Prevalence of latex allergy in spina bifida: genetic and environmental risk factors.

AIM OF STUDY: To evaluate the prevalence of latex allergy in a population of children with spina bifida (SB) and to assess the role of early exposure to latex products and others risk factors. INTRODUCTION: SB is related with an higher incidence of latex allergic reactions. These patients received repeated surgical procedures, implant of latex-containing materials and catheterization. MATERIALS AND METHODS: Eighty consecutive subjects affected with SB besides answering a questionnaire, underwent a skin-prick test (SPT) to latex and the determination of the specific serum IgE (RAST CAP) to latex. 40% (32/80) of the patients showed a latex sensitization with specific IgE > 0.7 kU/I but only twelve of the 32 sensitized patients (40%) suffered from clinical reactions to latex (urticaria, conjunctivitis, angioedema, rhinitis, bronchial asthma). Number of surgical procedures, but particularly early exposure to latex and familiarity for allergy are correlated with latex allergy (p < 0.01). CONCLUSION: Latex allergy in SB children is multifactorial situation related with a disease-associated propensity for latex sensitization, early exposure and number of surgical procedures. Prophylactic measures to avoid the exposure, not only in the sanitary environment, through the institution of latex-safe routes and every day, prevent potentially serious allergic reactions.

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.

[Diagnosis of type I hypersensitivity by laboratory test]. / Diagnostic au laboratoire de l'hypersensibilite immediate.

Type I hypersensitivity is a major problem in public health, often requiring numerous investigations which aim to diagnose atopy and identify the causative allergen. Among these investigations, several blood tests, mostly using immunoenzymatic methods, can be performed to measure total and specific IgE levels. Type I hypersensitivity can also be investigated with assays which quantitate several mediators released after cellular activation induced by allergens.

Human antibodies to immunoglobulin A (IgA). A radioimmunological method for differentiation between anti-IgA antibodies and IgA in the serum of IgA deficient individuals.

In the sera of 12 out of 27 individuals with IgA deficiency (serum level below 0.02 mg IgA/ml) class-specific anti-IgA antibodies were demonstrated by haemagglutination. These sera showed false-positive results in a solid-phase inhibition radioimmunoassay (RIST) (apparent IgA concentration between 0.6 and 13.7 microgram IgA/ml) indicating that the RIST is not an appropriate test for the analysis of serum of IgA deficient individuals. A modification of the RIST is proposed (titration RIA) that permits differentiation between low levels of IgA and class-specific anti-IgA antibodies. With this test IgA deficient individuals could be classified as those with low but detectable levels of IgA and those with class-specific anti-IgA antibodies. A computer procedure was developed to calculate both the amount and the avidity (K) of the anti-IgA antibodies and to simulate the assay system. The K value calculated from experimental points proved to be an overestimation of the K value which fitted most adequately in the simulation. The comparison of the results with clinical findings indicated a possible correlation between the amount and the avidity of the anti-IgA antibodies and the appearance of anaphylactic reactions after transfusion of IgA.

A paper radioimmunosorbent test (PRIST) for the detection of larva-specific antibodies to Toxocara canis in human sera.

A paper radioimmunosorbent test (PRIST) was shown to be sensitive and reproducible when used with excretory/secretory antigen of Toxocara canis second stage larvae. Whatman No. 50 filter paper (5 mm discs) gave the most consistent and clear results with antigen at a concentration of 100 micrograms/ml, and could be stored for up to 3 weeks in vacuo at -70 degrees C. Antigen coated discs were incubated with test sera at 1:10 dilution for 3 h at room temperature (21 degrees C), reacted with [125I]anti-human IgG for 1 h and counts determined in a gamma counter. Sera from patients with fascioliasis, taeniasis, schistosomiasis, oxyuriasis, trichinellosis and ancyclostomiasis gave counts similar to cord serum controls. Sera from patients with ascariasis gave counts of up to twice as great as controls, but sera from patients with toxicariasis produced counts of 7,000-13,000, a 4-6-fold increase.

A solid-phase fluoroimmunoassay for human IgE.

A fluoroimmunoassay (FIA) for the measurement of immunoglobulin E (IgE) is described. The method involves a sandwich technique in which antiserum to human IgE is adsorbed on a cellulose acetate/nitrate disc which is attached to a plastic StiQTM sampler. The prepared sampler is reacted with serum and antigen is bound specifically. After buffer wash and treatment with goat serum to block non-specific binding, the samplers are reacted with monospecific fluorescein conjugated antisera to human IgE. Two buffer washes remove unbound material and the fluorescence which is directly proportional to the IgE concentration is measured in a FIAX fluorometer. Assay standards range from 2 to 400 IU/ml. The method gives within run coefficients of variation (CV) from 3.3 to 8.4% and between run CV from 6.2 to 16.1% being less precise at low analyte concentrations. IgE concentrations of a group of 74 sera determined by FIA using StiQsTM prepared with antisera from 2 sources correlated well with results found by radioimmunoassay.

Characterization of metabolically labelled antigen following immunoprecipitation with antibody bound on ELISA plates.

Immunoprecipitation is a powerful technique for the immunochemical characterization of antigens. In combination with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) a number of features, e.g., presence of antigen, rate of synthesis, relative molecular weight of the polypeptide chain or post-translational modifications can be determined. Four different steps are basically involved in the immunoprecipitation procedure: (1) metabolic labelling of the antigen by incubation of viable cells with a radioactive precursor, (2) harvesting of the labelled antigen from the cells by lysis or in the case of secretory proteins from the supernatant, (3) formation and (4) purification of antibody-antigen complexes. The last step relies on secondary agents which bind to the antibody.

Quantitative measurement of human immunoglobulin E using monoclonal antibodies to distinct epitopes.

Monoclonal antibodies (MAb) which recognize distinct epitopes on human immunoglobulin E have been used to develop two-site sandwich radio- and enzyme-linked immunoassays for the quantitation of human IgE. In the first step, a purified anti-IgE MAb coated to polyvinyl or polystyrene microtiter plates specifically bound the IgE contained in the samples. In the second step, another anti-IgE MAb (either iodinated or conjugated to beta-galactosidase) directed to a different antigenic determinant was used to estimate the amount of bound IgE. This simple method permitted the determination of IgE concentrations of 10 ng/ml and greater in about 3 h. Coefficients of variation on a single day did not exceed 7.5% for IgE levels, covering a wide range of the standard curve. The values obtained on serum samples showed a good correlation with those obtained using the paper radioimmunosorbent test (PRIST).

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.

Slide immunoenzymatic assay for human IgE (SIA-IgE).

A rapid and inexpensive method is described for determination of human serum IgE using 5-microliter samples. The slide immunoenzymatic assay for IgE (SIA-IgE) is carried out on a glass slide in which up to 24 samples can be tested including controls and standards; up to four of these slides can be read automatically in conventional vertical beam readers. Flat circles on the slide are covered with a layer of biotinylated antibody specific for human IgE (trapping antibody). Five microliters of serum sample is dropped to cover each circle, and the slide is incubated. The circles are washed with water, dried, incubated under 10 microliters of enzyme-labeled antibody to human IgE, then washed again, and covered with 10 microliters of enzyme substrate. The intensity of color generated is measured at the proper wavelength. The method is simple, accurate and non-radioactive and can be completed within 2 h.

Evidence of a high-activity C type of carbonic anhydrase in human ciliary processes.

Carbonic anhydrase activity was found in the ciliary process of fresh human donor eyes, originating from an enzyme antigenically similar to the erythrocyte high-activity isoenzyme HCA C. It was sensitive to inhibition by acetazolamide and resistant to inhibition by halides like HCA C. The enzyme is probably identical with HCA C. Its tissue concentration was one fifth to one tenth of that in the human kidney. The erythrocyte low-activity isoenzyme HCA B was also found in the processes as a contaminant.

Two-site assay of bovine parathyroid hormone.

A two-site assay has been developed for bovine PTH. This technique involves reaction of the antigen with two antibody molecules with the purpose of increasing specificity. In paratice the hormone was extracted form plasma samples on to plastic tubes coated with antibody specific for PTH (1-34). Uptake was then measured using 125I-labelled antibodies specific for PTH (53-84). In this way a sensitive assay was obtained for PTH (1-84) which did not recognize molecular fragments. This technique was used in conjunction with immunoradiometric assays specific for either NH2-terminal (1-34) or CO2H-terminal (53-84) molecular fragments to study the clearance of infused PTH in the cow. Preliminary results support the hypothesis that the intact molecule is rapidly degraded in the peripheral circulation with the preferential disappearance of an NH2-terminal fragment. Studies on endogenous secretion during calcium and EDTA infusions indicated that there was little intact hormone present at the times when CO2H-terminal immunoreactivity was readily measured.

The occurrence of immunoglobulin E in Human seminal plasma.

The presence of IgE in human seminal plasma has been explored using two different radioimmunoassay methods. Samples were obtained from 84 patients under investigation for involuntary infertility. Low levels were recorded in seminal plasma (less than 0.05-805 kIU/1) together with a wide scatter of serum values (less than 0.5-956 kIU/1). The incidence of detectable seminal plasma IgE was related to high serum IgE values (P less than 0.001; chi 2-test). Among men reporting allergy problems and who has serum IgE levels above 50 kIU/1 detectable semen IgE was seen in 15 out of 18 patients. Sperm agglutination was observed in 18 men of whom 11 had detectable seminal plasma IgE. The question of whether or not seminal plasma IgE is a plasma transudate or is locally produced as well as its possible function in seminal plasma is discussed.

Detection of false seroconversions in cytomegalovirus seronegative platelet donors by serial complement fixation or IgG-specific radioimmunosorbent tests.

A panel of 421 cytomegalovirus (CMV) seronegative-platelet donors was followed using complement-fixation (CFT) and IgG-specific radioimmunosorbent tests (RIST) to detect seroconversion. During 4623 person-months of observation 2452 serum specimens were tested, and the annual rate of seroreactivity detected by RIST was 56%. However, most of the positive RIST results were followed by negative results with later serum samples. CFT seroreactivity was a poor predictor of RIST seroreactivity. The high rate of transient seroreactivity detected by RIST may have reflected the lack of absolute specificity of the test. Alternatively, the results could illustrate the difficulty of determining whether an individual harbours latent CMV infection using antibody tests when some seronegative individuals appear to be latently infected. In the light of our observations recommendations for CMV antibody testing of blood donors are proposed.

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.

Protein A as an immunoadsorbent in radioimmunoassay: usefulness in a rapid assay for urinary luteinizing hormone.

A staphylococcal cell wall protein, designated protein A, is known specifically and rapidly to bind to immunoglobulin G. We have utilized this protein as an immunoadsorbent to separate antigen-antibody complexes from free antigen in a rapid 6-hour radioimmunoassay for urinary human luteinizing hormone (hLH). We have further documented the efficacy of this system and the optimal conditions for this assay. The ability of this assay to provide rapid and reliable measurements of hLH in timed urinary specimens provides a useful tool for determination of the preovulatory LH surge and prediction of the time of ovulation in women.

Serum carbonic anhydrase III in neuromuscular disorders and in healthy persons after a long-distance run.

A radioimmunosorbent technique was used for the assay of the skeletal muscle specific enzyme, carbonic anhydrase III (CA III). The usefulness of serum CA III determinations for detecting skeletal muscle damage was evaluated by comparing the serum levels of this enzyme and of myoglobin and creatine kinase in 64 patients with neuromuscular disorders and in 13 healthy volunteers before and after a long-distance run. Increased serum CA III levels were found in all patients with muscular dystrophy, chronic polymyositis and amyotrophic lateral sclerosis and in many with myasthenia gravis. In patients with polymyositis who were followed up with repeated blood sampling, the time courses of serum CA III levels, myoglobin levels and clinical symptoms were closely related. In all the runners the serum CA III level immediately after the run was increased. In the present study serum CA III and myoglobin seemed to be equally sensitive as biochemical markers of muscular damage and more sensitive than creatine kinase.

Comparison of serum IgE determination by conventional radioimmunoassay (RIST) and by a modified (sandwich) technique (PRIST).

It has been reported that the sandwich technique (PRIST) is a more accurate method for determining serum IgE levels than the conventional radioimmunoassay (RIST), especially for low IgE levels. By using half the volumes prescribed for RIST and by introducing a 3-h preincubation step at room temperature, IgE values in the range of 30-4000 I.U./ml could accurately be established. It is concluded that the proposed modification may replace the conventional RIST for routine purposes, because it offers comparable accuracy, is more exonomical, and provides better results in the low IgE range. Discordant results between RIST and PRIST cannot be correctly interpreted, because interfering serum factors may cause either exaggerated IgE values with RIST, or falsely low values with PRIST.