Two-step Radioimmunoassay Research Articles

Development of two-step radioimmunoassay (RIA) for the measurement of free triiodothyronine in human serum based on antibody coated tubes

Development of two-step radioimmunoassay (RIA) for the measurement of free triiodothyronine in human serum based on antibody coated tubes

DEVELOPMENT AND VALIDATION OF A TWO-STEP FREE THYROXINE RADIOIMMUNOASSAY BASED ON ANTIBODY COATED TUBES

We describe a two-step radioimmunoassay procedure (RIA) for the measurement of free thyroxine (fT4) in human serum. A commercial antibody with the affinity of 5.5 × 109 L/M was used for this study. The anti-T4 antibody was immobilized on the inner walls of the polystyrene tubes by passive adsorption through normal rabbit IgG and anti-rabbit IgG as immunobridges. The method developed uses very small amounts of antibody with minimum dilution of the sample. The assay covers the range of 0 to 87.5 pmol/L with sensitivity of 0.9 pmol/L. The intra-assay precisions were 6.3% and 7% at 6.7 and 20.7 pmol/L, respectively, for 15 replicates. The inter-assay precision at 6.4 and 24.4 pmol/L were 13% and 11.6%, respectively. The normal range established by analyzing 54 healthy volunteers was 15.3–24.7 pmol/L and that of 69 ambulatory subjects was 14.8–23.3 pmol/L. Progressive dilution of euthyroid and hypothyroid samples up to 50-fold yielded virtually constant values, whereas a registered decrease in free T4 was observed with hyperthyroid samples. Free T4 estimated for 75 samples, including samples from 16 pregnant female subjects, correlated well with the values obtained by a reputed commercial kit with the linear correlation coefficient of 0.84.

Traque des interférences en immunoanalyse : la coopération entre le laboratoire et le fabricant est indispensable. L'exemple de la T3 libre Elecsys ®

Interference from heterophilic antibodies, generally suspected by clinicians faced with discrepant clinical and biological data, remains a problem as regards immunoassays. In the laboratory, we checked for interference in a FT3 assay by studying associations between increased Elecsys FT3 values and normal Elecsys TSH results. Out of 10,000 sera analysed between May 2004 and October 2005 we found 38 such cases. In 14 out of these 38 sera, the increased Elecsys FT3 level was confirmed by a two-step radioimmunoassay, but in the remaining 24 sera interference was suspected. Screenings for anti-T3 or heterophilic antibodies and for drug interference were unsuccessful. Out of these 24 sera, 16 were sent to the manufacturer's laboratory for further investigations. Interference from anti-idiotype antibodies was evidenced in 3 sera and interference from anti-ruthenium antibodies in the other 13 samples. The manufacturer developed a reagent to eliminate interference due to anti-ruthenium antibodies. Interference due to anti-idiotype antibodies remains difficult to eliminate. In this study its frequency (around 0.03%) is in accordance with the state-of-the-art (< 0.05%) in immunoassays incorporating unspecific and specific interference blocking reagents.

The T4/TBG ratio in healthy subjects and in patients with disorders of thyroid function: correlation with FT4 concentrations

Summary: The proportion of free hormones can be estimated in the presence of protein-bound hormones by a variety of direct assay approaches or by a two test strategy to calculate a free hormone »index«. The T4/TBG ratio, determined from measurements of total thyroxine (TT4) and thyroxine-binding globulin (TBG) concentrations, is an appropriate index with improved diagnostic accuracy compared with T4. In this study, we calculated the T4/TBG ratio after determining serum TT4 and TBG by radioimmunoassay (INEP, SCG) in euthyroid persons (n=102), in patients with hypothyrosis (n=33) and hyperthyrosis (n=66), as well as in healthy pregnant women (n=346). The reference range for each group of patients was determined. Values obtained for the T4/TBG ratio in healthy subjects varied from 2.1 to 4.4. They were lower in hypothyrosis (0.5 ’2.2) and elevated in hyperthyrosis (4.7–13.1). The reference intervals of the T4/TBG ratio were also determined for each trimester of pregnancy. A significant correlation was obtained between the values for T4/TBG ratio and FT4 concentration, determined by the two-step radioimmunoassay (CIS-bio international, France), in healthy subjects and patients with thyroid disorders (r=0.93), as well as during pregnancy (0.59). In conclusion, we determined reference intervals for the T4/TBG ratio, confirming its practical usefulness in the assessment of thyroid function.

Hyperproinsulinemia is associated with increased beta cell demand after hemipancreatectomy in humans.

The cause of disproportionate hyperproinsulinemia in patients with type II diabetes is controversial. To examine whether increased beta cell demand might contribute, we measured proinsulin and insulin concentrations in clinically healthy humans who had undergone hemipancreatectomy for the purpose of organ donation, a procedure previously demonstrated to increase beta cell demand and diminish insulin secretory reserve capacity. Subjects were studied at least 1 yr after hemipancreatectomy. Seven donors were followed prospectively and serves as their own controls. Nine additional donors were matched with normal controls (cross-sectional group). Fasting serum concentrations of intact proinsulin and conversion intermediates (total) were measured by a two-step radioimmunoassay; independent determinations of intact proinsulin and 32,33 split proinsulin were performed using an immunoradiometric assay. Serum total proinsulin values were significantly greater in hemipancreatectomized groups than controls (prospective group: predonation = 6.24 +/- 1.14 pM, postdonation = 34.63 +/- 17.47 pM, P < 0.005; cross-sectional group: controls = 5.78 +/- 1.12 pM, donors = 15.22 +/- 5.20 pM, P < 0.025). The ratio of total proinsulin to immunoreactive insulin was directly correlated with fasting plasma glucose and showed a significant inverse relationship to secretory reserve capacity. Both absolute and relative hyperproinsulinemia is found in hemipancreatectomized donors. These data demonstrate that partial pancreatectomy with its associated increase in beta cell demand raises measures of proinsulin in humans.

Hapten-heterologous conjugates evaluated for application to free thyroxine immunoassays

We evaluated the effect of hapten heterology on free thyroxine (FT4) immunoassays involving the biotin-streptavidin system and time-resolved fluorometry. We compared protein derivatives of thyroxine (T4) and triiodothyronine (T3) as solid-phase antigen or biotinylated protein-tracer conjugate for competitive (or sequential) binding to a mouse anti-T4 monoclonal antibody. In both one- and two-step assays, the heterologous combination of the antibody and T3 conjugates showed superior standard curve sensitivity but up to eightfold lower zero standard signal (B(o)) when the same amounts of antibody and conjugates were used. The improved sensitivity was not altered when the amount of coupled T3 was increased to obtain a B(o) value similar to that of the homologous combination of antibody and T4 conjugates. In the two-step format, the sensitivity of the homologous assay was insufficient for routine use, consistent with displacement of bound T4 during the antibody back-titration step (demonstrated in the T4 displacement experiment with excess conjugate). Results from the one-step (labeled antibody) heterologous assay for approximately 85 clinical samples correlated well with those from an immunofluorometric assay and a two-step radioimmunoassay. The assay was not affected by a wide variation in endogenous serum concentrations of T4-binding globulin and albumin.

Two new two-step immunoassays for free thyroxin evaluated: solid-phase radioimmunoassay and time-resolved fluoroimmunoassay

We measured concentrations of free thyroxin (FT4) in serum by using two new two-step FT4 assays--a solid-phase two-step radioimmunoassay. Spectria, and a time-resolved fluoroimmunoassay. Delfia--and compared the results with those by a two-step FT4 assay (RIA-gnost), a one-step FT4 analog assay (Amerlex-M), and FT4 measured after equilibrium dialysis. The new FT4 assays classified 30 hypothyroid and 43 hyperthyroid patients (untreated) well. In 138 patients with nonthyroidal illness (NTI) and in late pregnancy (n = 36), fewer subnormal FT4 values were reported by Spectria (P less than 0.001), Delfia (P less than 0.001), and RIA-gnost (P less than 0.01) than by Amerlex-M. The results of the Spectria and Delfia methods correlated with the results of the dialysis method (r = 0.76) in NTI patients and pregnancy, and were in better agreement with the clinical state than was FT4 by Amerlex-M. The FT4 values by Amerlex-M, but not by other methods, correlated with albumin concentration. We conclude that these new two-step methods present good alternatives for FT4 analysis.

One-step chemiluminescent immunoassay of free thyroxin with acridinium-ester-labeled thyroxin evaluated and compared with a two-step radioimmunoassay.

This new one-step chemiluminescent immunoassay of free thyroxin (FT4) involving a thyroxin-immunoglobulin conjugate labeled with acridinium ester (Magic Lite System; Ciba Corning Diagnostics Corp., Medfield, MA) is rapid (one 1-h incubation), requires two calibrators per run, and takes 10 s per sample for the quantification step. Analytical performances were excellent: within- and between-run CVs of less than 10% in the working range, no significant effect of hemolysis, bilirubin, or lipemia, and no significant interaction between the conjugate and the thyroxin-binding proteins. Magic Lite results (y) correlated well with those obtained by the Sclavo (x) two-step radioimmunoassay (Sclavo, Siena, Italy): y = 1.35x + 1.32 (r = 0.94, n = 267, P less than 0.001, Sxy = 6.29). Clinical sensitivities (diagnostic efficiencies) for hypothyroidism and hyperthyroidism were 0.91 and 0.98 for normal interval limits of 12 and 21.5 pmol/L (95% confidence interval). Magic Lite results in situations where patient therapy, treatment, or unusual conditions can result in a lack of correlation between the clinical status and the FT4 values were qualitatively the same as those obtained by the Sclavo assay.

New one-step enzyme immunoassay for free thyroxin.

This enzyme immunoassay for free thyroxin (FT4) involves simultaneous incubation of sample and thyroxin conjugated to horseradish peroxidase (EC 1.11.1.7) in antibody-coated tubes at room temperature. The test can be performed in 90 min. It is not influenced by variations in concentrations of thyroxin-binding globulin (TBG), thyroxin-binding prealbumin, or albumin. With increased concentrations of non-esterified fatty acids, the measured FT4 equivalent concentrations increase immediately, indicating displacement of endogenous thyroxin from its binding sites. FT4 values obtained with this assay for 110 samples, including sera with low albumin and high TBG as well as sera of patients treated with heparin, agreed well with those measured by a two-step radioimmunoassay and with the T4/TBG ratio. The measurable range of FT4 with our assay is 2.5 to 65 ng/L. Intra-assay CVs range from 3.4% to 2.2%, interassay CVs from 6.6% to 2.6% at concentrations of 9 to 45 ng of FT4 equivalent per liter.

A two-step radioimmunoassay for free triiodothyronine in serum.

Using a high-affinity solid-phase-bound antibody (Ka = 1.2 X 10(11) L/mol), we have standardized a two-step radioimmunoassay for free triiodothyronine (FT3) in serum, based on immunoextraction. The method was validated by comparison with an equilibrium-dialysis procedure (r = 0.96) involving RIA of T3 in the dialysate standardized with the same antibody and by a commercial (Liso-Phase, International-CIS) method. The two-step RIA could detect as little as 0.2 pg per milliliter. The mean CVs within and between assays were 9% and 12%, respectively. FT3 values measured in 30 normal adults ranged from 1.77 to 4.77 ng/L. Comparison with ratios of total T3 to thyroxin-binding globulin showed good agreement in normal subjects, pregnant women, and hypothyroid and hyperthyroid patients.

On the albumin dependence of measurements of free thyroxin. I. Technical performance of seven methods.

We evaluated three one-step (analog) and two two-step radioimmunoassay for free thyroxin (FT4), and a FT4 index calculated from the total T4 (TT4) and thyroxin-binding globulin (TBG) ratio for technical performance, for correlation with the reference FT4 method (equilibrium dialysis), and for dependence on TBG and albumin concentrations. The one-step methods (Amerlex, Coat-A-Count, and GammaCoat) showed greater precision than the two-step procedures (GammaCoat and Spiria). Results by the latter two techniques, however, correlated better with those by equilibrium dialysis than did those by the analog methods or by TT4/TBG. Only the GammaCoat two-step method had a slight but statistically significant (inverse) correlation with TBG concentration. All three analog methods and TT4/TBG showed a marked dependence on albumin concentration, whereas the Spiria technique showed only a slight dependence. Only equilibrium dialysis was independent of both the TBG and albumin concentration. Thus, despite their good precision, the analog (one-step) FT4 methods and the TT4/TBG approach cannot be expected to produce valid results when the concentration of albumin in serum is abnormally low or high.