COMPETITIVE IMMUNOFLUORESCENCE ASSAY AND TEST KIT

Abstract: Competitive immunofluorescence assays for antigens in which immune complexes are precipitated with a nonfluorescent, nonlight-scattering precipitant such as polyethylene glycol and the resulting immunoprecipitate is dissolved with a nonfluorescent solvent of low ionic strength that maintains the pH of the solution substantially constant for immunofluorescence intensity reading. The assays are carried out by incubating the sample with fluorescent-labeled antigen, anti-antigen antibody, and a secondary antibody to the anti-antigen antibody followed by addition of the precipitant to form an immunoprecipitate. The precipitate is separated by centrifuging, dissolved in the solvent, and the immunofluorescence intensity of the solution is read with a fluorometer and compared to a standard curve.

Description: COMPETITIVE IMMUNOFLUORESCENCE ASSAY AND TEST KIT Description Technical Field The invention is in the field of immuno- diagnostics. More particularly it involves an improved competitive immunofluorescence test for antigens. Background Art Competitive immunoassays for antigens or haptens are known in the immunodiagnostic art. They are based on competition for specific antibody against the antigen between labeled antigen (known) and unla- beled antigen (unknown) in the sample being assayed. Immune complexes that form between the antigen/hapten and antibody are separated and the amount of label therein detected by appropriate label detection means. The concentration of unknown (unlabeled) antigen/hap¬ ten in the sample is determined by comparison with the effect of standards. US Pats Nos 3,981,982 and 4,298,592 describe a type of competitive radioiminunoassay called a "double antibody separation" assay. In this technique the sample suspected of containing antigen is first incubated with a first specific antibody and known radiolabeled antigen. Following this a second anti¬ body that is specific to the first antibody is added together with polyethylene glycol. The second anti¬ body and polyethylene glycol form a complex with the antigen-first antibody reaction product that agglom- erates and precipitates from the incubation mixture. The precipitate is separated from the supernatant -2- containing free antigen. The radioactivity of the separated precipitate is read directly with a scintil¬ lation counter and the concentration of antigen in the sample is determined by comparing the reading to a 5 standard radioactivity-antigen concentration curve. These radioimmunoassays are sensitive but have the disadvantage of all radioimmunoassays of involving radioactive reagents that must be handled, used, and disposed of with extreme care by highly trained 10 personnel. Corresponding immunofluorescence assays in which the fluorescence of a solid phase is read are known. Such assays suffer from high background fluorescence from the solid phase itself or from scattered light. 15 Chard, T. , and Sykes, A., Clin Che (1979) 26/6: 973-976 teach a fluoroimmunoassay for human choriomammotro in in which a plasma sample is incu¬ bated with fluoreseein-labeled human choriomammotropin and sheep antiserum to the hormone. Following the 20 incubation immune complexes are separated from free hormone by addition of aqueous polyethylene glycol. The mixture is centrifuged and the fluorescence of the supernatant is read and compared to a standard. Such assays in which the fluorescence of the supernatant is 25 read have the problem of fluoresence due to interfer¬ ing materials in the supernatant (plasma or serum). Pourfarzaneh, M., et al, Clin Chem (1980) 26/6: 730-733, describes a competitive immunofluores¬ cence assay for cortisol in serum. The serum sample 30 is incubated with fluoreseein-labeled cortisol and anti-cortisol antibody coupled to magnetizable cellu¬ lose/iron oxide particles. After the incubation the solid phase is separated, washed to remove free, labeled antigen, and eluted with an equivolume mixture of ethanol and 0.02 M NaOH. The fluorescence of the eluate is read with a fluorometer and compared to a standard curve to determine the concentration of cortisol in the sample. Japanese Patent Application no 150547/79 describes direct and indirect immunofluorescence assays for various antigens. The sample is first incubated with antibody (fluorochrome-labeled in the direct assay, unlabeled in the indirect assay). The mixture is then incubated with an immunoadsorbent. The indirect assay involves an additional incubation of the immunoadsorbent with a fluorochrome-labeled antibody against the first antibody. In both types of assay, immune complexes are eluted from the immuno- adsorbent with 0.02-0.04 N NaOH and the fluorescence of the eluate is read and compared to a standard. These prior assays involving immunoadsorption and immobilization have several shortcomings. Firstly, the reproducibility of the immobilization is often inconsistent and affects the precision of the assay adversely. The adsorption may also affect the sta¬ bility of the immune complexes adversely and cause artificially low immunofluorescence intensity read¬ ings. Elution inefficiency and/or interference from spurious eluted materials may also cause artificially low fluorescence intensity readings. In contrast to the above described prior competitive immunofluorescence assays, the invention assay does not involve solid phase readings or read- ings of supernatants or eluates that may contain interfering materials. Instead the invention assay involves precipitating the immune complex with a nonfluorescent, non-light scattering immunoprecipi- tant, dissolving the resulting immunoprecipitate -with a solvent that does not add background fluorescence or light scatter, and reading the solution. Compared to the prior competitive immunofluorescence assays, the invention assay is highly sensitive and has improved reproducibility and dose-response and precision characteristics, and requires less antibody reagent. Disclosure of the Invention One aspect of the invention is a competition immunofluorescence assay for determining the amount of an antigen in a sample suspected of containing the antigen comprising incubating the sample with a solution of fluorescent-labeled antigen, anti-antigen antibody, and an antibody against the anti-antigen antibody, and adding a nonfluorescent nonlight-scat- tering immunoprecipitant to the incubation mixture to form an immunoprecipitate, characterized in that the immunoprecipitate is separated and dissolved in a nonfluorescent solvent that has a low ionic strength and maintains the pH of the resulting solution sub- stantially constant and the fluorescence intensity of the resulting solution is measured and compared to a standard curve. Another aspect of the invention is a test kit for carrying out the above-described competition immunofluorescence assay comprising in association: fluorescent-labeled antigen reagent, anti-antigen antibody reagent, an antibody against the anti-antigen antibody, and a nonfluorescent, nonlight-scattering immunoprecipitant characterized in that the kit includes a nonfluorescent solvent for dissolving immu- noprecipitates of the immunoprecipitant that has a low ionic strength and the ability to maintain the pH of the immunoprecipitate solution substantially constant. Modes for Carrying Out the Invention The sample that is analyzed by the invention method is a body fluid such as blood, blood serum blood plasm, urine, lymph fluid, bile, spinal fluid or the like. The particular body fluid analyzed may vary with the antigen being assayed. In most instances blood serum will be used. About 0.1 to about 500 μl of fluid will be used per assay. Samples may be cryo- preserved pending analysis if necessary. Substances that may be assayed by the inven¬ tion method include antigens (molecules that elicit an immune response when introduced into the bloodstream of a vertebrate host) and haptens that are not immuno- genic per se but may be conjugated to a protein car- rier to form a conjugate that is immunogenic and capable of raising antibodies against the hapten. The term "antigen" is used herein to generically denote both antigenic and haptenic compositions. Such sub¬ stances include drugs, hormones, pesticides, toxins, vitamins, human, bacterial, and viral proteins, and the like. Examples of antigens that may be assayed by the invention method are thyroxine (T4), triiodo- thyronine (T3) , digoxin, gentamicin, amikacin, tobra- micin, kanamycin, netilmicin, cortisol, luteinizing hormone, digitoxin, vitamin B12, progesterone, human chorionic gonadotropin, theophylline, angiotensin, human growth hormone, and the like. The reagents that are incubated with the sample suspected of containing antigen to form immune complexes are (1) fluorescent-labeled antigen, (2) anti-antigen antibody, (3) antibody against the anti-antigen antibody, and (4) a nonfluorescent non- light-scattering immunoprecipitant. The fluorescent- O PI labeled antigen may be made by coupling the antigen with a reactive derivative of a fluorogen such as fluorescein (eg, fluorescein isothiocyanate (FITC) and fluorescein amine), rhodamine or dansyl using multifunctional coupling agents such as aldehydes, carbodiimides, dimaleimides, imidates, and succinimides. Fluorescein is a preferred label. Antibody against the antigen may be made by inoculating a host vertebrate with the antigen, typically repeatedly at several day intervals, bleed¬ ing the host and separating Ig from the blood. Anti¬ body against the antibody to the antigen may be made by immunizing another host vertebrate species with said Ig and collecting Ig from the second host. The nonfluorescent, nonlight-scattering immunoprecipitant is a compound that causes an immune complex to precipitate or increases the rate of immune complex precipitation. Compounds that are conventionally used to precipitate proteins and that have the required nonfluorogenic properties may be used. Examples of such materials are polyethylene glycol having a molecular weight in the range of about 3000 to about 12000, preferably about 4000 to 10000, and inorganic salts such as ammonium sulfate. Polyethylene glycol is a preferred immunoprecipitant. The absolute amounts of the reagents used will, of course, depend upon the volume of the sample. A known amount of -labeled antigen will be use . The labeled antigen may be diluted to provide a reagent that is stoichiometrically ideal for the antiserum used. Common aqueous buffers such as Tris (an aqueous solution of tris (hydroxymethyl) amino methane), bar- bital, borate, phosphate, and the like may be used to dilute the labeled antigen. The antiserum against the -7- antigen will normally be added in excess of the total amount of antigen in the reaction medium. It, too, may be diluted with an aqueous buffer. The second anti¬ serum will be also added in excess relative to the 5 antiserum against the antigen. The second antiserum should be one that does not cross react with the anti¬ gen. The immunoprecipitant will normally be added as a dilute aqueous solution (about 5% to 8% by weight) in amounts in the range of about 2% to about 20%, 10 preferably 5% to about 8% by weight of solution based on the second antiserum. The sequence in which the reagents are added to the sample is generally not critical. Usually the labeled antigen, first antiserum, and second antiserum 15 will be incubated with the sample separately and the immunoprecipitant added later. The incubation will be carried out at a temperature and pH that permits for¬ mation of immune complexes. Moderate temperatures in the range of about 15°C to about 55 β C, conveniently 20 room temperature, and pHs in the range of about 6 and 9, preferably 7 to 8, will normally be used. The duration of the incubation will depend to some extent upon the sequence in which the reagents are added to the sample. In the case where the labeled antigen and 25 two antisera are added together at the above condi¬ tions, incubation times of about one min to two hr will be sufficient time to allow the antigen and anti- sera to react and form immune complexes. The com¬ plexes formed by the antigen (labeled and unlabeled in 30 the sample) and the two antisera remain in solution and may not agglomerate and precipitate until the immunoprecipitant is added. In instances where the immunoprecipitant is included in the incubation, im unoprecipitation occurs concurrently with the. formation of the immune complexes. When immunopreci- pitation is carried out in a separate step by adding the precipitant later, about one in to one hr are allowed for the precipitant to be incorporated into the complex and form a precipitate. Following the immunoprecipitation, the prec¬ ipitate is separated from the supernatant, such as by centrifuging, and the supernatant is discarded. The precipitate may be washed with buffer if desired. The precipitate ,is then dissolved in a nonfluorescent sol¬ vent that has a low ionic strength (ie, typically less than about 0.2 and more usually between about 0.001 and 0.06) and that maintains the pH of the resulting solution substantially constant (ie, ±0.005). Pre- ferably the pH is maintained above 8, more preferably above 9. Examples of such solvents are aqueous solu¬ tions of alkali metal (eg, Na, K) hydroxides and borates, barbital, and Tris. Preferably the solvent has the capacity to buffer the solution at the speci- fied pH. The ionic strength that provides maximum fluorescence intensity will vary depending upon the particular solvent involved. For instance, in the case of sodium hydroxide the ionic strength is less than about 0.04, usually 0.02 to 0.04, and preferably approximately 0.03. For sodium borate the ionic strength should be less than about 0.2, usually 0.01 to 0.1, and preferably approximately 0.06. A minimum volume of the solvent (ie, that amount that just dis¬ solves the precipitate) is preferably used. That amount will usually range between about 100 and 500 μl for samples of the above described volumes. The dissolution may be done at moderate temperatures, conveniently at ambient temperature. The resulting-, solution is then transferred to an appropriate •5-" "* ** . - δHEAl O PI container, eg, a flow cell, for fluorescence intensity reading. The fluorescence intensity is compared to a standard curve of fluorescence intensity vs antigen concentration derived by assaying a series of samples containing known amounts of antigen. From this com¬ parison, the quantity of antigen in the sample is determined. The basic ingredients of the test kit for carrying out the invention assay are: (1) the fluores- cent-labeled antigen, (2) the first antiserum against the antigen, (3) the antiserum against the first anti¬ serum, (4) the immunoprecipitant, and (5) the solvent. These ingredients are preferably packaged separately in the kit and will typically be provided in amounts sufficient to carry out multiple assays. The test kit may also include appropriate buffers, unlabeled anti¬ gen reagent for preparing standard and control samples, equipment or vials for performing the assay, and instructions for carrying out the assay. The kit components may be packaged in manners conventionally used in diagnostic kit manufacturing. The kits will be stored at reduced temperatures, preferably 2 β C to 6°C. The following examples further illustrate the invention and its advantages over alternative assay techniques. These examples are not intended to limit the invention in any manner. Unless indicated otherwise, percentages are by weight. Example 1: T4 Assay by Invention Method and Prior Art Twenty-five μl aliquots of serum containing known amounts of T4 were each incubated at 25 * C for 20 min with 25 μl of FITC-labeled T4 in carbonate buffer, 0.01M, pH 8.6, 25 μl of a mixture of rabbit ant.i-T4 serum and goat anti-rabbit Ig serum in phosphate buf¬ fered saline. Following incubation, one μl of a cold 6.5% aqueous solution of polyethylene glycol (8000 mw) was added to the mixture. The resulting precipitates were separated by centrifugation and the supernatants were discarded. The precipitates were each dissolved in 200 μl of 0.03 N aqueous NaOH. The solutions were transferred to flow cell cuvettes and their fluores¬ cence intensities were read with a fluorometer. duplicate set of T4-containing aliquots were assayed by the procedure of Example 1 of Japanese Patent Application 150547/79. The results of both assays are reported in Table 1 below. Table 1 T^/Tube Relative Fluorescence Intensity (RFI) (ng) Normalized for Comparison Prior Art Invention 0 45 340 46 342 1.25 42 278 41 282 2.50 31 217 34 217 7.25 34 151 28 141 As indicated by the data of Table 1, the effective assay range in RFI is about ten-fold greater in the invention method relative to the prior art method. The coefficient of variation in RFI for the invention method was less than 2% as compared to about 10% for the prior art method. This indicates that the OMPI precision of the invention T4 assay is substantially better than the precision of the comparison prior art T4 assay. Example 2: T3 Assay T3 assays were carried out on standards and unknowns using the general procedure described in Example 1. The reagents and incubation conditions were: Item Description Serum sample size 100 μl FITC-labeled T3 reagent 25 μl rabbit anti-T3 antibody - goat anti-rabbit Ig antibody 25 μl incubation temp 3. rc incubation time 1 hr The results of these assays are reported in Table 2 below. Table 2 Concentration (ng %) Standard/Sample . RFI Obtained Actual 1. Standard 1 731 - 0.0 2. Standard 1 762 - 0.0 3. Standard 2 712 - 50 4. Standard 2 713 - 50 5. Standard 3 684 - 100 6. Standard 3 697 - 100 7. Standard 4 583 - 400 8. Standard 4 601 - 400 9. Standard 5 528 - 800 10. Standard 5 526 _ 800 O PI -12- Concentration (ng %) Standard/Sample RFI Obtained Actual 11. Sample a 705 68 62 12. Sample a 712 52 62 13. Sample b 678 132 172 14. Sample b 672 143 172 15. Sample c 569 523 486 16. Sample c 574 470 486 Example 3: Digoxin Assay 10 Digoxin assays were carried out on standards and unknowns using the general procedure described in Example 1. The reagents and incubation conditions were: Item Description 15 Sample size 100 μl FITC-labeled digoxin reagent 25 μl rabbit anti-digoxin antibody - goat anti-rabbit Ig antibody 25 μl incubation temp 37 β C 20 incubation time 1 hr -^REAT OMPI V WIPO '^RNA1\ The results of these assays are reported in Table 3 below. Table 3 Concentration (ng/ml) Standard/Sample RFI Obtained Actual 1. Standard 1 2920 - ' 0.0 2. Standard 1 2937 - 0.0 3. Standard 2 2736 - 1.0 4. Standard 2 2801 - 1.0 5. * Standard 3 2463 - 2.0 6. Standard 3 2543 - 2.0 7. Standard 4 1979 - 4.0 8. Standard 4 2020 - 4.0 9. Standard 5 1803 - 8.0 10. Standard 5 1792 - 8.0 11. Sample a 1821 7.05 6.7 12. Sample a 1833 6.6 6.7 13. Sample b 2532 1.90 2.0 14. Sample b 2541 1.86 2.0 15. Sample c 1922 4.82 5.1 16. Sample c 1910 5.00 5.1 Example 4: Gentamicin Assay Gentamicin assays were carried out on stan¬ dards and unknowns using the general procedure des¬ cribed in Example 1. The reagents and incubation conditions were: Item Description sample size 25 μl FITC-labeled reagent 10 μl rabbit anti-gentamicin antibody - goat anti-rabbit Ig antibody 25 μl incubation temp 25 β C incubation time 10 min The results of these assays are reported in Table 4 below. - • REi OMPI ^ Table 4 Concentration (μg/ml) Standard/Sample RFI Obtained Actual 1. Standard 0 7086 - 0.0 2. Standard 1 6793 - 0.5 3. Standard 2 4453 - 2.0 4. Standard 3 2822 - 4.0 5. Standard 4 1234 - 16.0 6. Sample 1 1902 7.9 8.0 7. Sample 2 2791 4.15 4.0 8. Sample 3 1200 17.0 16.0 9. Sample 4 1853 8.2 8.0 10. Sample 5 6480 0.66 0.5 11. Sample 6 3828 2.45 2.0 12. Sample 7 5814 0.99 1.0 13. Sample 8 6921 0.5 0.0 14. Sample 9 5666 1.08 1.0 15. Sample 7 5800 1.00 1.0 16. Sample 7 5821 0.99 1.0 17. Sample 7 5762 1.03 1.0 18. Sample 7 5748 1.03 1.0 19. Sample 7 5819 0.99 1.0 20. Sample 7 5709 1.05 1.0 21. Sample 7 5798 1.00 1.0 22. Sample 7 5810 0.99 1.0 23. Sample 4 1870 8.1 8.0 24. Sample 4 1854 8.2 8.0 25. Sample 4 1900 7.9 8.0 r 26. Sample 4 1888 8.0 8.0 27. Sample 4 1909 7.9 8.0 28. Sample 4 1844 8.3 8.0 29. Sample 4 1865 8.2 8.0 30. Sample 4 1866 8.2 --8.0. Example 5: Amikacin Assay Amikacin assays were carried out on stan¬ dards and unknowns using the general procedure described in Example 1. The reagents and incubation conditions were: Item Description Serum sample size 25 μl FITC-labeled amikacin 10 μl rabbit anti-amikacin antibody - .goat anti-rabbit Ig- antibody 25 μl incubation temp 25'C incubation time 10 in The results of these assays are reported in Table 5 below. - • RE Al OMPI ^^/ A TlO Table 5 Concentration (μg/ml) Standard/Sample RFI Obtained Actual 1. Standard 1 2815 - 0.0 2. Standard 2 2128 - 3.0 3. Standard 3 1644 - 10.0 4. Standard 4 1357 - 20.0 5. Standard 5 1011 - 50.0 6. Sample 1 1640 10.1 10.0 7. Sample 2 1142 36.0 35.0 8. Sample 3 2562 3.0 0.0 9. Sample 4 1008 51.0 50.0 10. Sample 5 1180 32.5 35.0 11. Sample 6 2169 2.7 3.0 12. Sample 7 1400 18.7 20.0 13. Sample 2 1150 35.2 35.2 14. Sample 2 1161 34.3 35.0 15. Sample 2 1154 35.1 35.0 16. Sample 2 1205 38.0 35.0 17. Sample 2 1172 33.3 35.0 18. Sample 2 1131 37.0 35.0 19. Sample 2 1141 36.0 35.0 20. Sample 2 1139 36.0 35.0 21. Sample 1 1652 9.9 10.0 22. Sample 1 1639 10.1 10.0 23. Sample 1 1639 10.0 10.0 24. Sample 1 1646 10.0 10.0 25. Sample 1 1661 9.7 10.0 26. Sample 1 1632 10.3 10.0 27. Sample 1 1642 10.0 10.0 28. Sample 1 1658 9.8 10.0 29. Sample 1 1640 10.1 10.0 - QREX O Pl \ ~ WIPO ?N A τt Example 6: Tobramicin Assay Tobramicin assays were carried out on stan¬ dards and unknowns using the general procedure of Example 1. The reagents and incubation conditions were: Item Description Sample size 25 μl FITC-labeled reagent 10 μl rabbit anti-tobramicin antibody - goat anti-rabbit Ig antibody 25 μl incubation temp 25 β C incubation time 10 min The results of these assays are reported in Table 6 below. OMPI fa, WIPO &?NATlθ Table 6 Concentration (μg/ml) Standard/Sample RFI Obtained Actual 1. Standard 0 1957 - 0.0 2. Standard 1 1882 - 0.5 3. , Standard 2 1502 - 2.0 4. Standard 3 1057 - 4.0 5. Standard 4 610 - 16.0 6. Sample 1 801 8.00 8.0 7.- Sample 2 1752 0.99 1.0 8. Sample 3 621 15.70 16.0 9. Sample 4 782 8.40 8.0 10. Sample 5 1872 0.55 0.5 11. Sample 6 1733 1.07 1.0 12. Sample 7 1452 2.22 2.0 13. Sample 8 1095 3.73 4.0 14. Sample 9 1949 0.5 0.0 15. Sample 2 1748 1.00 1.0 16. Sample 2 1753 0.99 1.0 17. Sample 2 1740 1.03 1.0 18. Sample 2 1739 1.03 1.0 19. Sample 2 1751 0.99 1.0 20. Sample 2 1745 1.01 1.0 21. Sample 2 1748 1.00 1.0 22. Sample 2 1749 1.00 1.0 23. Sample 1 799 8.0 8.0 24. Sample 1 820 7.4 8.0 25. Sample 1 819 7.4 8.0 26. Sample 1 806 7.9 8.0 27. Sample 1 788 8.3 8.0 28. Sample 1 793 8.1 8.0 29. Sample 1 825 7.3 8.0 30. Sample 1 800 8.0 8..0 Example 7: Tobramicin Assay Using Sodium Borate Tobramicin assays were carried out on standards using the procedure of Example 6 except that 0.03 M sodium borate was used as a solvent instead of 0.03 N sodium hydroxide. Eight hundred μl of the borate solution was used for each precipitate. For comparison purposes assays using 0.03 N sodium hydroxide were also carried out. The results of these assays are reported in Table 7 below. Table 7 Concentration RFI Standard μg/ml NaOH Borate 1 0 2500 3094 2 1.0 2230 2513 3 2.0 1950 1653 4 4.0 1360 1519 5 8.0 990 1106 6 16.0 760 793 Example 8 The usefulness of the sodium borate solution of Example 7 as a solvent in assays of T4 and genta¬ micin was shown as follows. Known amounts of FITC- labeled antigen in the carbonate buffer of Example 1 were incubated with rabbit serum against the antigen and goat anti-rabbit serum as in Example 1. The labeled T4 solution contained 0.82 ng T4 whereas the labeled gentamicin solution contained 0.5 ng genta¬ micin. The resulting immune complexes were precipi¬ tated with aqueous polyethylene glycol, the precipi- tates were separated, and the supernatants were dis- * -_. - * —— carded as in Example 1. The resulting precipitates were dissolved in 0.03 N sodium hydroxide or 0.03 M sodium borate and the fluorescence intensities were read with a fluorometer. These tests were run in duplicate and are reported below. Antigen NaOH Borate 1658 1835 T4 1690 1882 1962 2377 Gentamicin 1973 2418 Modifications of the above described modes for carrying out the invention that are obvious to those of ordinary skill in the immunodiagnostics art and/or related arts are intended to be within the scope of the following claims.

Claims: Claims 1. A competitive immunofluorescence assay for determining the amount of an antigen in a sample suspected of containing the antigen comprising incu- bating the sample with a solution of fluorescent- labeled antigen, anti-antigen antibody, and an anti¬ body against the anti-antigen antibody, and adding a nonfluorescent, nonlight-scattering immunoprecipitant to the incubation mixture to form an immunoprecipi- tate, characterized in that the immunoprecipitate is separated and dissolved in a nonfluorescent solvent that has a low ionic strength and maintains the pH of the resulting solution substantially constant, and the fluorescence intensity of the resulting solution is measured and compared to a standard curve. 2. The competitive immunofluorescence assay of claim 1 further characterized in that the solvent maintains the pH of the solution above about 8. 3. The competitive immunofluorescence assay of claim 1 or 2 further characterized in that the solvent is a buffer. 4. The competitive immunofluorescence assay of claim 1 further characterized in that the solvent is an aqueous solution of an alkali metal hydroxide or art alkali metal borate. 5. The competitive immunofluorescence assay of claim 1 further characterized in that the solvent is aqueous sodium hydroxide having an ionic strength less than about 0.04. 6. The competitive immunofluorescence assay of claim 5 further characterized in that the ionic strength is approximately 0.03. 7. The competitive immunofluorescence assay of claim 1 further characterized in that the solvent is an aqueous solution of sodium borate having an ionic strength less than about 0.2. 8. The competitive immunofluorescence assay of claim 1 further characterized in that a minimum volume of the solvent is used to dissolve the immuno¬ precipitate. 9. The competitive immunofluorescence assay of claim 1, 2, 3, 4, 5, 6, 7, or 8 further character¬ ized in that the fluorescent-labeled antigen is a fluorescein-labeled antigen and the immunoprecipitant is polyethylene glycol. 10. A test kit for carrying out a competi¬ tive immunofluorescence assay for determining the amount of an antigen in a sample suspected of contain- ing the antigen comprising in association fluorescein- labeled antigen reagent, anti-antigen antibody rea¬ gent, antibody against the anti-antigen antibody, and a nonfluorescent, nonlight-scattering immunopreci¬ pitant characterized in that the kit includes a non- fluorescent solvent for dissolving immunoprecipitates of the immunoprecipitant that has a low ionic strength and the ability to maintain the pH of the immunopreci¬ pitate solution substantially constant. -24- 11. The test kit of claim 9 further characterized in that the solvent has the ability to maintain said pH above about 8. 12. The test kit of claim 10 further characterized in that the solvent is aqueous sodium hydroxide having an ionic strength less than about 0.04 or an aqueous solution of sodium borate having an ionic strength less than about 0.2. 13. The test kit of claim 10, 11 or 12 further characterized in that the fluorescent-labeled antigen is a fluorescein-labeled antigen and the immunoprecipitant is polyethylene glycol.

Inventors: KAMEDA NAOMI, KAMEDA, NAOMI
Applicants: PARAGON DIAGNOSTICS
Application year: 1983
Main classification: G01N31
IPC codes: G01N33