comparison to other more reactive materials when measured against their ability to adsorb protein.
• the physical form of the solid phase is such that the inter-stices or pores within such solid phase are sufficiently small so that the reaction fluids are retained and transported therebetween by capillary action.
• the solid phase is advantageously composed of a mat of compressed fibers, such as glass or synthetic fibers.
• This solid phase may also 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 this invention because of its inert characteristics and because of its ability to adsorb the soluble immunocomplex of this invention in quantities sufficient for the sensitization of the glass fibers so as to render it suitable in immunoassay.
• the multi-test immunochemical reagent can be used in a wide variety of analytical protocols for analysis of a variety of biological and industrial fluids. It should be noted that a single glass fiber filter paper can be. used for any assay and may contain antiserum for a number of species. For instance, these reagents can be advantageously used for the immunoassay of blood or urine in the rapid and quantitative analysis of such fluid for the presence of therapeutic drugs, natural or synthetic steroids, hormones, antibodies and other analytes of interest.
• Exemplary of therapeutic drugs which can be analyzed in such a protocol include digoxin, dilantin, phenobarbital, theophylline, gentamicin, quinidine, and the like.
• Multi-test immunochemical reagent prepared in the foregoing manner can also be utilized in the immunoassay for the detection of steroids, such as cortisol, aldosterone, testosterone, progesterone, and estriol or serum protein, such as ferritin.
• Hormone levels are also capable of determination through the use of appropriate immunochemical reagents, immobilized as described hereinabove, on an appropriate solid phase.
• hormones include the thyroid hormones, i.e. triiodo-thyronine or thyroid stimulating hormone (TSH) ; the peptide hormones, i.e. insulin, corticotropin, gastrin, angiotensin, and proangiotensin; the polypeptide hormones, i.e. thyrotropin, levteotropin, and somatotropin human chorionic gonadotropic hormone (HCG) .
• TSH thyroid stimulating hormone
• peptide hormones i.e. insulin, corticotropin, gastrin, angiotensin, and proangiotensin
• polypeptide hormones i.e. thyrotropin, levteotropin, and somatotropin human chorionic gonadotropic hormone (HCG) .
• the assay format includes both competitive and sandwich assays.
• the conjugate may be added simultaneously with the sample containing the analyte and the specific antibody.
• Estradiol conjugate an estradiol conjugate of .estradiol (E2) and alkaline phosphatase (ALP) were prepared using a standard mixed anhydride method. (B.K. Van Weeman, FEBS LETTERS Vol.24, No.l, July 1972) . The resulting E2-ALP conjugate was diluted to .04 ⁇ g/mL in 50 mM Tris, pH 8.0, 50 M NaCl containing 0.1% protein with 0.1% azide added as a preservative.
• Estradiol Calibrators An estradiol stock solution of 100 ⁇ g/mL of estradiol in 80% ethanol was prepared.
• estradiol calibrators are a standard that has a specific amount of analyte.
• Substrate/Wash A solution containing 1 mM 4- methylumbelliferyl phosphate in diethanolamine buffer, pH 9.0, containing stabilizers, surfactant and blue dye, with 0.1% sodium azide added as a preservative.
• Rat anti-estradiol monoclonal antibody (Rat anti- F2) was obtained from OEM Concepts, Inc.
• Goat anti-mouse monoclonal antibody was prepared using conventional procedures. Preparation and Optimization of Goat Anti-Mouse Multi- Test Reagent
• the tabs spotted with the incremental solutions of GAM were evaluated as follows: One hundred microliters of a 1/100 dilution of Rat anti-E2 were added to 100 ⁇ L of 0(A) calibrator. One hundred and fifty microliter aliquots of the mixture were added to the tabs in the zone containing the GAM (reaction zone) . During a brief (two minute) incubation the Rat anti-E2 binds to the GAM. Next, 50 ⁇ L of E2-ALP conjugate were added to about the center of the reaction zone. The E2-ALP binds to the Rat anti-E2. Next, about 75 ⁇ L of substrate/wash were applied to about the center of the reaction zone. The substrate/wash elutes any unbound material away from the reaction zone and simultaneously the reaction between the bound ALP and the substrate is initiated forming a fluorescent product. The fluorescent response from a finite portion of the reaction zone was determined using a front surface fluorometer.
• the immunoglobulin spotted on the tab could be from the rat with the estradiol specific antibody coming from a mouse, the reverse of the method described above, which again increases the choice of available antibodies.
• Optimization of the Rat Anti-Estradiol Concentration The optimal rat anti-estradiol antibody (rat anti-E2) was determined using the 2.5% GAM tabs prepared as described above. The rat anti-E2 was diluted to various concentrations in 50mM Tris, pH 8.0, containing 0.1% bovine serum albumin. The dilutions were as follows: 1:50, 1:60 and 1:75.
• dilutions may be evaluated (i.e., 1:10, 1:100, 1:1000 and 1:10,000 or 1:2, 1:3, 1:5 respectively) depending on the antibody being evaluated and the nature of that antibody.
• concentrations were evaluated as follows: One hundred microliters of each dilution of rat anti-E2 were added to cups containing 100 ⁇ L of A(0) calibrator and cups containing 100 ⁇ L of B(20 pg/mL) calibrator. The mixtures were incubated at room temperature for fifteen minutes. The time and temperature for incubation may be varied according to the requirements for the assay. Preferred ranges are from 0 to 4 hours and 18 to 37 degrees C.
• the optimal dilution of rat anti-E2 was selected by measuring two parameters.
• the first parameter is fluorescent response generated when the A(0) calibrator is assayed with each dilution of rat anti- E2.
• the fluorescent response is directly proportional to the amount of rat anti-E2 captured by the GAM. Accordingly, these responses are indirectly proportional to the amount of E2 in the sample (characteristic of a competitive assay) .
• the second parameter measured is the ratio of the fluorescent response from the B calibrator (20 pg/mL) over the fluorescent response from the A(0) calibrator (B/A ratio) . Low B/A ratios indicate that there is a measurable difference between these two calibrator levels.
• the highest Calibrator A rate with the lowest Calibrator B/A ratio occurs using the 1:50 dilution of Rat anti- E2.
• the other dilutions show no differentiation between these two levels of calibrators hence are not useful in a diagnostic assay of Estradiol.
• the 1:50 dilution was chosen to perform assay of Estradiol.
• the antibody-antigen complex is formed by adding 100 ⁇ l of the 1:50 dilution of Rat estradiol monoclonal antibody in solution and 100 ⁇ l of serum ' sample or calibrator to a STRATUS* (Baxter Diagnostics Inc.) cup.
• This mixture which now contains antigen-antibody complex as well as uncomplexed antibody, is then added directly to the center of the multi-test goat anti- mouse reagent matrix which captures the free estradiol antibody as well as estradiol-antibody complex that was formed in the STRATUS* (Baxter Diagnostics Inc.) cup.
• the reagent matrix is then moved to a second work station where 50 ⁇ l of the E2 conjugate is added to the center of the matrix.
• the conjugated E2 combines with any unoccupied binding sites of the immobilized estradiol antibody.
• enzyme activity is initiated at the same time that bound and free material partitioned by addition of 78 ⁇ l of substrate/wash solution III.
• Example II Goat-anti-rabbit Multi-test Reagent Used in an Assay for Estradiol
• multi-test reagent materials and assay format using goat-anti-rabbit antibody with normal rabbit serum And a rabbit anti- estradiol antibody.
• STRATUS Boxter Diagnostics Inc.
• E2 conjugate an £2-ALP conjugate was prepared using the standard mixed anhydride method. It was then diluted to a concentration of 1.2 ⁇ g/mL in a Tris buffer at pH 7.0.
• Substrate/Wash a solution containing ImM 4- ⁇ thylumbellif ⁇ ryl phosphate in diethanolamine buffer, pH 9.0.
• Rabbit anti-E2 antibody from Diagnostic Systems Laboratories, Inc. Tab Optimization Solution A was prepared by diluting normal rabbit serum to 1% in a 50 M Tris buffer, pH 8, containing 0.5% Zonyl FSN.
• Solution B was prepared by dilution goat anti-rabbit immunoglobulin to 1.2% in the above buffer.
• One mL of Solution A was added to each of 13test tubes, labeled 1 to 13. Then, beginning with 0.5mL in tube 1, Solution B was added in 0.1 mL increment to tubes 2 to 13 (Titration tubes) . After an overnight incubation at 25°C to effectuate formation of a goat anti-rabbit/normal rabbit serum complex the solutions were filtered through .8 ⁇ m filter to remove particulates.
• the rabbit anti-estradiol antibody was titered using the optimal goat anti-rabbit/normal rabbit serum tabs described above.
• the titer was accomplished by serially diluting the rabbit anti-estradiol antibody in a 50 mM Tris buffer, pH 8, containing 0.1% bovine serum albumin and 0.1% (3-[chloamidopropyl)- dimethylammonio]-2-hydroxy-l-propane sulfonate) and were assayed as described below.
• the optimal dilution of rabbit anti-estradiol was selected by measuring two parameters. The first parameter is fluorescent response generated when the A(0) calibrator is assayed with each dilution of rabbit anti-estradiol.
• the fluorescent response is directly proportional to the amount of rabbit anti-estradiol captured by the GAR. Accordingly, these responses are indirectly proportional to the amount of estradiol in the sample (characteristic of a competitive assay) .
• the second parameter measured is the result of subtracting the fluorescent response of the F (2500 pg/mL) calibrator from the fluorescent response of the B (50 pg/mL) calibrator. (Cal B - Cal F) .
• the dilution which gives the maximal difference is a measure of sensitivity.
• the dilution which produced maximal Calibrator A response and maximal Calibrator B response minus Calibrator F response was determined and this dilution was chosen as the optimal dilution for the rabbit anti-estradiol.
• An example of the titration is shown in Fig.3; the 1:7500 dilution was selected as optimal.
• serum estradiol is extracted from endogenous binding proteins by adding 50 ⁇ l of E2 Assay Diluent to 25 ⁇ l of patient sample, control or calibrator. Then, the binding reaction is initiated by addition of 25 ⁇ l of the 1:7500 dilution of rabbit anti-estradiol to the extracted sample followed by a 15 minute incubation of the samples at 25°C. Following the incubation about 76 ⁇ l of the sample is immediately applied to the glass-fiber paper tab which had been prepared with the goat anti-rabbit/normal rabbit serum complex that gave the highest response. Sample application is directed to the center of the dried complex. The tab is moved to a second work- station where about 60 ⁇ l of the estradiol conjugate is added to about the center.
• the conjugate combines with unoccupied binding sites of immobilized estradiol antibody.
• the tab is moved to a third work station and enzyme activity is initiated and simultaneously free and bound material are radially partitioned by addition of about 70 ⁇ l of sub ⁇ strate/wash solution III to the center.
• front surface fluorometry is used to measure fluorescent rates. Responses are inversely pro- portional to the concentration of sample E2 and are converted to clinical units by comparison to a stored standard curve.
• Example 3 Comparative Tab Performance In Fig.4 the current STRATUS* (Baxter Diagnostics Inc.) and multi-test tab calibration curves are compared. There is significant improvement in curve slope (sensitivity) of estradiol values less than 500 pg/ml with the multi-test tabs. However, the current STRATUS* (Baxter Diagnostics Inc.) tab's performance is better (slope is greater) for estradiol values of greater than 1000 pg/mL. Fig.5 compares the response per unit of estradiol for values of less than 100 pg/mL; the greater the change the more sensitive the assay. Here, the multi-test tab demonstrates improved performance over STRATUS* (Baxter Diagnostics Inc.) .
• Example 4 Goat Anti-mouse Multi-test Reagent Used in An Assay for hCG
• Anti-hCG conjugate an anti-hCG antibody conjugate of alkaline phosphatase (ALP) and antibody to hCG were prepared using a standard method using sulfo-SIAB (sulfo-succimimidyl (4-iodoacetyl)- aminobenzoate sodium salt and coupled to reduced anti- hCG Fab' antibody. The resulting conjugate was diluted to about 180 ng/mL in buffer with 0.1% sodium azide as a preservative.
• hCG Calibrators an hCG stock solution of 12 million mlU/mL in distilled water was prepared. Aliquots of the hCG stock were added to normal hCG free human serum to produce a set of hCG calibrators at 0(A), 5(B), 15(C), 50(D), 150(E), and 500(F) miu/mL.
• the glass fiber filters spotted with the incremental solutions of GAM are evaluated as follows: about a one hundred microliter aliquot of about a 1:100 dilution of mouse anti-hCG antibody are added to about 100 ⁇ l of an (F) calibrator. In a brief incubation, the hCG in the F calibrator binds to the anti-hCG antibody. About 100 ⁇ l of the incubation mixture are added to the tabs in the zone containing the GAM (reaction zone) . During a brief (about two minutes) incubation the mouse anti-hCG binds to the GAM. Next, about 50 ⁇ l of anti-hCG antibody conjugate are added to about the center of the reaction zone.
• the conjugate binds to the hCG which is bound to the anti-hCG antibody, which in turn is bound to the GAM on the tab of glass fiber filter paper.
• substrate-wash is applied to about the center of the reaction zone.
• the substrate-wash elutes any unbound material away from the reaction zone and simultaneously the reaction between the bound ALP and the substrate is initiated forming a fluorescent product.
• the fluorescent response from a finite portion of the reaction zone is determined using a front surface fluorometer.
• the solution of maximum Calibrator F response is selected as being optimal since the most antibody is captured per unit GAM. Multiple tabs of the glass fiber filter paper are spotted using the solution which gives the maximal response in the method described above.
• the optimal mouse anti-hCG antibody is determined using the GAM tabs prepared as described above.
• the mouse anti-hCG antibody is diluted to various concentrations in 50 mM Tris, pH 8.0, containing 0.1% bovine serum albumin.
• concentrations evaluated are as follows: 1:100, 1:200 and 1:500. It is readily apparent that wider or narrower concentrations may be evaluated (ie., 1:10, 1:100, 1:1000 and 1:10,000 or 1:2, 1:3, and 1:5 respectively) depending on the antibody being evaluated and the nature of that antibody.
• the dilutions are evaluated as follows: about 100 ⁇ l of each dilution of mouse anti-hCG antibody are added to cups containing 100 ⁇ l of F (500 mlU/mL) calibrator, cups containing 100 ⁇ l of B (5 mlU/mL) calibrator and 100 ⁇ l of A (0) calibrator.
• the mixtures are incubated at room temperature for fifteen minutes. The time and temperature for incubation may be varied according to the requirements for the assay. Pre ⁇ ferred ranges are from 0 to 4 hours and 18 to 37 degrees Celsius. After the incubation, 100 ⁇ l aliquot of the mixtures are added to the tabs in the reaction zone.
• mouse anti-hCG antibody (bound and unbound) binds to the GAM.
• 50 ⁇ l of anti-hCG conjugate is added t ⁇ about the center of the reaction zone.
• the conjugate binds to any hCG in the reaction zone.
• about 75 ⁇ l of substrate-wash are applied to about the center of the reaction zone.
• the substrate-wash elutes any unbound material away from the reaction zone and simultaneously the reaction between the bound ALP and the substrate is initiated forming a. fluorescent product.
• the fluorescent response from a finite portion of the reaction zone is determined using a front surface fluorometer.
• the optimal concentration of mouse anti-hCG antibody is selected by measuring two parameters.
• the first parameter is fluorescent response generated when the F(500 mlU/mL) calibrator is assayed with each concentration of mouse anti-hCG antibody.
• the fluorescent response is directly proportional to the amount of anti-hCG antibody captured by the GAM. Accordingly, these responses are also directly proportional to the amount of hCG in the sample (characteristic of a sandwich assay) .
• the second paramater measured is the ratio of the fluorescent response from the B calibrator (5 miu/mL) over the fluorescent response from the A(0) calibrator (B/A ratio) . High B/A ratios indicates that there is a measurable difference between these two calibrator levels.
• the anti-hCG antibody concentration which gives the highest fluorescent response using the F(500 miu/mL) calibrator with the highest B/A ratio is chosen as optimal.
• the anti-hCG antibody can be optimized first by using tabs of glass fiber filter paper that have been prepared with 76 ⁇ l of an excess amount of GAM (ie. , % of GAM that is greater than the % of GAM which gave the maximal Calibrator A response) .
• the GAM can be optimized in the procedure described above.
• the antibody-antigen complex is formed by adding 100 ⁇ L of the optimum anti-hCG antibody in solution and 100 ⁇ L of serum sample, calibrator or control to a STRATUS" (Baxter Diagnostics Inc.) cup.
• STRATUS Boxter Diagnostics Inc.
• One hundred microliters of this mixture which now contains antigen-antibody complex as well as uncomplexed antibody, is then added directly to about the center of multi-test goat anti-mouse reagent matrix which captures the antigen-antibody complex that was formed as well as an uncomplexed antibody.
• the reagent matrix is then moved to a second work station where 50 ⁇ L of the conjugate is added to about the center of the matrix.
• the conjugate combines with the bound hCG.
• Example 5 Example 2 is repeated except that the antibody on the glass fiber filter paper comprises about a 76 ⁇ l aliquot of the proper final concentrations of the GAR of Example 2 and the GAM of Example 4.
• Example 2 The optimi ⁇ zation of concentration of the GAR and GAM which are applied to the glass fiber filter paper are determined by the procedures set forth in Example 2 (for the GAR) and Example 4 (for the GAM) . Optimization of the rat anti-estradiol antibody concentration is determined as per Example 2. The concentration of serum samples, calibrators, and controls are determined as per the assay format of Example 2.
• Example 6 The optimi ⁇ zation of concentration of the GAR and GAM which are applied to the glass fiber filter paper are determined by the procedures set forth in Example 2 (for the GAR) and Example 4 (for the GAM) . Optimization of the rat anti-estradiol antibody concentration is determined as per Example 2. The concentration of serum samples, calibrators, and controls are determined as per the assay format of Example 2. Example 6
• Example 4 is repeated except that the antibody on the glass fiber filter paper comprises the GAR of Example 2 and the GAM of Example 4.
• the optimization of concentrations of the GAR and GAM which are applied to the glass fiber filter paper are determined by the procedures set forth in Example 2 (for the GAR) and Example 4 (for the GAM) .
• Optimization of the mouse anti-hCG antibody concentration is determined as per Example 4.
• the concentration of serum sample, calibrator, or control is determined as per the assay format of Example 4. Examples 7-11
• Examples 2 and 4-6 are repeated respectively except that the specific antibody (ie., anti-hCG antibody, anti-E2 antibody etc.) is added to the multi-test reagent prior to the addition of the sample containing the analyte.
• the specific antibody ie., anti-hCG antibody, anti-E2 antibody etc.

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• 1992
  • 1992-12-30 JP JP5512469A patent/JPH06505803A/ja active Pending
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• 1993
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