Journal of Immunological Methods, 131 (1990) 137-142
137
Elsevier JIM05634
Homogeneous latex immunoassay for thyroid hormone testing Determination of thyroxine and triiodothyronine J.C. M a r e s c h a l , C.L. C a m b i a s o , J. J a n s s e n s , J . N . L i m e t , N . T a s i a u x a n d D. C o l l e t - C a s s a r t Unit of Experimental Medicine, Faculty of Medicine, Catholic University of Louvain, 75, Avenue Hippocrate, B-1200 Brussels, Belgium
(Received 2 October 1989, revised received 11 December 1989, accepted 6 April 1990)
We describe here two latex immunoassays for total thyroxine (T4) and total triiodothyronine (T3). These homogeneous 60 min assays are quantified by optically counting the monomeric particles remaining after agglutination. When precision is assessed, both methods display coefficients of variation of 3-7% for within-run assays and 4-10% for between-run assays. The accuracy of the methods, as tested by dilution and spike recovery experiments, was found to be satisfactory. Two correlation studies were carried out to compare the present method with leading commercial methods. The coefficients obtained were: r = 0.92 and r = 0.93 with 150 sera for T 3, and r = 0.95 (150 sera) and r = 0.93 (108 sera) for T 4. Key words: Latex immunoassay; Latex agglutination; Thyroid hormone; Non-isotopic immunoassay
Introduction
Accurate determination of T3 and T 4 in serum is important for assessing thyroid status. These hormones are conventionally assayed by radioimmunoassays using an 125I label (Chopra, 1972; Mitsuma et al., 1972). However the increasing need for non-isotopic methods has led scientists and companies to develop alternative technologies for thyroid hormone testing e.g., enzyme immunoassays (O'Sullivan et al., 1978; Plomp et al., 1979), fluorescence immunoassays (Smith, 1977; Bellet et al., 1981) and luminescence immunoassays (Schroeder et al., 1983).
We present here two homogeneous latex particle systems based on the agglutination of polystyrene particles coated with T 3- or Ta-protein conjugate by anti-T 3 or anti-T 4 antibodies followed by a second antibody. Inhibition of agglutination by the hormones, after a short enzymatic treatment of the serum, permitted us to set up within the concentration ranges 0.5-6/~g/1 for T3, and 3 0 - 2 5 0 / ~ g / l for T 4. The assay principles and optical reading conditions have been described in full elsewhere (Masson et al., 1981).
Materials and methods Materials
Correspondence to: D. CoUet-Cassart,Unit of Experimental Medicine, Faculty of Medicine, Catholic University of Louvain, 75, Avenue Hippocrate, B-1200 Brussels, Belgium. Abbreviations: GBS, giycine-buffered saline; PBS, phosphate-buffered saline; BSA, bovine serum albumin; RIA, radioimmunoassay; SDS, sodium dodecyl sulfate; SD, standard deviation.
Reagents GBS. This consisted of 0.1 mol of glycine
(Merck, Darmstadt, F.R.G.), 0.17 mol of sodium chloride (Merck) and 400 mg of sodium azide (Merck)/1. The p H was adjusted to 8.5 with sodium hydroxide.
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
138 PBS. This consisted of 0.02 mol of potassium dihydrogen phosphate (Merck)/1, adjusted to pH 7.5 with 0.02 mol of di-sodium hydrogen phosphate (Merck)/1. The buffer contained 9 g of sodium chloride, 1 g of sodium azide and 1 g of Thimerosal (Sigma Chemical Co., St. Louis, MO, U.S.A.)/1. GBS-BSA. This consisted of GBS twice diluted and containing 5 g of BSA (RIA grade) (U.S. Biochemical Corp., Cleveland, OH 44122) and 1 g of Thimerosal/1. PBS-BSA. This consisted of PBS twice diluted and containing 5 g of BSA (RIA grade)/1. Tris-SDS. This contained 1 tool of Tris (Merck), 2 g of SDS (Merck) and 5 g of BSA (RIA grade)/1. Pepsin-HCl for T3 assay. This consisted of 0.4 mol of HC1 and 5 g of porcine pepsin (EC 3.4.23.1. twice crystallized; Sigma Chemical, St. Louis, MO 63178)/1. Pepsin-HCl for T4 assay. This consisted of 0.3 mol of HC1 and 5 g of porcine pepsin/1. Albumin-T 3 (BSA-T3) and albumin-T4 (BSA-T4) conjugates. These were prepared by coupling 3,3',5-triiodo-L-thyronine (Sigma Chemical Co.) and L-thyroxine (Sigma Chemical Co.) to BSA (Fraction V, Boehringer, Mannheim, F.R.G.) via the cardodiimide method (Chopra et al., 1972) using 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (Sigma). B S A - T 3 latex. Carboxylated latex particles, 800 nm in diameter (Estapor K150; Rhbne-Poulenc, Courbevoie, France) were covalently coated with BSA-T3 conjugate as described elsewhere (Collet-Cassart et al., 1981). The working suspension of latex particles contained 250 mg of coated particles/1 of PBS-BSA. This reagent was stable for at least several years when stored at - 2 0 °C or freeze dried and for at least 3 months when kept at 4 o C. B S A - T 4 latex. The same procedure was followed as for BSA-T3 latex, except that the suspension was diluted in GBS-BSA buffer at a concentration of 250 mg/1 and stored in a lyophilized form. Prior to use the particles were rehydrated with a solution containing 0.7 mol of Tris (Merck), 0.5 g of SDS and 100/~1 of Tween 20 (Technicon Instruments Corp., Tarrytown, NY 10591)/1. Once
reconstituted, the latex reagent was stable for three months at 4 ° C. Anti-T 3. A sheep was immunized three times at 2 week intervals with 100 /~g BSA-T3 in complete Freund's adjuvant. Each month, the sheep was boosted with the same amount of BSA-T3 and bled 10 days after each injection. The blood was collected, tested and pooled. A dilution of 1/4000 in PBS-BSA buffer was used in the T 3 assay. Anti-T 4. Two antisera were mixed for the present assay. The first one was obtained by injecting three rabbits intradermally with 100/~g of BSA-T4 conjugate in complete Freund's adjuvant, three times at 2 week intervals. Each month, the rabbits were boosted with the same dose and bled 10 days after each injection. The blood was collected, tested and pooled. A second antiserum, which was raised in a sheep was purchased form the Benenden Chest Hospital (Cranbrook, England). A mixture of one part of rabbit antiserum and two parts of sheep antiserum was used at a dilution of 1/400 in GBS-BSA buffer. Second antibody. A human rheumatoid factor serum from a patient who was undergoing plasmapheresis, with a latex agglutination titer (latexrheumatoid factor reagent; Behringwerke, Marburg/Lahn, F.R.G.) of 1:1250 was used as second antibody. A dilution of 1/300 in PBS-BSA was prepared for the T 3 assay and of 1/500 in GBS-BSA for the T4 assay. Serum calibrators. L-thyroxine and 3,3',5-triiodothyronine were solubilized in 0.15 mol/1 of NaOH at 500 mg/1 concentrations. Both hormones were further diluted in T3- and Ta-free delipidated human serum purchased from CBR Laboratories (Boston, MA 02115) to obtain standards. Lyphochek control sera (Bio-Rad, Anaheim, CA 92806) were used to validate the assays. Sera Serum samples were obtained from the thyroid clinic of Dr. Schoutens, H6pital Erasme, Universit6 Libre de Bruxelles, Belgium. Samples were stored at - 2 0 °C until assay.
Instruments We used the Multipact system (Acade Instruments, 1348 Louvain-la-Neuve) as described elsewhere (Wilkins et al., 1988).
139
Methods Assay protocol for T3 To 20 #1 of serum sample, calibrator, or control, 25/~1 of pepsin-HC1 reagent were added. The mixture was incubated for 15 rain at ambient temperature. Then 25 #1 of Tris-SDS solution, followed by 25 #1 of BSA-T3 latex and 50 #1 of a mixture, consisting of equal volumes of anti-T 3 and second antibody, were added. The resulting mixture was incubated for 1 h in the system's agitator. To stop the reaction, 500 #1 of GBS were added, containing 0.1 mol of ethylenedinitrilotetraacetic acid, disodium salt dihydrate ( M e r c k ) / 1. The number of unreacted particles was then quantified in the system's reader. Assay protocol for T4 To 20 #1 of serum sample, calibrator, or control, 50 #1 of pepsin-HC1 reagent were added. The mixture was incubated for 10 min at ambient temperature. Then 50 #1 of latex BSA-T4 were added, followed by 50 #1 of a mixture consisting of equal volumes of anti-T4 and second antibody. The resulting mixture was incubated for 1 h in the system's agitator. The reaction was stopped with 7 5 0 / t l of the above mentioned solution and read in the system's reader. Comparison methods T 3 assays were performed with the Abbot T3 Riabead (North Chicago, IL 60064) and Amersham T3 Amerlex (Arlington Heights, IL 60005)
methods. T4 assays were performed with the Amersham T 4 Amerlex and T4 clinical assays (Cambridge, MA 02139) methods. All methods were performed according to the manufacturer's recommendations.
Results
Calibration curves Calibration curves were constructed by plotting the percentage of remaining unreacted particles (monomers) versus the log of the analyte concentration. Fig. 1 shows calibration curves for T 3 ( a ) and for T 4 (b) assay. The detection limits, defined as two times the standard deviation of the unreacted particle counts at the zero dose (Rodbard, 1978), were ~< 0.2 #g/1 for the T 3 assay and ~< 11/~g/ml for T4 assay. Precision profile Within-run precisions were determined with three sera measured 20 times on the same day, and between-run precisions were performed by measuring three sera ten times on 10 different days. Table I shows precision data for the T 3 and T4 assays. Accuracy The accuracy of the tests was assessed by two methods. In a dilution recovery test sera were diluted 1 / 2 and 1 / 4 in T 3 and T a free serum. The recovery was calculated by establishing the per-
a
co uJ
100
100
LU
_...I
0 I-rr 13.. a
._1
0 8O
I--
ncL
60
IJJ
a
6o
LLI
4O
LU I-0 ,
/2/~g/l: 5% ~/2.0 ~tg/l: 16% ~ 60/.t g/l: 20% 60-120/~g/l: 67% >/120/~g/l: 13% ~/120/~g/l: 6.5%
y = 0.99x + 3.19
0.93
141 commercial RIAs. These results are shown in Table IV.
Discussion Although there is a trend towards the use of free T 3 and free T 4 assays, the measurements of total T 3 and total T 4 are still widely performed in the clinical laboratory. Most T 3 and T 4 assays are based on radioimmunological principles, but alternative classical techniques based on enzyme activity, fluorescence or luminescence signals have also been applied. The major advantages of the techniques presented here reside in their non-isotopic and homogeneous features. The agglutination of haptencoated particles by a double antibody system had already been used for the determination of digoxin several years ago (Collet-Cassart et al., 1981). This reaction involved the fixation of a first antibody (anti-hapten), which did not itself agglutinate the latex particles, followed by agglutination due to the second antibody. The latter was a h u m a n IgM RF, known to have low affinity for monomeric I g G but high avidity for I g G involved in antigen-antibody complexes or fixed to solid supports (Masson et al., 1981). Since IgM R F acts as a second antibody, different R F sera can be used in such an assay, provided the R F dilution is adapted to the particular test. Moreover, in another publication (Poncelet et al., 1989), we showed that a rabbit anti-mouse I g G could be used as second antibody when a monoclonal mouse I g G was selected as the primary antibody. In both of the assays described all operations are performed in the same tube and 5 - 6 pipetting steps are necessary to complete each test. Since no separation steps are required the techniques could be readily automated. However, even with manual pipetting steps, an acceptable precision has been obtained with values reaching 10% for between-run assays at the lower ends of the curves. The validation of both assays by dilution and spike recovery experiments (Tables II and III) gives a good indication of the accuracy of the methods at different concentrations of analyte. In none of the correlations did any serum show a major discrepancy from the values obtained by
R I A and all of the correlations confirm the accuracy of the latex particle methods. Moreover, we believe that the pepsin pretreatment of sera should eliminate interference b y autoantibodies to thyroxine and triiodothyronine (Bhagal et al., 1983). The wide applicability of pepsin treatment, even for protein antigens, was demonstrated by Wilkins et al. (1988) in a high sensitivity T S H assay. In another study (Galanti et al., 1987), similar pepsin treatment of serum was found to be successful in releasing a hidden hepatitis B antigen from its complex with antibodies. Since pepsin digestion is not a specific treatment for latex particle agglutination techniques, it could be applied in any other immunoassay subject to interference from autoantibodies to T 3 and to T 4. The performance of the T 3 and T 4 assays presented here demonstrates the validity of latex particle agglutination and optical counting techniques. This technique is potentially applicable to a large spectrum of analytes and once it is fully automated, could become a useful tool in the clinical laboratory.
References Bellet, N., Winfrey, L., Horton, A., et al. (1981) Development of homogeneous fluorescencerate immunoassaysfor thyroid function testing (abstract). Clin. Chem. 27, 6, 1071. Bhagal, C.I., Garcia-Webb, P., Watson, F. and Beilby, J.P. (1983) Interference in radioimmunoassay of total serum thyroxin and free thyroxin due to thyroxin binding autoantibodies (Letter). Clin. Chem. 29, 1324. Chopra, l.J. (1972) A radioimmunoassay for measurement of thyroxin in unextracted serum. J. Clin. Endocrinol. Metab. 34, 938. Chopra, I.J., Nelson, J.C., Solomon, D.H. and Beall, G.N. (1972) Production of antibodies specifically binding triiodothyronine and thyroxine. J. Clin. Endocrinol. Metab., 32, 299. Collet-Cassart, D., Magnusson, C.G.M., Cambiaso, C.L., et al. (1981) Automated particle counting immunoassay for digoxin. Clin. Chem. 27, 7, 1205. Galanti, L.M., Cambiaso, C.L., Cornu, C.J., et al. (1987) Immunoassay of hepatitis B surface antigen by particle counting after pepsin digestion. J. Virol. Methods 18, 215. Masson, P.L., Cambiaso, C.L., Collet-Cassart, D., et al. (1981) Particle counting immunoassay (PACIA). Methods Enzymol. 74, 106. Mitsuma, T., Colucci, J., Shenkman, L. and Hollander, C.S. (1972) Rapid simultaneous radioimmunoassay for tri-
142 iodothyronine and thyroxine in unextracted serum. Biochem. Biophys. Res. Commun. 46, 2107. O'Sullivan, M.J., Gnemmi, E., Morris, D., et al. (1978) An enzyme immunoassay for triiodothyronine. In: S.B. Pol (Ed.), Enzyme Labelled Immunoassay of Hormones and Drugs. Walter de Gruyter, Berlin, p. 301. Plomp, T.A., Drost, R.H. and Thyssen, J.H.N. (1979) Evaluation of the manual enzyme immunoassay (EMIT) procedure for the determination of serum thyroxin. J. Clin. Chem. Clin. Biochem. 17, 315. Rodbard, D. (1978) Statistical estimation of the minimal detectable concentration ('sensitivity') for radioligand assays. Anal. Biochem. 90, 1.
Schroeder, H.R., Yeager, F.M., Bogulaski, R.C. and Vogelhut, P.O. (1983) Immunoassay for serum thyroxin by solid-phase chemiluminescence immunoassay. Ann. Clin. Biochem. 20, 275. Smith, D.S. (1977) Enhancement fluoroimmunoassay of thyroxin. FEBS Lett. 77, 25. Wilkins, T.A., Brouwers, G., Mareschal, J.C.M. and Cambiaso, C.L. (1988) High sensitivity, Homogeneous particle-based immunoassay for thyrotropin (MultipactTM). Clin. Chem. 34, 1749.
137
Elsevier JIM05634
Homogeneous latex immunoassay for thyroid hormone testing Determination of thyroxine and triiodothyronine J.C. M a r e s c h a l , C.L. C a m b i a s o , J. J a n s s e n s , J . N . L i m e t , N . T a s i a u x a n d D. C o l l e t - C a s s a r t Unit of Experimental Medicine, Faculty of Medicine, Catholic University of Louvain, 75, Avenue Hippocrate, B-1200 Brussels, Belgium
(Received 2 October 1989, revised received 11 December 1989, accepted 6 April 1990)
We describe here two latex immunoassays for total thyroxine (T4) and total triiodothyronine (T3). These homogeneous 60 min assays are quantified by optically counting the monomeric particles remaining after agglutination. When precision is assessed, both methods display coefficients of variation of 3-7% for within-run assays and 4-10% for between-run assays. The accuracy of the methods, as tested by dilution and spike recovery experiments, was found to be satisfactory. Two correlation studies were carried out to compare the present method with leading commercial methods. The coefficients obtained were: r = 0.92 and r = 0.93 with 150 sera for T 3, and r = 0.95 (150 sera) and r = 0.93 (108 sera) for T 4. Key words: Latex immunoassay; Latex agglutination; Thyroid hormone; Non-isotopic immunoassay
Introduction
Accurate determination of T3 and T 4 in serum is important for assessing thyroid status. These hormones are conventionally assayed by radioimmunoassays using an 125I label (Chopra, 1972; Mitsuma et al., 1972). However the increasing need for non-isotopic methods has led scientists and companies to develop alternative technologies for thyroid hormone testing e.g., enzyme immunoassays (O'Sullivan et al., 1978; Plomp et al., 1979), fluorescence immunoassays (Smith, 1977; Bellet et al., 1981) and luminescence immunoassays (Schroeder et al., 1983).
We present here two homogeneous latex particle systems based on the agglutination of polystyrene particles coated with T 3- or Ta-protein conjugate by anti-T 3 or anti-T 4 antibodies followed by a second antibody. Inhibition of agglutination by the hormones, after a short enzymatic treatment of the serum, permitted us to set up within the concentration ranges 0.5-6/~g/1 for T3, and 3 0 - 2 5 0 / ~ g / l for T 4. The assay principles and optical reading conditions have been described in full elsewhere (Masson et al., 1981).
Materials and methods Materials
Correspondence to: D. CoUet-Cassart,Unit of Experimental Medicine, Faculty of Medicine, Catholic University of Louvain, 75, Avenue Hippocrate, B-1200 Brussels, Belgium. Abbreviations: GBS, giycine-buffered saline; PBS, phosphate-buffered saline; BSA, bovine serum albumin; RIA, radioimmunoassay; SDS, sodium dodecyl sulfate; SD, standard deviation.
Reagents GBS. This consisted of 0.1 mol of glycine
(Merck, Darmstadt, F.R.G.), 0.17 mol of sodium chloride (Merck) and 400 mg of sodium azide (Merck)/1. The p H was adjusted to 8.5 with sodium hydroxide.
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
138 PBS. This consisted of 0.02 mol of potassium dihydrogen phosphate (Merck)/1, adjusted to pH 7.5 with 0.02 mol of di-sodium hydrogen phosphate (Merck)/1. The buffer contained 9 g of sodium chloride, 1 g of sodium azide and 1 g of Thimerosal (Sigma Chemical Co., St. Louis, MO, U.S.A.)/1. GBS-BSA. This consisted of GBS twice diluted and containing 5 g of BSA (RIA grade) (U.S. Biochemical Corp., Cleveland, OH 44122) and 1 g of Thimerosal/1. PBS-BSA. This consisted of PBS twice diluted and containing 5 g of BSA (RIA grade)/1. Tris-SDS. This contained 1 tool of Tris (Merck), 2 g of SDS (Merck) and 5 g of BSA (RIA grade)/1. Pepsin-HCl for T3 assay. This consisted of 0.4 mol of HC1 and 5 g of porcine pepsin (EC 3.4.23.1. twice crystallized; Sigma Chemical, St. Louis, MO 63178)/1. Pepsin-HCl for T4 assay. This consisted of 0.3 mol of HC1 and 5 g of porcine pepsin/1. Albumin-T 3 (BSA-T3) and albumin-T4 (BSA-T4) conjugates. These were prepared by coupling 3,3',5-triiodo-L-thyronine (Sigma Chemical Co.) and L-thyroxine (Sigma Chemical Co.) to BSA (Fraction V, Boehringer, Mannheim, F.R.G.) via the cardodiimide method (Chopra et al., 1972) using 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (Sigma). B S A - T 3 latex. Carboxylated latex particles, 800 nm in diameter (Estapor K150; Rhbne-Poulenc, Courbevoie, France) were covalently coated with BSA-T3 conjugate as described elsewhere (Collet-Cassart et al., 1981). The working suspension of latex particles contained 250 mg of coated particles/1 of PBS-BSA. This reagent was stable for at least several years when stored at - 2 0 °C or freeze dried and for at least 3 months when kept at 4 o C. B S A - T 4 latex. The same procedure was followed as for BSA-T3 latex, except that the suspension was diluted in GBS-BSA buffer at a concentration of 250 mg/1 and stored in a lyophilized form. Prior to use the particles were rehydrated with a solution containing 0.7 mol of Tris (Merck), 0.5 g of SDS and 100/~1 of Tween 20 (Technicon Instruments Corp., Tarrytown, NY 10591)/1. Once
reconstituted, the latex reagent was stable for three months at 4 ° C. Anti-T 3. A sheep was immunized three times at 2 week intervals with 100 /~g BSA-T3 in complete Freund's adjuvant. Each month, the sheep was boosted with the same amount of BSA-T3 and bled 10 days after each injection. The blood was collected, tested and pooled. A dilution of 1/4000 in PBS-BSA buffer was used in the T 3 assay. Anti-T 4. Two antisera were mixed for the present assay. The first one was obtained by injecting three rabbits intradermally with 100/~g of BSA-T4 conjugate in complete Freund's adjuvant, three times at 2 week intervals. Each month, the rabbits were boosted with the same dose and bled 10 days after each injection. The blood was collected, tested and pooled. A second antiserum, which was raised in a sheep was purchased form the Benenden Chest Hospital (Cranbrook, England). A mixture of one part of rabbit antiserum and two parts of sheep antiserum was used at a dilution of 1/400 in GBS-BSA buffer. Second antibody. A human rheumatoid factor serum from a patient who was undergoing plasmapheresis, with a latex agglutination titer (latexrheumatoid factor reagent; Behringwerke, Marburg/Lahn, F.R.G.) of 1:1250 was used as second antibody. A dilution of 1/300 in PBS-BSA was prepared for the T 3 assay and of 1/500 in GBS-BSA for the T4 assay. Serum calibrators. L-thyroxine and 3,3',5-triiodothyronine were solubilized in 0.15 mol/1 of NaOH at 500 mg/1 concentrations. Both hormones were further diluted in T3- and Ta-free delipidated human serum purchased from CBR Laboratories (Boston, MA 02115) to obtain standards. Lyphochek control sera (Bio-Rad, Anaheim, CA 92806) were used to validate the assays. Sera Serum samples were obtained from the thyroid clinic of Dr. Schoutens, H6pital Erasme, Universit6 Libre de Bruxelles, Belgium. Samples were stored at - 2 0 °C until assay.
Instruments We used the Multipact system (Acade Instruments, 1348 Louvain-la-Neuve) as described elsewhere (Wilkins et al., 1988).
139
Methods Assay protocol for T3 To 20 #1 of serum sample, calibrator, or control, 25/~1 of pepsin-HC1 reagent were added. The mixture was incubated for 15 rain at ambient temperature. Then 25 #1 of Tris-SDS solution, followed by 25 #1 of BSA-T3 latex and 50 #1 of a mixture, consisting of equal volumes of anti-T 3 and second antibody, were added. The resulting mixture was incubated for 1 h in the system's agitator. To stop the reaction, 500 #1 of GBS were added, containing 0.1 mol of ethylenedinitrilotetraacetic acid, disodium salt dihydrate ( M e r c k ) / 1. The number of unreacted particles was then quantified in the system's reader. Assay protocol for T4 To 20 #1 of serum sample, calibrator, or control, 50 #1 of pepsin-HC1 reagent were added. The mixture was incubated for 10 min at ambient temperature. Then 50 #1 of latex BSA-T4 were added, followed by 50 #1 of a mixture consisting of equal volumes of anti-T4 and second antibody. The resulting mixture was incubated for 1 h in the system's agitator. The reaction was stopped with 7 5 0 / t l of the above mentioned solution and read in the system's reader. Comparison methods T 3 assays were performed with the Abbot T3 Riabead (North Chicago, IL 60064) and Amersham T3 Amerlex (Arlington Heights, IL 60005)
methods. T4 assays were performed with the Amersham T 4 Amerlex and T4 clinical assays (Cambridge, MA 02139) methods. All methods were performed according to the manufacturer's recommendations.
Results
Calibration curves Calibration curves were constructed by plotting the percentage of remaining unreacted particles (monomers) versus the log of the analyte concentration. Fig. 1 shows calibration curves for T 3 ( a ) and for T 4 (b) assay. The detection limits, defined as two times the standard deviation of the unreacted particle counts at the zero dose (Rodbard, 1978), were ~< 0.2 #g/1 for the T 3 assay and ~< 11/~g/ml for T4 assay. Precision profile Within-run precisions were determined with three sera measured 20 times on the same day, and between-run precisions were performed by measuring three sera ten times on 10 different days. Table I shows precision data for the T 3 and T4 assays. Accuracy The accuracy of the tests was assessed by two methods. In a dilution recovery test sera were diluted 1 / 2 and 1 / 4 in T 3 and T a free serum. The recovery was calculated by establishing the per-
a
co uJ
100
100
LU
_...I
0 I-rr 13.. a
._1
0 8O
I--
ncL
60
IJJ
a
6o
LLI
4O
LU I-0 ,
/2/~g/l: 5% ~/2.0 ~tg/l: 16% ~ 60/.t g/l: 20% 60-120/~g/l: 67% >/120/~g/l: 13% ~/120/~g/l: 6.5%
y = 0.99x + 3.19
0.93
141 commercial RIAs. These results are shown in Table IV.
Discussion Although there is a trend towards the use of free T 3 and free T 4 assays, the measurements of total T 3 and total T 4 are still widely performed in the clinical laboratory. Most T 3 and T 4 assays are based on radioimmunological principles, but alternative classical techniques based on enzyme activity, fluorescence or luminescence signals have also been applied. The major advantages of the techniques presented here reside in their non-isotopic and homogeneous features. The agglutination of haptencoated particles by a double antibody system had already been used for the determination of digoxin several years ago (Collet-Cassart et al., 1981). This reaction involved the fixation of a first antibody (anti-hapten), which did not itself agglutinate the latex particles, followed by agglutination due to the second antibody. The latter was a h u m a n IgM RF, known to have low affinity for monomeric I g G but high avidity for I g G involved in antigen-antibody complexes or fixed to solid supports (Masson et al., 1981). Since IgM R F acts as a second antibody, different R F sera can be used in such an assay, provided the R F dilution is adapted to the particular test. Moreover, in another publication (Poncelet et al., 1989), we showed that a rabbit anti-mouse I g G could be used as second antibody when a monoclonal mouse I g G was selected as the primary antibody. In both of the assays described all operations are performed in the same tube and 5 - 6 pipetting steps are necessary to complete each test. Since no separation steps are required the techniques could be readily automated. However, even with manual pipetting steps, an acceptable precision has been obtained with values reaching 10% for between-run assays at the lower ends of the curves. The validation of both assays by dilution and spike recovery experiments (Tables II and III) gives a good indication of the accuracy of the methods at different concentrations of analyte. In none of the correlations did any serum show a major discrepancy from the values obtained by
R I A and all of the correlations confirm the accuracy of the latex particle methods. Moreover, we believe that the pepsin pretreatment of sera should eliminate interference b y autoantibodies to thyroxine and triiodothyronine (Bhagal et al., 1983). The wide applicability of pepsin treatment, even for protein antigens, was demonstrated by Wilkins et al. (1988) in a high sensitivity T S H assay. In another study (Galanti et al., 1987), similar pepsin treatment of serum was found to be successful in releasing a hidden hepatitis B antigen from its complex with antibodies. Since pepsin digestion is not a specific treatment for latex particle agglutination techniques, it could be applied in any other immunoassay subject to interference from autoantibodies to T 3 and to T 4. The performance of the T 3 and T 4 assays presented here demonstrates the validity of latex particle agglutination and optical counting techniques. This technique is potentially applicable to a large spectrum of analytes and once it is fully automated, could become a useful tool in the clinical laboratory.
References Bellet, N., Winfrey, L., Horton, A., et al. (1981) Development of homogeneous fluorescencerate immunoassaysfor thyroid function testing (abstract). Clin. Chem. 27, 6, 1071. Bhagal, C.I., Garcia-Webb, P., Watson, F. and Beilby, J.P. (1983) Interference in radioimmunoassay of total serum thyroxin and free thyroxin due to thyroxin binding autoantibodies (Letter). Clin. Chem. 29, 1324. Chopra, l.J. (1972) A radioimmunoassay for measurement of thyroxin in unextracted serum. J. Clin. Endocrinol. Metab. 34, 938. Chopra, I.J., Nelson, J.C., Solomon, D.H. and Beall, G.N. (1972) Production of antibodies specifically binding triiodothyronine and thyroxine. J. Clin. Endocrinol. Metab., 32, 299. Collet-Cassart, D., Magnusson, C.G.M., Cambiaso, C.L., et al. (1981) Automated particle counting immunoassay for digoxin. Clin. Chem. 27, 7, 1205. Galanti, L.M., Cambiaso, C.L., Cornu, C.J., et al. (1987) Immunoassay of hepatitis B surface antigen by particle counting after pepsin digestion. J. Virol. Methods 18, 215. Masson, P.L., Cambiaso, C.L., Collet-Cassart, D., et al. (1981) Particle counting immunoassay (PACIA). Methods Enzymol. 74, 106. Mitsuma, T., Colucci, J., Shenkman, L. and Hollander, C.S. (1972) Rapid simultaneous radioimmunoassay for tri-
142 iodothyronine and thyroxine in unextracted serum. Biochem. Biophys. Res. Commun. 46, 2107. O'Sullivan, M.J., Gnemmi, E., Morris, D., et al. (1978) An enzyme immunoassay for triiodothyronine. In: S.B. Pol (Ed.), Enzyme Labelled Immunoassay of Hormones and Drugs. Walter de Gruyter, Berlin, p. 301. Plomp, T.A., Drost, R.H. and Thyssen, J.H.N. (1979) Evaluation of the manual enzyme immunoassay (EMIT) procedure for the determination of serum thyroxin. J. Clin. Chem. Clin. Biochem. 17, 315. Rodbard, D. (1978) Statistical estimation of the minimal detectable concentration ('sensitivity') for radioligand assays. Anal. Biochem. 90, 1.
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