CA1218596A - Competitive immunofluorescence assay and test kit - Google Patents

Abstract

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 immuno-precipitate 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 fluorecent-labeled antigen, anti-antigen antibody, and a secondary antibody to the anti-antigen antibody followed by addition of the pre-cipitant to form an immunoprecipitate. The precipi-tate 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

CO~PETITIVE IMMUN FLUORE~SCEl~CE ASSAY AND TEST KIT

Description Technical Fle~d The invention is in the field of immuno-5 diagnostics. More particularly it involves an improved com~etitive immunofluorescence test for antigens.

t Competitive immunoassays for anti~ens or 10 haptens are known in the immunodiagnostic art. They are based on competition for specific antibody against the antic~en between labeled anti~en (known) and unla-beled antigen (unknown) in the sample being assayed.
Immune complexes that form between the antigen/hapten 15 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 determinecl by comparison with the effect of standards.
US Pats Nos 3,981,982 and 4,298,592 describe a type of competitive radioimmunoassay called a "double antibody separation" assay. In this technique the sample suspected of containing antic3en is first incubated with a first specific antibody and known radiolabeled antigen. Followin~ tllis a second anti-body th~t is specific to the first antibody is added `i"

5~ -together with polyethylene gly~ol. The second anti-body and polyethylene gIycol form a complex with the antigen-first antibody reaction product that agglom-era~es and precipitates from the incubation mixture.
The precipitate is separated from the supernatant 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 compariny the reading to a 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 personnel. Corresponding immunofluorescence assays in which the fluorescence of a solid phase is read are known. Such assays suffer from hi~h background fluorescence from the solid phase itself or from scattered light.
Chard, T., and Syl~es, A., Clin Chem (1979) 26/6: 973-976 teach a fluoroimmunoassay for human choriomammotropin in which a plasma sample is incu-bated with fluorescein-labeled human choriomammotropin and sheep antiserum to the hormone. Following the incubation immune complexes are separated from free hormone by addition of aqueous polyethylene glycol.
The mixture is centrifuged a~d the fluorescence of the supernatant is read and compared to a standard. Such assays in which the fluorescence of the supernatant is read have the problem of fluoresence due to interfer-ing materials in the supernatant (plas~a or serum).
Pourfarzaneh, M., et al, Clin Cllem (1980) 26/6: 730-733, describes a competitive immunofluores-cence assay for cortisol in serum. The serum sample is incubated with fluorescein-^labeled cortisol and anti-cortisol antibody coupled to magnetizable cellu-lose/iron oxide particles. After the incubation the solid ph~se is separated, washed to remove free, labeled antigen, and eluted with an equivolume mixture of methanol 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 15n547/79 describes direct and indirect immunofluorescence assays for various antigens. The sample is first incubated with antibody (fluorochrome-labeled in the direct assay, unla~eled in the indirect assay). The mixture is then incubated with an immunoadsorbent.
The indirect assay involves an additional incubation of the immunoadsorbent ~ith 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 ~ 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 complex~s adversely and cause artificially low immunofluorescence intensity read-ings. Elution inefficiency and/or interference ~rom 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 o supernatants or eluates that may containinter~erinc3 materials. Instead the invention assay involves precipitating the immune complex with a nonfluorescent, non-light scattering immunoprecipi-tant, dissolving the resulting immunoprecipitate witha solvent t'nat 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 reproducihility and dose-response and precision eharaeteristics,. and requires less antibody reagent.

Disclosure of the Invention One aspect of -the invention is a competitive assay Eor determining the amount o an antigen in a sample suspected of eontaining the antigen eomprising:
(a) incubating the sample with a sol~tion of a fluo-rescent-labeled antigen, anti-antigen antibody, and an antibody against the anti-antigen antibody; (b) adding a nonfluorescent nonlight-scatterinc3 immunopreeipitant to the incubation mixture to form an immunopreeipi-tate; (e) separatinc3 the immunopreeipitate and dissol-ving the immunoprecipitate in a nonfluorescent solvent that has a low ionic strength and maintains the p~ of the resulting solution substantially eonstant; and (d) ~easurinc3 the fluoreseence intensity of the solu-tion of step (c) and compari~ng said fluorescence intensity to a standard curve..
Another aspeet of the invention is a test kit or carrying out the above-described competition immunofluorescence assay comprisin~ in association:
(a) a fluorescent-labeled anti~en reagent; (b) anti-antigen antibody reagent; (c) an antibody against the anti-anti~en antibody; and (d) a nonfluorescent sol-35~6 vent for dissolvin~ immunoprecipitates of the immuno-precipitant that has a low ionic stren~th and the ability to maintain the p~ of the immunoprecipitate solution substantially constant.

~od_s ~or Carryiny Out the Invention The sample that is analyzed by the invention method is a body fluid such as blood, blood serum, ~J~ ~/g5 blood plas~, urine, lymph Eluid, 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 ~1 of 1uid will be used per assay. Samples may be cryo-prescrved pendin~ analysis if necessary.
Substances that may be assayed by the inven-tion method include anti~ens (molec~les that elicit animmune response when introduced into the b~oodstream of a vertebrate host) and haptens that are not immuno-~enic per se but may be conju~ated to a protein car-rier to form a conjugate that is immuno~enic and capable of raisin~ antibodies against the hapten. The term "anti~en" is use~ herein to ~enerically 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 anti~ens that may be assayed by the invention method are thyroxine (T4), triiodo-thyronine (T3), di~oxin, ~entamicin, amikacin, tobra-micin, kanamycin, netilmicin, cortisol, luteinizing hormone, digitoxin, vitamin B12, progesterone, human chorionic gonadotropin, theophylline, an~iotensin, human ~rowth hormone, and the like.

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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-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 a-Jents such as aldehydes, carbodiimicles, dima]eimides, imidates, and succinimides. Fluorescein is a preEerred label.
Antibod~ against the antic~en may be made by inoculating a host vertebrate with the antigen, typically repeatedly at several day intervals, bleed-ing the host and separatiny 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 rom tl~e second host. The non luorescent, 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 polyethyl~ne 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.
~ he absolute amounts of the reagents used will, of course, depend upon the volume of the sample.
A known amount of labeled antigen will be used. The labeled antigen may be diluted- to provide a reagent that is stoichiometrical]y 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 li~e may be used todilute the labeled antigen. The antiserum against the 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-lG serum will be also added in excess relative to theantiserum against the anti~en. The second antiserum sho~ld be one that does not c-ross react with the anti-gen. The immunoprecipitant will normally be added as a dilute aqueous solution (about 5~ to 8~ by weight) in amo~nts in the ran~e of about 2% to about 20%, 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. Vsually the labeled antigen, first antiserum, and second antiserum 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 15C to about 55C, conveniently room temperature, and pHs in the range of about 6 and 9, preferably 7 to 8, will n~rmally 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 two antisera are added to~ether at the above condi-tions, incubation times of about one min to two days will be sufficient time to allow the antigen and anti-sera to react and form immune complexes. The com-, ~ ~1$~36 plexes formed by the antigen ~labeled and unlabeled inthe sample) and the two antisera remain in solution and may not ag~lomerate and precipitate until the immunoprecipitant is added. In instances where the immunoprecipitant is included in the incubation, immunoprecipitation occurs concurrently with the formation of the immune complexes. When immunopreci-pitation is carried out in a separate step by adding the precipitant later, about one min to one hr are allowed for the precipitant to be incorporated into the complex and form a precipitate.
Following the immunoprecipitation, the prec-ipitate is separated from the supernatant, such as by centri~uging, and the supernatant is discarded. The precipitate may be washed with buffer if desired. The precipitate is then dissolved in a nonfluorescent sol-vent that has a lo~ ionic strength (ie, typically less than about 0.2 and more usually between about 0.001 and 0.06) and that maintains the pH of the resulting solution substantially constant (ie, l0.005). Pre-ferably the pH is maintained above 8, more preerably above 9. Examples of such solvents are aqueous solu-tions of alkali metal (eg, Na, K) hydroxides and borates, barbital, and Tris. Preferably the solvent has the capacity to buffer the solution at the speci-fied p~. The ionic strength that provides maximum fluorescence intensity will ~ary depending upon the particular solvent involved. For instance, in the case of sodium hydroxide the ionic strength is less than about 0.04, usually 0.02 to 0.04, and preferably approximately 0.03. For sodium borate the ionic strength should be less than about 0.2, usually 0.01 to 0.1, and preferably approximately 0.06. A minimum volume of the solvent (ie, that amount that just dis-., 121~3S~$~

solves the precipitate) is pre^ferably used. That amount will usually range between about lO0 and 500 ~1 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 container, eg, a flow cell, for fluorescence intensity reading. The fluorescence intensity is compared to a standard curve of fluorescence intensity vs antic3en concentration derived by assaying a series of samples containing known amounts of antigen. From this com-parison, the quantity of antic3en in the sample is determined.
The basic ingredients of the test kit for carryinc3 out the invention assay are: (l) the fluores-cent-labeled antigen, (2) the first antiserum against the anti~en, (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 ~lill typically be provided in amounts sufficient to carry out multiple assays. The test kit may also include appropriate buffers, unlabeled anti-gen reagent for preparinc3 standard and control samples, equipment or vials for performing the assay, and instructions for carryinc3 out the assay. The kit components may be packaged in manners conventionally used in diagnostic kit manu~acturin~. The kits will be stored at reduced temperatures, preferably 2C to 6C.
The following examples further illustrate the invention and its advantages over alternative assay techniques. These cxamples are not intended to limit the invention in any manner. Unlcss indicated otherwise, percentages are by weight.

lZ~5 ~

--1 o--Example 1: T4 Assay by _ vention Method and Prior Art Twenty-five ~1 aliquots of serum containing Xnown amounts of T4 were each incubated at 25 C for 20 min with 25 ~1 of FITC-labeled T4 in carbonate buffer, O.OlM, pH 8.6, 25 ~1 of a mixture of rabbit anti-T4 seru~ and goat anti-rabbit Ig serum in phosphate buf-fered saline. Following incubation, one ~1 of a cold 6.5~ aqueous solution of polyethylene glycol (8000 mw) was added to the mixture. The resulting precipitates were separated by centrifu-~ation and the supernatants were discarded. The precipitates were each dissolved in 200 ~1 of 0.03 N aqueous NaOH. The solutions were transferred to flow cell cuvettes and their fluores-cence intensities were read with a fluorometer.
A du~licate set of T4-containing aliquots ~ere assayed by prior art procedure.

The results of both assays are reported in Table 1 below.
Table 1 20T4/TubeRelative Fluorescence Intensity (RFI) (ng)Normalized for Comparison Prior Art Invention 251.25 42 278 2.50 31 - 217 7.25 34 151 :

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 precision of the invention T4 assay is substantially better than the precision of the comparison prior art T4 assay.

l~xam~le 2: T3 Assay T3 assays were carried out on standards and unknowns using the general procedure described in Exam~le 1. The reagents and incubation conditions were:
15 Item Description Serum sample size 100 ~1 FITC-labeled T3 reagent 25 ~1 rabbit anti-T3 antibody -goat anti-rabbit Ig antibody 25 ~ll 20incubation temp 37C
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 J~

^ Concentration (ng %) Standard/Sample RFIObtainedActual

7. Standard ~ 583 - 400

8. Standard 4 601 - 400 5 9. Standard 5 528 - 800 10. Standard 5 526 - 800 11. Sample a 705 68 62 12. Sample a 712 52 62 13. Sample b 678132 172 1014. Sample b 672143 172 15. Sample c 569523 486 16. Sample c 574470 486 Example 3 DicJo in Assa~
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_ Sample size 100 ~1 FITC-labeled digoxin reagent 25 ~1 20 rahbit anti-di(Joxin antibody -goat anti-rabbit Ig antibody 25 ~1 incubation temp 37C
incubation time 1 hr ~8~96 The results of these assays are reported in Table 3 below.
_able 3 Concentration (ng/ml) Standard/Sample RFI Ohtained 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 10 6. Standard 3 2543 _ 2.0 7. Standard 4 :L979 - 4.0 ~. Standard 4 2020 - 4.0

9. Standard 5 1803 - 8.0

10. Standard 5 1792 - 8.0 1511. 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 2016. Sample c 1910 5.00 5.1 ~Z1~5~

Exam~le ~: 5entamicin 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 ~1 FITC-labeled reagent 10 ~1 rabbit anti-gentamicin 10antibody -goat anti-rabbit Ig antibody25 ~1 incubation temp 25C
incubation time 10 min The results of these assays are reported in Table 4 below.

~i85~6 Table ~
- Concentration (~g/ml) Standard/Sample RFI ObtainedActual 1. Standard 0 7086 - 0.0 2. Standard 1 6793 - 0.5 5 3. Standard 2 4453 - 2.0 4. Standard 3 2822 - 4.0 5. Standa~d 4 1234 - 16.0 6. Sample 1 1902 7.9 8.0 7. Sample 2 2791 4.15 4.0 10 8. Sample 3 120n 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 1513. 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 2018. Sample 7 5748 1.03 1.0 l9. 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 2523. Sample 4 18~70 8.1 8.0 24. Sample 4 1854 8.2 8.0 25. Sample 4 1900 7.9 8.0 26. Sample 4 1888 8.0 8.0 27. Sample 4 1909 7.9 8.0 3028. Sample 4 1844 8.3 8.0 29. Sample 4 1865 8.2 8.0 30. Sample 4 1866 8.2 8.0 s~

Example 5: Amikacin As say Amikacin assays were carried out on stan-dards and unknowns using the general procedure described in Example l. The reagents and incubation 5conditions were:
Item Description Ser~m sample size 25 ~l FITC-labeled amikacin lO ~1 rabbit anti-amikacin antibody -10goat anti-rabbit Ig antibody 25 ~ll incubation temp 25C
inc~bation time lO min The results o~ these assays are reported in Table 5 below.

12~

Table 5 Concentration (~g/ml) Standard/Sample RFIOhtainedActual 1. Standard 1 2815 - 0.0 2. Standard 2 2128 - 3.0 5 3. Standard 3 1644 - 10.0 4. Standard 4 1357 - 20.0 5. Standard 5 1011 - 50.0 6. Sample 1 164010.1 10.0 7. Sample 2 114236.0 35.0 10 8. Sample 3 25623.0 0.0 9. Sample 4 100851.0 50.0 10. Sample 5 118032.5 35.0 11. Sample 6 21692.7 3.0 12. Sample 7 140018.7 20.0 1513. Sample 2 115035.2 35.2 14. Sample 2 116134.3 35.0 15. Sample 2 115435.1 35.0 16. Sample 2 120538.0 35.0 17. Sample 2 117233.3 35.0 2018. Sample 2 113137.0 35.0 19. Sample 2 114136.0 35.0 20. Sample 2 113936.0 35.0 21. Sample 1 16529.9 10.0 22. Sample 1 163910.1 10.0 2523. Sample 1 16~910.0 10.0 24. Sample 1 1646,10.0 10.0 25. Sample 1 16619.7 10.0 26. Sample 1 163210.3 10.0 27. Sample 1 164210.0 10.0 3028. Sample 1 16589.8 10.0 29. Sample 1 164010.1 10.0 lZ185~

Fxample 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 ~1 FITC-labeled reagent 10 ~1 rabbit anti-tobramicin antibody -10goat anti-rabbit Ig antibody 25 ~1 incubation temp 25C
incubation time 10 min The results of these assays are reported in Table 6 below.

~able 6 Concentration (~g~ml) Standard/Sample RFI OhtainedActual 1. Standard 0 1957 - 0.0 2. Standard 1 1882 - 0.5 53. 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 1.0 108. Sample 3 621 15.7016.0 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 1513. Sample 8 ln95 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 2018. 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 2523. Sample 1 79~ 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 3028. Sample 1 793 8.1 8.0 29. Sample 1 825 7.3 S.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 ~1 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~g/ml NaOH Borate 2 1.0 2230 2513 3 2.0 1950 1653 4 4.0 1360 151g 8.0 990 1106 6 16.0 760 793 F,xample 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 s~rum 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 resultin~ 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 3S$6 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 an~ are reported below~
Anti~ NaOH Borate Gentamicin 1973 2418 Modifications of the above described modes for carrying out the invention that are obvious to those of ordinary skill in the immunodia~nostics art and/or related arts are intended to be within the scope of the followin~ claims.

Claims (15)

The embodiments of the invention in which an exclusive property or privilege is claimed as defined as follows:

1. A competitive immunofluorescence assay for determining the amount of an antigen in a sample suspected of containing the antigen comprising:
(a) incubating the sample with a solution of a fluorescent-labeled antigen, anti-antigen antibody, and an antibody against the anti-antigen antibody;
(b) adding polyethylene glycol to the incubation mixture in an amount effective to form an immunoprecipitate;
(c) separating the immunoprecipitate and dissolving the immunoprecipitate in a nonfluorescent solvent that has a low ionic strength and maintains the pH of the resulting solution substantially constant, and (d) measuring the fluorescence intensity of the solution of step (c) and comparing said fluores-cence intensity to a standard curve.

2. The competitive immunofluorescence assay of claim 1 wherein the antigen is T3, T4, digoxin, gentamicin, amikacin, tobramycin, kanamycin, netilmicin, or theophylline.

3. The competitive immunofluorescence assay of Claim 1 wherein the fluorescent-labeled antigen is a fluorescein-labeled antigen.

4. The competitive immunofluorescence assay of claim 1 wherein the solvent maintains the pH of the solution above about 8.

5. The competitive immunofluorescence assay of claim 1 wherein the solvent is a buffer.

6. The competitive immunofluorescence assay of claim 1 wherein the solvent is an aqueous solution of an alkali metal hydroxide or an alkali metal borate.

7. The competitive immunofluorescence assay of claim 1 wherein the solvent is aqueous sodium hydroxide having an ionic strength less than about 0.04.

8. The competitive immunofluorescence assay of claim 7 wherein the ionic strength is approximately 0.03.

9. The competitive immunofluorescence assay of claim 1 wherein the solvent is an aqueous solution of sodium borate having an ionic strength less than about 0.2

10. The competitive immunofluorescence assay of claim 1 wherein a minimum volume of the solvent is used to dissolve the immunoprecipitate.

11. The competitive immunofluorescence assay of claim 2 wherein the fluorescent labeled antigen is fluorescein isothiocyanate labeled antigen, the solvent is aqueous sodium hydroxide having an ionic strength less than about 0.04 or sodium borate having an ionic strength less than about 0.2, said pH is above 8.

12. A test kit for carrying out the assay of claim 1, comprising:
(a) a container containing a fluorescent-labeled antigen reagent;
(b) a container containing anti-antigen antibody reagent;
(c) a container containing an antibody against the anti-antigen antibody;
(d) a container containing polyethylene glycol; and (e) a container containing a nonfluores-cent solvent for dissolving immunoprecipitates of the immunoprecipitant that has a low ionic strength and the ability to maintain the pH of the immunoprecipitate solution substantially constant.

13. The test kit of claim 12 wherein the solvent has the ability to maintain said pH above about 8.

14. The test kit of claim 13 wherein 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.

15. The test kit of claim 14 wherein the fluorescent-labeled antigen is a fluorescein-labeled antigen.