C. François-Gérard

An enzyme immunoassay for prostate-specific p30 antigen detection in the postcoital vaginal tract

A sandwich enzyme immunoassay test utilising a mouse monoclonal antibody has been adapted for the sensitive detection of the p30 prostatic antigen in semen and in postcoital vaginal swabs. In liquid semen, p30 was still detectable at a 1/1,000,000 dilution, and it could still be detected on vaginal swabs 24 h postcoitus.

Influence of affinity of antibodies upon their detection by liquid phase radiobinding assay and solid phase enzyme linked immunosorbent assay. Demonstration using monoclonal antibodies raised against rDNA human proinsulin

SummaryHybridomas producing proinsulin antibodies were cloned by limiting dilution of cell cultures obtained by fusion of splenocytes of immunized mice with immortal myeloma cells. Some proinsulin monoclonal antibodies crossreacted with labelled insulin but none did with labelled C-peptide indicating that the involved epitopes were at one of the insulin/C-peptide junctions or included in the insulin moiety. Hybridoma supernatants were assayed for IgG concentration by a solid phase assay and for ligand binding by a radiobinding assay and an enzyme linked immunosorbent assay. The half-life of immune complexes formed with radioligand was measured and, as expected, correlated with affinity as measured by the method of Scatchard. Antibody titres determined by enzyme linked immunosorbent assay did not correlate to those measured by radiobinding assay. IgG concentration correlated to enzyme linked immunosorbent assay titres but not to radiobinding assay titres. Finally, a significant correlation was found between radiobinding assay titre and the product of enzyme linked immunosorbent assay titre by the period of immune complexes. It is concluded that, except for very low affinity antibodies, enzyme linked immunosorbent assay is a capacity assay whereas radiobinding assay is influenced by both antibody concentration and affinity. The former assay is thus best suited to detecting low affinity antibodies whereas the latter is more efficient in the presence of low levels of high affinity antibodies.

Semi-quantitative assessment of anti-insulin total IgG and IgG sub-classes in insulin-immunized patients using a highly sensitive immunochemical micromethod

SummaryAn immunochemical micromethod was designed to estimate total IgG and IgG sub-classes of anti-insulin antibodies in immunized diabetic patients. Insulin, immobilized on a solid phase, was allowed to react with serum samples containing anti-insulin antibodies. Bound anti-insulin IgG interacted with mouse monoclonal antibodies specific for total IgG or for each IgG isotype. The fixation of mouse monoclonal antibody was subsequently detected using a horseradish peroxidase-conjugated rabbit anti-mouse IgG in the presence of a chromogenic substrate. The test was standardized by an immunocapture assay utilizing coated rabbit anti-human IgG and known concentrations of purified human myelomatous proteins of each sub-class. Results of anti-insulin IgG and anti-insulin IgG sub-classes assay could therefore be expressed in ng equivalent myelomatous proteins per ml of serum. Analysis of serum samples from 24 insulin-immunized diabetic patients revealed a quasi absence of IgG2 anti-insulin antibodies and an increase of the relative abundance of the other three anti-insulin IgG isotypes. In our series, anti-insulin IgG1 was predominant, followed by IgG3 (in 17/24 patients) or IgG4 (in 7/24). Insulin immunization was deduced to be of polyclonal nature, the isotype pattern of which is not representative of the relative proportion of IgG sub-classes in whole normal serum.

Detection of p30 antigen in sexual assault case material

A sandwich enzyme immunoassay for the detection of p30 prostate specific antigen was applied in 52 forensic cases. In each case, immunoassay results were compared with those of the search for spermatozoa and prostatic acid phosphatase analysis. The results indicate that the p30 assay gave no false positive and fewer false negative reactions than the acid phosphatase test. The sensitivities compared to the search for spermatozoa as a reference method were 94.8% for the p30 assay and 84.4% for the acid phosphatase test; the specificities were 95.6% and 90.0% respectively.

Characterization of a neuraminidase from Corynebacterium aquaticum responsible for Th polyagglutination

Abstract. Th polyagglutinability is characterized by the agglutination of the red blood cells (RBC) by Arachis hypogaea, Medicago disciformis, Vicia cretica but, in contrast to the T phenomenon, not by Glycine max (Glycine soja). Because Th transformation of RBC has been obtained in vitro, the mechanism of Th polyagglutinability expression has been studied and reproduced experimentally. An enzyme with neuraminidase specificity has been isolated from the culture supernatant of Corynebacterium aquaticum, and further characterized (MW = 55,600 kDa, pH = 5.5, Km = 0.138 μM, Kcat = 0.22 μg). Reversely, Th transformation of RBC could be obtained by using other neuraminidases but in very mild conditions of hydrolysis. From our results, it can be concluded that by the release of less than 20 μg of sialic acid per 1010 RBC, Th reactivity can be induced whereas hydrolysis of greater amounts of sialic acid (>20 μg/1010 RBC) give the classical T polyagglutinability.

Elucidation of non-parallel EIA curves

Quantitative determinations by EIA can be only obtained by reverse regression when linear portions of sample and standard curves are parallel. However, analysis of complex biological fluids often yields sigmoid curves displaying lower slopes, thus invalidating any quantitative interpretation. We hypothesized that this phenomenon was due to a competition effect between the target (for example an antigen) and related molecules for the binding sites (for example a capture antibody) immobilized onto the solid phase. This has been confirmed experimentally using various target-to-competitor ratios and formulated as a mathematical model. The slope decrease in target detection was related to the proportion of competitor, not in a linear, but in an exponential manner. This mathematical model has been computerized and can be used to correct aberrant sample curves provided the relevant parameters have been previously determined in the same systems.

Clonally restricted insulin autoantibodies in a cohort of 2200 healthy blood donors

SummarySerum samples of 2200 blood donors were screened for anti-insulin IgG by enzyme-linked immunosorbent assay. Specificity of detected antibodies was verified by competition with an excess of insulin and observation that saturated anti-insulin IgG were no longer measurable. The upper limit of measured signal in the population was defined as the mean plus three SD. In the direct assay, 32 sera were positive. Among these, 22 (1%) contained saturable insulin antibodies (true positive) and 10 were non-saturable and considered as non-insulin-specific. The positive blood donors were requested to answer a questionnaire and according to their answers, none had ever received insulin, was a first degree relative of a Type 1 (insulin-dependent) diabetic patient or had experienced a hypoglycaemic episode. None of the 22 true positive sera detected by enzyme-immunosorbent assay bound 125-I-insulin to any significant extent. The nine sera yielding the highest signal were further characterized with regard to heavy and light chains as well as species specificity of ligand. Anti-insulin IgG from healthy blood donors contained only one heavy (γ1 2/9; γ3 7/9) and one light (κ 8/9; λ 1/9) chain. Three sera were human insulin specific; two were non-species-specific; the other four bound insulin in the order human = porcine > bovine. These results indicate that low affinity clonally restricted antibodies against insulin are present in unselected blood donors with a prevalence of 1%.

A double-sandwich ELISA test for Glm(3) phenotyping of bloodstains with a mouse monoclonal antibody

Abstract ELISA procedures have been developed for the Gm(3) allotypic marker of human IgGI immunoglobulins, using a mouse monoclonal anti-Gm(3) antibody. The test has a high sensitivity since the monoclonal reagent, even diluted 1/32,000, still detects the Gm(3) antigen in serum samples diluted 1/128,000 to 1/2,000,000. On account of its sensitivity and its objective reading, the test is well adapted for bloodstain typing. The addition of a parallel test for IgG makes it possible to distinguish negative results due to Gm(−3) phenotypes from those due to a lack of IgG material. Gm(3) and IgG were detected in bloodstains more than 5 years old.

Absence of seroconversion in a PCR positive person 18 months after transfusion of HIV infected blood

The donor, a 25-year-old man was a regular blood donor who has been tested for HIV 1 antibodies and found to be negative till September 1987. Three months later, in December 1987, this donor had seroconverted. Much later, we heard that homosexual contacts, dated July 1987, were most probably at the origin of the HIV 1 contamination. In December 1987, no antigen could be detected in his serum by using the EIA neutralization test from Abbott. 7 months after contamination, in January 1988, the T4/T8 ratio of the donors lymphocytes was as low as 0.28 with a total T4 cell count of 657c/mm 3 . Further investigation of the donors immune function showed that lymphoblastic stimulation was lowered with PHA and PKW, whereas it was in the normal range with OKT3 as mitogen.

Application à la sérologie transfusionnelle du caractère antigénique P1 du blanc d'œuf de pigeon

Summary An antigen cross-reacting with the human blood group P1 has been discovered in turtle-dove and pigeons blood and egg-white. In the egg-white, The P1 antigenicity is carried by a glycoprotein called ovomucoid which is particularly rich in galactose residues. Turtle-dove ovomocoid is an acidic glycoprotein homogeneous in size but heterogeneous in charge. The protein consists in fact of five components whose isoelectric points are ranging from 3,6 to 4,1 and possessing all a strong P1 activity. Turtle-dove and pigeon ovomucoids are both monospecific anti-P1 inhibitors as it has been established by inhibiting the hemagglutination of a panel of anti-erythrocyte antibodies. This paper describes the use of ovomucoid as specific anti-P1 inhibitor in the cases where two or more antibodies must be elucidated in the serum of polyimmunized people. On account of its velocity and efficiency, the use of that new soluble blood group substance will probably and advantageously supplant the classical method of absorption with erythrocytes.