Bioconjugate Chem. 1994, 5, 459-462
459
Immunoassay Reagents for Thyroid Testing. 1. Synthesis of Thyroxine Conjugates Maciej Adamczyk,* Lynnmarie Fino, Jeffrey R. Fishpaugh, Donald D. Johnson, and Phillip G. Mattingly Divisional Organic Chemistry Research, Diagnostics Division, Abbott Laboratories, DSNM, Building AP20, One Abbott Park Road, Abbott Park, Illinois 60064-3500. Received April 5, 1994@
Immunoreagents were designed to improve the performance of a commercial fluorescent polarization immunoassay for thyroxine. The thyroxine immunogen was prepared by selective coupling of N-acetylL-thyroxine to BSA via a n aminocaproic acid spacer arm. The fluorescent tracer was prepared by a multistep reaction sequence which relied on extensive use of orthogonol protecting groups.
INTRODUCTION
The amino acid 3,5,3’,5’-tetraiodo-~-thyronine (thyroxine or T4, 11,is the predominant iodothyronine secreted from the thyroid gland. T4 is responsible for regulating diverse biochemical processes throughout the body, which are essential for normal metabolic and neural activity. The measurement of serum T4 concentration has become the common initial test in the diagnosis of altered thyroid function ( I ) . The concentration of thyroxine in the bloodstream is extremely low and can only be detected with very sensitive techniques. Approximately 0.05% of the total circulating thyroxine is physiologically active (i.e., free thyroxine). The remaining circulating thyroxine is bound to proteins, primarily thyroxine binding globulin (TBG). Thyroxine will also bind to other binding proteins, particularly, thyroxine binding prealbumin and albumin (1).
Radioimmunoassay (RIA)has proved to be a sensitive, specific technique for measuring T4 ( 2 , 3 ) . More recently, fluorescent polarization immunoassay (FPIA) has been used to assay for T4 ( 4 ) . Fluorescent polarization techniques are based on the principle that a fluorescent labeled compound when excited by linearly polarized light will emit fluorescence having a degree of polarization inversely related to its rate of rotation. Therefore, when a fluorescent labeled tracer-antibody complex is excited with linearly polarized light, the emitted light remains highly polarized because the fluorophore is constrained from rotating between the time light is absorbed and emitted. When a “free” tracer compound (Le., unbound to an antibody) is excited by linearly polarized light, its rotation is much faster than the corresponding tracer-antibody conjugate and the molecules are more randomly oriented; therefore, the emitted light is depolarized. Thus, fluorescent polarization provides a quantitative means for measuring the amount of tracer-antibody conjugate produced in a competitive binding immunoassay (5). FPIA has advantages over RIA in that there are no radioactive substances to dispose of and the assay is homogenous and can be easily automated. However, it has been reported that in isolated individuals, the commercially available Abbott TDx T4 FPIA ( 6 ) assay resulted in a low T4 value which did not conform to RIA measurement and the clinical symptoms of hypothyroidAbstract published in Advance ACS Abstracts, August 1, 1994. @
I043-1802/94/2905-0459$04.50/0
ism (7). It was postulated that the FPIA tracer used in the assay might be binding to endogenous immunoglobulin G in the patient sample. In this work we present the synthesis of new thyroxine conjugates which served in the development of a new TDxAMx Total T4 FPIA which more closely correlates with RIA and clinical symptoms (8, 9). EXPERIMENTAL PROCEDURES
General Comments. All reagents were purchased from Aldrich Chemical Go., Inc., Milwaukee, WI, and were used without further purification, except where noted. Solvents employed were of reagent or HPLC grade and were used as received. lH NMR spectra were recorded a t 200 MHz on a Chemagnetics A-200 spectrometer or a t 300 MHz on a Varian Gemini 300 in CDC13 with TMS as a standard. Mass spectra were recorded on a Nermag 3010 MS-50 mass spectrometer. HPLC was carried out using a Waters RCM C18 (8 x 10) reversed phase column eluting a t 1 m u m i n with the solvent indicated. Synthesis of the L-Thyroxine Immunogen (8). L-Thyroxine (1)as the sodium salt, pentahydrate (10 g, 11 mmol) was nearly completely dissolved in ethanoU2 N ammonium hydroxide (111, v/v, 400 mL) and filtered and the filtrate poured into 5% HC1 (425 mL). The resulting precipitate was isolated by vacuum filtration and dried under high vacuum to afford a white solid. This material was dissolved in dimethylformamide (160 mL); acetic anhydride (100 mL, 1.06 mol) was added. The reaction mixture was stirred for 1.5h, diluted with water (850 mL), and allowed to stand a t 4 “C for 16 h. The resulting precipitate was isolated by filtration, dissolved in ethanol (350 mL) containing 1 N NaOH (41 mL), and stirred for 2.5 h. HC1 (5%, 680 mL) was added and the mixture allowed to stand a t 4 “C for 16 h. The resulting precipitate was isolated by vacuum filtration and dried under high vacuum to yield 8.1 g (90%) of the desired N-acetyl-L-thyroxine ( 5 ) ( I O ) as a white solid: ‘H NMR (200 MHz, CD30D) (6) 7.8 (s, 2H), 7 . 1 ( s , 2H), 4.6-4.7 (m, lH), 2.8-3.0 (m, 2H), 2.0 (s, 3H); MS (FAB)(M + H)+ mlz 820. N-Acetyl-L-thyroxine ( 5 )(1.0 g, 1.2 mmol) was dissolved in tetrahydrofuran (50 mL). N-Hydroxysuccinimide (170 mg, 1.5 mmol) and 1,3-dicyclohexylcarbodiimide(300 mg, 1.5 mmol) were added and the reaction stirred under nitrogen for 3 days. The reaction mixture was then vacuum filtered to remove insoluble urea, affording 40 mL of filtrate. Half the filtrate volume (20 mL, 0.6 mmol) was combined with 6-aminocaproic acid (80 mg, 0.6 0 1994 American Chemical Society
Adamczyk et al.
460 Bioconjugate Chem., Vol. 5, No. 5, 1994
mmol). The pH was adjusted to 9 with triethylamine, and the reaction was allowed to stir under nitrogen for 2 days. The solvent was then removed in vacuo and the crude product purified by chromatography [Chromatotron, Harrison Research, Palo Alto, CAI, eluting with methylene chloride/methanol/acetic acid (90/10/0.2, v/v), t o yield 300 mg (54%) of the desired product (6) as a yellow oil: MS (FAB) (M H)+ m/z 933; lH NMR (200 MHz, CDCldCD30D, 9:l) 6 7.81 (s,2H), 7.13 (s,2H), 4.54 (t, l H , J = 13 Hz), 3.32-2.80 (m, 4H), 2.25 (t, 2H, J = 13 Hz), 1.99 (s, 3H), 1.70-1.20 (m, 6H); HPLC (Waters p-Porasil, 3.9 x 150;6% methanol in methylene chloride; 254 nm; 1 mL/min) retention time 8.07 min, 98.9%. The acid 6 (300 mg, 0.322 mmol) was dissolved in THF (25 mL); N-hydroxysuccinimide (45 mg, 0.39 mmol) and 1,3-dicyclohexylcarbodiimide(80 mg, 0.39 mmol) were added and the reaction mixture stirred for 16 h under nitrogen. The reaction mixture was then vacuum filtered to remove insoluble 1,3-dicyclohexylurea,affording 16 mL of filtrate. Then 4 mL (0.08 mmol) of the filtrate containing the active ester (7) was added to a stirred solution of bovine serum albumin (250 mg, 0.0037 mmol) dissolved in 0.05 M sodium phosphate (10 mL, pH = 8.0) and DMF (10 mL). After being stirred for 3 days the reaction was dialyzed against 0.05 M sodium phosphate (4 L, pH = 8.0) for 24 h and then water (4 L) for 24 h. The dialyzed solution was then lyophilized to afford the desired L-thyroxine immunogen ( 8 ) (297 mg). Synthesis of the L-Thyroxine Tracer (16). LThyroxine (1)sodium salt, pentahydrate (22.0 g, 24.7 mmoi) and sodium carbonate (7.85 g, 74.1 mmol) were stirred in THF/water (Vl,v/v, 960 mL). 9-Fluorenylmethyl chloroformate (7.04 g, 27.2 mmol) was added, and the reaction mixture was stirred for 30 min. The reaction mixture was then diluted with 1 N HC1 (170 mL) and extracted with ethyl acetate (3 x 700 mL). The organic extracts were combined, dried over anhydrous Na2S04, filtered, and evaporated in vacuo to afford N-FMOC-Lthyroxine (9,26.3 g) as a beige solid: lH NMR (300 MHz, DMSO-&) 6 9.29 (s, lH), 7.08-7.89 (m, 12H), 4.19-4.29 (m, 4H), 3.06-3.16 (m, lH), 2.82 (t, lH, J = 13 Hz); MS (FAB) (M Na)+ calcd for C30Hz1N0614Na 1021.7441, found 1021.7448;HPLC (15:85:0.4water:methanol:acetic acid; 220 nm) retention time 10.4 min, 98.9%. N-FMOC-L-thyroxine (9, 26.3 g, 23.2 mmol) was dissolved in THF (150 mL) and treated with acetic anhydride (3.28 mL, 34.8 mmol) and 4-(Nfl-dimethylamino)pyridine (283 mg, 2.32 mmol). The reaction was stirred under nitrogen for 45 min and then poured into water (400 mL) and extracted with chloroform (3 x 400 mL). The chloroform extracts were combined, dried over anhydrous NazS04, filtered, and evaporated in uacuo. The residue was then purified by silica gel column chromatography, eluting with methylene chloride/methanol/ acetic acid (90/10/0.4, v/v), to yield 0-acetyl-N-FMOC-Lthyroxine (10, 21.95 g, 91%) as a beige solid: lH NMR (300 MHz, DMSO-&) 6 7.12-7.91 (m, 12H), 4.07-4.31 (m, 4H), 3.07-3.19 (m, lH), 2.82 (t,l H , J = 13 Hz), 2.292.40 (m, 3H); MS (FAB) (M Na)+ m/z 1064; HPLC (Waters RCM p-porasil 8 x 10; 9 5 5 0 . 2 methylene ch1oride:methanol:acetic acid; 240 nm) retention time, 4.54 min, 90%. 0-Acetyl-N-FMOC-L-thyroxine (10,21.70 g, 19.17 "01) was dissolved in methylene chloride (250 mL), cooled to 0 "C, and treated with 0-tert-butyl-NJV'-diisopropylisourea (11, 12) (19.20 g, 95.85 mmol) in methylene chloride (50 mL) dropwise. The reaction mixture was stirred overnight under nitrogen, a t room temperature, and vacuum filtered to remove insoluble impurities, and the filtrate solvent was removed in uacuo. The resulting
+
+
+
residue was stirred in ethyl acetatehexane (300 mL, 40/ 60, v/v) for 4 h and vacuum filtered to remove insoluble impurities, and filtrate solvent was removed in vacuo. The residue was then purified by silica gel column chromatography, eluting with ethyl acetatehexane (40/ 60, v/v), to afford tert-butyl 0-acetyl-N-FMOC-L-thyroxine (11,9,64 g, 46%) (2)as a beige solid: lH NMR (300 MHz, CDC13) 6 7.18-7.82 (m, 12H), 4.21-4.57 (m, 4H), 3.05 (s, 2H), 2.39 (s, 3H), 1.33-1.54 (m, 9H); MS (FAB) (M H)+ m/z 1098; HPLC [Waters RCM p-Porasil8 x 10; 20: 80 ethyl acetate:hexane; 256 nml retention time, 9.13 min, 89%. tert-Butyl 0-acetyl-N-FMOC-L-thyroxine (11,9.59 g, 8.04 mmol) was dissolved in dimethylformamide (40 mL), triethylamine (1.12 mL, 8.04 mmol) was added, and the reaction mixture was stirred overnight under nitrogen. Ethyl bromoacetate (1.78 mL, 16.1 mmol) followed by triethylamine (1.12 mL, 8.04 mmol) were added. The reaction mixture was stirred an additional 2 h under nitrogen and then poured into water (200 mL) and extracted with ethyl acetate (3 x 200 mL). The ethyl acetate extracts were combined, dried over anhydrous MgS04, and evaporated in vacuo. The resulting oil was purified initially by silica gel column chromatography, eluting with ethyl acetatehexane (40/60, v/v), and then purified a second time by preparative silica gel HPLC, eluting with ethyl acetatehexane (20/80, v/v), to yield tert-butylO-acetyl-N-(carbethoxymethyl)-L-thyroxine (13, 3.77 g, 49%) as a white solid: 'H NMR (300 MHz, CDCl3) 6 7.75 (s,2H), 7.19 (s,2H), 4.20 (q,2H, J = 5 Hz), 3.393.49 (m, 3H), 2.80-2.98 (m, 2H), 2.39 (s, 3H), 1.42 (s, 9H), 1.27 (t,3H, J = 5 Hz); MS (FAB) (M H)+ calcd for C26H28N0714 961.8040, found 961.8046; HPLC [Waters RCM p-Porasil 8 x 10; 30:70 ethyl acetate:hexane; 260 nm, 1.5 mumin] retention time, 6.8 min, 98%. The ethyl ester intermediate (13, 3.73 g, 3.88 mmol) was dissolved in methanol (85 mL) containing 10% sodium hydroxide (12.4 mL, 31 mmol). The reaction mixture was stirred for 40 min and poured into water (250 mL). The pH of the solution was adjusted to 4 with 1N HC1 and then extracted with ethyl acetate (3 x 250 mL). The ethyl acetate extracts were combined, dried over anhydrous MgS04, and evaporated in uacuo to afford tert-butyl N-(carboxymethyl)-L-thyroxine(14,3.29 g, 95%) ( 3 )as a white solid; 'H NMR (300 MHz, DMSO-&) 6 7.81 (s, 2H), 7.07 (s, 2H), 3.52 (m, lH), 3.32 (s, 2H), 2.892.98 (m, 1H), 2.20-2.31 (m, lH), 1.32 (s, 9H); MS (FAB) (M H)+ calcd for C21H22N0614 891.7621,found 891.7622; HPLC [20:80:0.4 water:methanol:acetic acid; 240 nml retention time, 7.3 min, 95%. tert-Butyl N-(carboxymethyl)-L-thyroxine(14, 1.78 g, 2.00 mmol) was dissolved in dimethylformamide (20 mL) and treated with N-hydroxysuccinimide (230 mg, 2.00 mmol) and 1,3-dicyclohexylcarbodiimide(413 mg, 2.00 mmol). The reaction mixture was stirred for 16 h under nitrogen and then vacuum filtered. The filtrate was combined with 5-(aminomethyl)fluoresceinhydrobromide (13)(884 mg, 2.00 mmol) and triethylamine (1.8 mL, 13 mmol), and the reaction was stirred for 16 h, under nitrogen, in the dark. The solvent was removed in vacuo, and the residue was purified by preparative reversed phase C18 HPLC, eluting with water/methanol/acetic acid (25/75/0.4, v/v), to afford 1.51 g (61%)of the desired tert-butyl ester protected tracer as a n orange solid: 'H NMR (300 MHz, DMSO-ds) 6 10.13 (s,2H), 9.29 (s, lH), 8.43 (m, lH), 7.85 (s, l H ) , 7.83 (s, 2H), 7.68 (d, l H , J = 5 Hz), 7.12-7.28 (m, 2H), 7.07 (s, 2H), 6.68 (s, 2H), 6.54 (s, 4H), 4.36-4.61 (m, 2H), 3.26-3.50 (m, 3H), 2.94-3.04 (m, lH), 2.71-2.82 (m, lH), 1.33(s, 9H); MS (FAB)(MI+ calcd for C42H34N201014 1234.8466, found 1234.8465;
+
+
+
Bioconjugafe Chem., Vol. 5,No. 5, 1994 461
Synthesis of Thyroxine Conjugates
Scheme 2"
Scheme 1" HO I
HO I
I
I
R
e
R'
Rl
H
FMOC
OH
c10
COCH,
FMOC
OH
cll
COCH,
FMOC
0-1-C,H,
G12
COCH,
H
0-1-C,H,
COCH,
CHzCOzCzH5
O.t-C,H,
CHzCOzH
O-t-C,H,
9
c
13
fL1, H OH BSA
a Key: (a) AczO; (b) DCC, NHS, 6-aminocaproic acid; EDAC, NHS; (d) BSA.
(c)
HPLC [20:80:0.4 water:methanol:acetic acid; 240 nml retention time 12.7 min, 98%. The tert-butyl ester protected tracer (15,1.464 g, 1.19 mmol) was dissolved in methylene chlorideltrifluoroacetic acid (30 mL 1/1, v/v) and stirred for 5 h, and the solvent was removed in vacuo. The crude product was purified by preparative reversed phase C18 HPLC, eluting with waterlmethanollacetic acid (2517510.4, vlv) to yield 1.01 g (72%) of the desired L-thyroxine tracer (16)as an orange solid: 'H NMR (300 MHz, DMSO-de) 6 10.0-10.3 (broad s, 2H), 8.31 (t, l H , J = 2 Hz), 7.86 (s, 2H), 7.83 (s, lH), 7.64 (d, l H , J = 7 Hz), 7.15-7.30 (m, 2H), 7.08 (s, 2H), 6.68 (s, 2H), 6.55 (s, 4H), 4.31-4.59 (m, 2H), 3.28-3.55 (m, 3H), 2.82-2.99 (m, 2H); MS (FAB) (M H)+ calcd for C38H27N201014 1178.7840,found 1178.7834;HPLC [25: 750.4, water:methanol:acetic acid; 240 nm] retention time 8.1 min, 99%.
+
RESULTS The first step toward improvement of the T4 immunoassay was to prepare a well-defined immunogen (14, 15).This was accomplished by the selective conjugation of L - T to ~ BSA through the carboxyl group. Initially, the a-amino group of T4 was acetylated to give compound 5. Activation of the carboxyl group with DCC and NHS followed by coupling to 6-aminocaproic acid resulted in L - T hapten ~ 6. The terminal carboxyl group of the L-T4 hapten was finally activated with a water soluble carbodiimide (EDAC), and NHS in DMF then conjugated to BSA to produce the desired immunogen, 8. Analysis by TNBS titration showed 48% of the available amino groups of BSA had been substituted by the hapten (16). Contrary to the obvious practice of using an analogous tracer, the L - T tracer ~ was prepared by conjugating the fluorescent label through the amino group of L - T ~not , through the carboxyl group as done in the immunogen preparation. The multiple step synthesis of the L-T4 fluorescent tracer is shown in Scheme 2. The first step was the selective protection of the amino group with fluoromethyl chloroformate (FMOC-CI)to give N-FMOC-L-T~,9. The phenolic group was acetylated with acetic anhydride and the carboxyl group protected as the tert-butyl ester to give
(a) FMOC-Cl, NaZC03, H20PTHF; (b) AczO, DMAP, THF; CHzClz (d)EtsN, DMF; ( e ) BrCHzCOzEt, Et3N, DMF (0 10% NaOH, MeOH (g) NHS, DCC, DMF; (h) 5-(aminomethyl)fluorescein HBr, EtsN, DMF; (i) TFA, CHZC12. a
(c) 0-tert-butyl-N,"-diisopropylisourea,
11. Next the FMOC group was selectively removed by triethylamine in DMF. The unmasked amino group was alkylated in situ with ethyl bromoacetate to produce compound 13. Simultaneous removal of the acetyl and ethyl ester protecting groups was achieved in methanolic sodium hydroxide. tert-Butyl N-(carboxymethyl)-~-T4,14, was conjugated to 5-(aminomethyl)fluorescein (13)and the tert-butyl ester subsequently removed in the presence of trifluoroacetic acid to give the tracer, 16. DISCUSSION When preparing specific antibodies and complementary labeled haptens, one needs to consider the chemical structure of both the immunogen used to elicit the antibody response and the labeled hapten. Traditionally, one attaches the hapten to the carrier protein through a site on the hapten that is remote from the unique features of the hapten that are critical for achieving selective antibodies. Likewise, when preparing a labeled hapten able to bind to such antibodies, it is customary to attach the label to the hapten through the same site as the carrier protein. The immunogen and tracer prepared by this design are homologous. One reason for choosing the homologous approach in immunoreagent design is that the carrier protein sterically blocks access of the immune system to that part of the hapten closest to the point of attachment. Normally, the complementary labeled hapten is synthesized by attaching its label to the same site on the hapten as the immunogen uses for attachment of its carrier protein, so as not to interfere with antibody binding to the critical features of the
462 Bioconjugate Chem., Vol. 5, No. 5, 1994
hapten. In the case of T4 the unique feature important for antibody recognition is the tetraiodosubstituted diphenyl ether system. A second approach to immunoreagent design is the heterologous approach. In this approach, the immunogen and tracer do not share a common point of attachment on the hapten. The heterologous approach has sometimes been used successfully to improve assay performance (17). A heterologous free T4 assay has recently been described (18). The reason that this approach is superior only in some cases has not been fully elucidated. One rationalization might be that in some homologous systems the antibody binds the tracer too tightly, preventing effective competition with the analyte to be measured. A heterologous tracer which does not share the same hapten as the immunogen would be expected to bind less tightly, restoring effective competition and improving the assay performance. The Abbott TDx T4FPIA, used in the Levine (7) report, was developed using a n antibody to a n immunogen represented by structures 2-4, the result of a nonspecific coupling of L-T4 through both the carboxyl and amino groups to the carrier protein, bovine serum albumin (BSA). The FPIA was optimized using D-T, tracer 17. In a sense this tracer was heterologous to all the immunogen structures produced, since the D enantiomer was used.
0&OH
17
In the present work, the immunogen of structure 8 was produced in which the amino group of L-T4 was permanently blocked with an acetyl group. Thus, two modifications to the parent L-T4 structure were introduced in making the immunogen; Le., the carboxylic acid was converted to a n amide and the amino group was acetylated. Neither modification alters the critical tetraiodosubstituted diphenyl ether system. The tracer 16 was prepared from L-TI through a multistep sequence resulting in the fluorescent label attached to the a-amino group via a carboxymethyl spacer arm. Thus, in this paper we have described the synthesis of a new immunogen 8 and fluorescent tracer 16 for
Adamczyk et al.
thyroxine. We have successfully utilized these two reagents in the development of a new FPIA (9) on the TDx analyzer which has replaced the assay used in the Levine study and alleviates the problem noted in that study. We will describe the full details of the assay optimization and performance elsewhere. LITERATURE CITED
(1) Alexander, N. M. (1984) Thyroid function tests. Clin. Chem. 30, 827-828. (2) Siebert, G. R., and Armstrong, J. (1987) Monoclonal Antibodies recognizing L-Thyroxine. US Patent 4,636,478. (3) Siebert, G. R., and Armstrong, J. (1989) Monoclonal Antibodies recognizing L-Thyroxine. US Patent 4,888,296. (4) Fino, J . F., and Kirkemo, C. L. (1984) Substituted carboxyfluoresceins. US Patent 4,476,229. (5) Blecka, L. J., and Jackson, G. J. (1987) Immunoassays in therapeutic drug monitoring. Clin. Lab. Med. 7, 357-70. (6) (1992) Abbott Laboratories TDx Operation Manual. (7) Levine, S., Noth, R., Loo, A., Chopra, I. J., and Klee, G. G. (1990) Anomalous Serum Thyroxine Measurements with the Abbott TDx Procedure. Clin. Chem. 36, 1838-1840. (8) Kabadi, U. M., Fox, I. S., and Cook, P. (1994) Falsely Low Serum Thyroxine Concentration Measured with the Abbott TDx. Clin. Chem. 40, 337-338. (9) Lewis, C. A., and Clarisse, D. (1994) A response to “Falsely Low Serum Thyroxine Concentration Measured with the Abbott TDx”. Clin. Chem. 40, 338. (10) Pitt-Rivers, R. (1948) The Oxidation of Diiodotyrosine Derivatives. Biochem. J . 43, 223-231. (11) Mathias, L. J . (1979) Esterification and Alkylation Reactions Employing Isoureas. Synthesis 561-576. (12) Schmidt, E., and Moosmueller, F. (1955) Zur Kenntnis aliphatischer Carbodiimide, IX. Mitt. Liebigs Ann. Chem. 597, 235-240. (13) Mattingly, P. G. (1992) Preparation of 5 and &(Aminomethy1)fluorescein. Bioconjugate Chem. 3, 430-431. (14) Adamczyk, M., Fishpaugh, J . , Harrington, C., Hartter, D., Johnson, D., and Vanderbilt, A. (1993) Immunoassay reagents for psychoactive drugs. I. The method for the development of antibodies specific to amitryptyline and nortriptyline. J . Immunol. Method, 162, 47-58. (15) Adamczyk, M., Fishpaugh, J., Harrington, C., Johnson, D., and Vanderbilt, A. (1993) Immunoassay reagents for psychoactive drugs. 11. The method of the development of antibodies specific to imipramine and desipramine. J . Immunol. Methods 163, 187-197. (16) Shinoda, T., and Tsuzukida, Y. (1974) Identification of rapidly trinitrophenylating amino groups of human BenceJones proteins modified by incubating for 45 minutes at 37 “C before measuring W absorbance. J . Biochem. 75, 23. (17) Kasson, B. G., Bai, S., Liu, J., Tobin, C., and Kessel, B. (1993) Characterization of a rapid and sensitive enzyme immunoassay (EIA) for progesterone applied to conditioned cell culture media. J . Immunoassay 14, 33-49. (18) Khosravi, M. J., and Papanastasiou-Diamandi, A. (1993) Hapten-heterologous conjugates evaluated for application to free thyroxine immunoassays. Clin. Chem. 39, 256-262.
