Anti-dsDNA antibody avidity determination by a simple reliable ELISA method for SLE diagnosis and monitoring.

High avidity anti-dsDNA antibodies are more specific for SLE diagnosis, and more closely associated with renal involvement than intermediate or low-affinity anti-dsDNA antibodies. ELISA methods are largely used to detect anti-dsDNA, but their high sensitivity is inversely related to specificity because they also detect low avidity antibodies. We developed an ELISA assay based on the law of mass action and the competitive binding of dsDNA in solution and coated to microwells with anti-dsDNA antibodies. A simplified Scatchard plot analysis system was used to measure anti-dsDNA antibody avidity which was expressed as apparent affinity constant (Kaa), and quantified in liters per unit (I/U). We prospectively studied 101 consecutive SLE patients, who were followed for 3 years; three serum samples were sequentially collected from each patient during follow-up for determination of IgG anti-dsDNA antibody concentration, and anti-dsDNA avidity. SLE disease activity was estimated using the European Consensus Lupus Activity Measure (ECLAM) index. Sera from 100 healthy subjects and 133 patients with other connective tissue diseases or infectious diseases were also assayed as controls. The mean Kaa in SLE patients was 65.2 +/- 47.3 l/U, with no variations over time. Anti-dsDNA-positive SLE patients had higher Kaa values (79.1 +/- 46.8) than anti-dsDNA negative patients (27.2 +/- 20.1; P < 0.001). No correlation emerged between anti-dsDNA avidity and the ECLAM activity index score. Avidity was significantly higher in patients with renal involvement vs patients without this complication (78.2 +/- 50 vs 59.9 +/- 45.6 l/U; P = 0.0013). This simple ELISA method could be very useful in the diagnostic phase to differentiate high avidity anti-dsDNA autoantibodies that are characteristically found in SLE patients from low avidity antibodies that can also be found in other inflammatory diseases. Moreover, our data confirm the predictive value of high avidity anti-dsDNA antibodies for the development of lupus nephritis.

Evaluation of free thyroxine methods in the presence of iodothyronine-binding autoantibodies.

Circulating iodothyronine-binding autoantibodies interfere with total T4 and T3 RIAs, giving falsely high or falsely low values depending on the assay separation method used. Direct serum free T4 (FT4) measurement should compensate for such abnormal binding if the method is free of artefact. We assessed 5 different FT4 methods in 12 patients with immunoglobulin binding of iodothyronines, with limited assessment of a sixth method in 2 subjects. Thyroid status was assessed clinically and by measurement of TSH and its response to TRH. T4 methods which use analog tracers, i.e. Amerlex, and Clinical Assays One-step FT4, gave spuriously high values in almost all hypothyroid and some euthyroid patients, due to immunoglobulin binding of the tracers used in these techniques. The kinetic indirect method (Corning Immunophase FT4) gave inappropriately high values in 4 of 6 hypothyroid patients. FT4 by equilibrium dialysis or by adsorption chromatography and RIA, accurately assessed thyroid status. These findings suggest that FT4 methods are valid in patients with circulating iodothyronine-binding immunoglobulins only if the free hormone fraction is physically separated from serum binding proteins before the assay procedure.

Circulating antitriiodothyronine autoantibodies in two euthyroid patients: apparent lack of interference in total T3 radioimmunoassay based on second antibody or solid phase separation techniques.

Two clinically euthyroid patients were noted to have low total T3 levels as assessed by RIA using either dextran-charcoal (DC) or polyethylene glycol (PEG) for separation of bound from unbound T3, in spite of normal free T3, total and free T4 and basal and TRH-stimulated TSH concentrations. The presence of circulating substances binding T3 was suggested by high nonspecific binding in total T3 RIA system using either DC or PEG separation. The presence of anti-T3 autoantibodies was then suspected and confirmed by the presence of [125]-T3 bound to patients' gammaglobulins, precipitated with rabbit anti-human immunoglobulins. Serum T3 concentration determined by extracting T3 from patients' sera with methanol was 166 and 226 ng/dl. Similar or even lower values were unexpectedly obtained in RIA systems with solid phase or second antibody (anti-rabbit) separation and with competitive protein binding assay. To face this paradoxical finding, simulated experiments were carried out by incubating T3- and T4-free sera added with various amounts of stable T3 and T4 in the presence of goat anti-T3 or anti-T4 serum. These samples were then radioimmunoassayed. The DC separation caused a consistent underestimation of the actual T3 and T4 concentration. The second antibody separation caused a T3 and T4 overestimation for actual levels below 200 ng/dl and 10 micrograms/dl, respectively, while at the higher T3 or T4 concentrations, an overlap or, even, an underestimation of actual T3 or T4 levels were found. These data provide evidence that, with second antibody or solid phase separation methods, there could be an apparent lack of interfering effect of endogenously occurring antibodies.

Measurement of free thyroid hormones in serum by column adsorption chromatography and radioimmunoassay.

A new method for the assay of free thyroid hormones in human serum is described. This method is based on a chromatographic adsorption process of thyroid hormones onto a Sephadex LH-20 resin column. Protein fractions are eliminated by washing columns, adsorbed hormones are eluted with methanol and determined by radioimmunoassay. It was demonstrated that under the experimental conditions adopted the presence of the resin R does not significantly change the free hormone level in the serum, and the amount of hormone adsorbed onto the resin HR is exclusively in function of the free hormone concentration [H], according to a linear relationship: HR = phi [H], where phi is the resin adsorption constant K ads multiplied by the number of resin binding sites nR. The phi value, experimentally determined, was 32 ml for T3 and 58 ml for T4, when 150 mg resin were used. The method sensitivity was 0.3 pg/ml for FT3 and 0.6 pg/ml for FT4. The within-assay reproducibility was about 5% (CV) and the between-assay reproducibility was about 6% (CV), both for FT3 and FT4. FT3 and FT4 levels, in 96 normal subjects, were 3.9 +/- 0.7 pg/ml (mean +/- SD) and 11.1 +/- 1.9 pg/ml (mean +/- SD) respectively.