Sequential adsorption analysis of antibodies to neuroectodermal cells by an indirect quantitative method.

An indirect radioimmunoassay was developed for the sequential adsorption analysis of rabbit antibodies raised against rat neuroectodermal cells. The quantitativee relationship between primary adsorbed rabbit antitumor antibodies and secondary adsorbed goat antibodies to rabbit IgG was explored by paired radioiodine analysis. In the final indirect method, the amount of unlabeled rabbit antibody removed by cultured monolayers of cells at each step in a sequence of cells could be determined from an equation relating the unlabeled amount to values of bound 125I-labeled goat antirabbit IgG. To obtain the total quantity of rabbit antibody in a particular antiserum reagent, a sequential adsorption analysis was done with successive steps of homologous cells. To obtain the amount of antibody that was cross-reactive for other cell lines, we included those lines in the first several steps of the sequence. The sequential adsorption profile was considered as a more important indicator of the quality of a particular antiserum reagent than was the total amount of antibody contained in it. Neuroectodermal cell lines used as illustrative examples included subclones of the C6 astrocytoma and of the RN-2 schwannoma.

Measurement of DNA-binding immunoglobulins in systemic lupus erythematosus.

A radioimmunoadsorbant assay for quantitating DNA antibody in the IgM, IgG, and IgA classes is described. The method was standardized allowing expression of results in absolute terms. Effects of rheumatoid factor on the results were investigated. The new assay was found to correlate well with the Farr assay for DNA antibody. Serum levels of DNA antibody in all three immunoglobulin classes reflected the degree of clinical activity of SLE.

Immunoradiometric assay for cytomegalovirus-specific IgG antibodies: assay development and evaluation in blood transfusion practice.

An immunoradiometric assay (radio-immunosorbent test; RIST) for the detection of IgG antibodies to human herpesvirus 4 [human cytomegalovirus (CMV)] has been developed. The technique utilizes CMV antigen passively adsorbed to a polyvinyl microtitration plate and a radiolabelled murine monoclonal anti-human IgG antibody to detect binding of human antibody to the 'solid phase' reagent. The assay was optimized, and its specificity confirmed by testing paired acute and convalescent sera from patients with acute CMV or other human herpesvirus infections. To determine the assay's sensitivity 1433 blood donor sera were examined. The RIST was more sensitive than a standard complement fixation (CFT), in that 53% of these sera were positive by RIST and 48% positive by CFT. There were 1303 concordant results, 88 sera positive only by RIST and 19 sera were only positive by CFT. These discrepant results remained after an attempt to exclude false positive reactivity; their significance is discussed. Use of a monoclonal anti-human IgG antibody in the RIST reduced non-specific binding to the control uninfected cell antigen such that blood donor sera could be tested in the assay using only a CMV antigen without generating an unacceptable false positive rate.

Determination of total IgE by ELISA in tubes and plates compared with PRIST.

IgE was measured by ELISA in tubes and in microtiter plates, the results being compared with PRIST data. The recommended readings of the tube contents in a spectrophotometer (SPM) were compared with results using a multi-channel photometer (MCP). Geometric mean values (International Units) and standard deviations of 44 normal sera examined by the 3 different methods were: PRIST 16.2 +/- 4.0; SPM 15.6 +/- 4.9 and MCP 18.4 +/- 4.4. Correlation coefficients were: PRIST-SPM r = 0.98; PRIST-MCP r = 0.98; and SPM-MCP r = 0.97. Intra- and inter-assay coefficients of variation were smaller for MCP than for SPM. In addition, reading in microtiter plates was much faster, while having little effect on sensitivity.

Simple and rapid radioimmunoassay for the routine determination of vasopressin in plasma.

A simple, rapid and sensitive radioimmunoassay for plasma arginine-vasopressin (AVP) has been developed for routine use. AVP is first extracted from plasma with use of an octadecasilyl silica cartridge. The mean (+/- SEM) recovery is 73.1 +/- 2.1% (n = 24). The antibody and the 125I-AVP are both obtained from commercial sources. Following a 48 h incubation time, bound and free fractions of AVP are separated by dextran-charcoal. The reproducibility of the method is acceptable (between- and within-assay CV of 9.5 and 7.6%). This technique allows the detection of 0.39 pg/tube of AVP. This assay is applicable to determination of human plasma AVP levels; mean (+/- SEM) plasma AVP levels in normal human subjects in standing or sitting positions, or after an oral water load, were respectively 5.2 +/- 0.7, 3.6 +/- 0.4 and 2.7 +/- 0.4 pg/mL. This method has also been validated by determinations of plasma AVP levels in rabbits and hamsters in various conditions. The commercial availability of the antibody and radioactive AVP, and the simplicity of the method, make this technique suitable for clinical and research purposes.

A modified sandwich technique for the measurement of IgE.

The paper radioimmunosorbent test (PRIST) has been described as the method of choice for determining low serum IgE levels; the radioimmunosorbent test (RIST) as the method of choice for determining normal and elevated serum IgE levels. By replacing the 125I-labelled anti-IgE antibody used in the paper radioimmunosorbent test by the 125I-labelled anti-IgE reagent used in radioallergosorbent test (RAST) and by changing the serum dilution and the incubation time, this modified sandwich technique (MST) became comparable to the RIST in the normal and elevated IgE-region and showed results similar to the PRIST in the very low IgE-region. The affinity of the 125I-labelled anti-IgE of the RAST proved to be about 2.6 times higher than the antibody used in the PRIST, which explains the improved results in the normal and the good results in the very low IgE-region. The lowest serum IgE level measurable by this method was as low as 0.05 I.U./ml, as determined in 20 cord sera. The mean IgE level in cord sera was 0.45 I.U./ml (range 0.05--2.63 I.U./ml). The results of this study suggest replacement of the antiserum used in PRIST by the one available for RAST.

Reutilization of 125I-labelled anti-IgE antibody and paper discs in PRIST and RAST IgE determination.

In the paper radioimmunosorbent test (PRIST) anti-human IgE coupled paper discs are used for the estimation of total IgE in blood serum: in the radioallergosorbent test (RAST) allergen-coupled paper discs are used for the estimation of specific IgE in blood serum. The bound IgE or specific IgE is quantified by a 125I-labelled anti-human IgE. The non-bound 125I-labelled anti-human IgE can be collected and used in new assay. By a 4-h incubation of the used paper discs with 1 M glycine-HCl buffer (pH 2.7) the IgE-labelled anti-IgE complex can largely be removed. The paper discs treated in this manner can be used in a new assay.

Optimum conditions for the radioimmunoprecipitation assay for the major internal protein of bovine leukemia virus.

The sensitivity of the radioimmunoprecipitation assay for the major internal protein (p24) of bovine leukemia virus (BLV) was examined. Conditions were varied for the assay by 1) using larger amounts of serum and 2) increasing the amount of second antibody. Sensitivity for the p24 radioimmunoprecipitation test was greatest when 10 microliter of test serum and 200 microliter of rabbit anti-bovine IgG were used. Under these conditions there were no discrepancies between the p24 radioimmunoprecipitation test and the radioimmunoprecipitation test for the surface glycoprotein (gp51) in 380 cattle from commercial dairy herds.

Determination of beta2-microglobulin in biological samples using an immunoenzymometric assay (chemiluminescence detection) or an immunoturbidimetric assay: comparison with a radioimmunoassay.

Monitoring beta2-microglobulin (beta2M) in biological fluids has gained considerable interest in pathologies such as haematologic malignancies, renal diseases, and chronic inflammatory diseases. Due to limitations of the RIA in the routine laboratory, we measure beta2M with non-isotopic methods. 189 patients suffering from myeloma (n=66), end stage renal failure (n=54) or inflammation (n=69) were included in this study. beta2M was determined in serum, urine and dialysate using an immunoenzymometric assay with chemiluminescence detection [Immulite Diagnostic Products Corporation (DPC), La Garenne Colombes, France] and an immunoturbidimetric assay (Olympus, Rungis, France). The data were compared with a radioimmunoassay (Immunotech, Marseille, France) taken as a reference. Using serum samples, the immunoenzymometric assay with chemiluminescence detection and the immunoturbidimetric assay have reliable analytical performances. Values obtained with serum samples are highly correlated with the radioimmunoassay (DPC/RIA r2=0.84; Olympus/RIA r2=0.94) whatever the type of pathology; however an over-estimation which could be related to cross reactivity with beta2M fragments was observed with the RIA method as suggested by crossover calibration and recovery studies. Values obtained with urinary samples (n=96) are closely related to those obtained with the RIA (DPC/RIA r2 = 0.98; Olympus/RIA: r2=0.99). Despite the low levels observed in dialysate (n=57) good correlations were observed between Olympus vs DPC (r2=0.85). By contrast, the two non-isotope methods are poorly related with the RIA method (DPC vs RIA r2=0.47 and Olympus vs RIA r2=0.54). In conclusion, the immunoenzymometric assay with chemiluminescence detection or the immunoturbidimetric assay could be used in the routine laboratory in order to determine beta2M in plasma, urine and dialysate.

Diagnostic efficacy of in vitro methods vs. skin testing in patients with inhalant allergies.

The purpose of our study was to investigate the diagnostic efficacy of two selected methods of in vitro allergy testing. Specifically, the PRIST/modified RAST I125 isotope systems and the Quantizyme/modified EAST alkaline phosphatase method were compared. The time, expense, convenience, and diagnostic efficacy of the two procedures are discussed. Special attention is given to the practicality of each method for the practicing physician.

Use of insulin immunoassays in clinical studies involving rapid-acting insulin analogues: Bi-insulin IRMA preliminary assessment.

BACKGROUND: In clinical studies involving rapid-acting analogues (RAAs), insulin immunoreactivity is frequently measured, including endogenous, regular insulin (RI) and RAA immunoreactivities. Such a procedure implies equivalent cross-reactivities of all insulins present in serum. Commercially available human insulin immunoassays have been widely used, but their limitations (including hemolysis and anti-insulin antibodies) were not fully investigated. The aims of our study were to compare cross-reactivities of RI and RAAs in buffer and in serum and to investigate insulin immunoassay pitfalls. METHODS: Cross-reactivities were assessed using Bi-insulin IRMA (Schering Cis-Bio International) in phosphate-buffered saline (PBS)-1% bovine serum albumin (BSA) and in pools of sera spiked with RI and RAAs (lispro and aspart). To investigate the influence of hemolysis, a pool of sera spiked with RAA was mixed with a concentrated hemolysate (final hemoglobin concentration 10 g/L) and incubated for 3 h at room temperature. To determine interference by anti-insulin antibodies, insulin was removed using charcoal from 18 sera with anti-insulin antibodies and from 17 sera without detectable anti-insulin antibodies. These insulin-free samples were then spiked with RI and RAAs and the immunoreactivity was determined. RESULTS: Compared with buffer, cross-reactivity in serum for RI, lispro and aspart was lower (35%, 29% and 26% lower, respectively). Hemolysis degraded almost all RI and RAAs contained in the serum (>or=95%). Anti-insulin antibody interference was significant for RI and RAAs (p

[RIST or PRIST?]. / RIST oder PRIST?

In 240 patients the radioimmunosorbent (RIST) and paper-radioimmunosorbent (PRIST) tests were used parallel for determination of serum IgE levels. Whilst the values achieved with PRIST generally were somewhat lower than those with RIST, a very good correlation was found between these two methods. Taking its procedural simplicity into account, PRIST thus appears better suited for routine diagnostic purposes than RIST.

[IgE concentration in healthy children of various age groups]. / Stezenie IgE u dzieci zdrowych w róznych grupach wiekowych.

Serum IgE was estimated in 181 healthy children aged 2 months-17 years. The concentration of IgE was measured by radioimmunosorbent technique (RIST). To eliminate serum interference the IgE levels were calculated using the correction factors: 0,96 and 0,91 (for the sera diluted respectively 1:10 and 1:5). Statistical calculations were made after logarithmic transformation of the particular results. On the base of the mean geometric values linear and parabolic regression lines have been made. On the grounds of the received regression equations it has been found that parabolic variant of the changes of the IgE concentration with respect to age, shows more accurately the real state. The index of the parabolic correlation (r = 0,98) shows a close and positive relations between IgE concentration and the age of children. The lowest concentrations of IgE were found in the youngest group of infants and then the level increased during childhood. The parabolic regression line and 95% confidence limits for the geometric mean values ar inserted in the paper.

[Differences in the levels of serum immunoglobulin E determined by the competitive radio-immuno-sorbent and the paper-radioimmuno-sorbent methods: Removal of these differences by pretreatment of sera with polyethylene glycol (author's transl)]. / Unterschiede für Immunglobulin E im Serum, bestimmt mit kompetitivem bzw. Papier-Radio-immunosorbenttest: Beseitigung durch Vorbehandlung mit Polyethylenglycol.

The serum IgE of patients with chronic nonspecific lung diseases was determined with the competitive radio-immuno-sorbent and the paper-radio-immuno-sorbent methods (Phadebas). Some samples showed markedly higher values with the competitive radio-immuno-sorbent than with the paper-radio-immuno-sorbent method. These discrepancies were abolished by pretreatment of the sera: polyethylene glycol 6000 (final concentration 35 g/l) was added, followed by centrifugation at 0 degrees C for 30 min at 20 000 g, and IgE was determined in the supernatant. This treatment had no effect on the values of the paper-radio-immuno-sorbent method, but the values of the competitive radio-immuno-sorbent method were decreased so that the two values agreed within the experimental confidence limits.