Study of β‐casein denaturation by microparticle‐enhanced nephelometric immunoassay

Rabbit anti‐native bovine ß‐casein antiserum reacted with native ß‐casein and fragments f( 1–105/7) and f( 106–209) formed during ß‐casein proteolysis by plasmin. Agglutination of ß‐casein‐coated microparticles by anti‐native ß‐casein antiserum was weakly inhibited by ß‐casein f(1–105/7) and ß‐casein f( 106–209) (0·04 and 1·4%, respectively, compared with native ß‐casein). Immunoreactivity of these ß‐casein peptides in microparticle‐enhanced nephelometric immunoassay was more preserved in the whole ß‐casein than in its isolated fragments. The protein concentration producing 50% inhibition of the ß‐casein‐coated microparticle agglutination with anti‐native ß‐casein antiserum increased during ß‐casein denaturation. A microparticle‐enhanced nephelometric immunoassay, quantifying changes of this inhibiting protein concentration, permitted detection of alteration of the immunoreactivity of ß‐casein during its plasmin proteolysis and heat denaturation, providing an adequate test for the integrity of the whole molecule.     

Priming effect of RANTES on eosinophil oxidative metabolism

Background RANTES has been shown to possess chemotactic activity for eosinophils, which have also been considered to play a role in allergic inflammation through reactive oxygen species. Thus, in this study, we examined the effect of RANTES on radical oxygen products from eosinophils.
Methods Purified eosinophils by CD16-negative selection or an eosinophilic cell line (EoL-1) were incubated with or without RANTES (2.5 x 10⁻⁶). To the mixture of eosinophils and luminol, calcium ionophore (A23187) or opsonized zymosan (OZ) was added, and radical oxygen products were determined by luminol-dependent chemiluminescence for 600 s.
Results Eosinophil-mediated radical oxygen products of untreated eosinophils produced with A23187 gave a peak value of 14.09 + 2.40 (mean±SE, n = 12) relative light units (RLU) and an integrated value of 3232.20 + 513.09 RLU. However, with treatment with RANTES, a peak value of 18.66 + 2.40 RLU and an integrated value of 5301.05 ±561.02 RLU were obtained. Eosinophil oxidative metabolism-induced A23187 or OZ was apparently augmented by the preincubation with RANTES. In addition, the radical oxygen products of EoL-1 showed similar results.
Conclusions Thus, we concluded that RANTES may play an important role the pathogenesis of allergic inflammation through its involvement in eosinophil activation, as evidenced by oxygen products, as well as in selective eosinophil infiltration as selective eosinophil chemoattractant.     

Simultaneous quantitation of diphtheria and tetanus antibodies by double antigen, time-resolved fluorescence immunoassay

A dual, double antigen, time-resolved fluorescence immunoassay (DELFIA) for the simultaneous detection and quantitation of diphtheria (D) and tetanus (T) antibodies in sera has been developed. In the double antigen format one arm of the antibody binds to antigen coated microtitre wells and the other arm binds to labelled antigen to provide a fluorescent signal. This assay was found to be functionally specific for IgG antibodies and showed a good correlation with established toxin neutralization assays. Furthermore, the double antigen set-up was species independent, permitting the direct use of existing international references of animal origin to measure protective antibody levels in humans in international units (IU/ml). The detection limit corresponded to 0.0003 IU/ml with Eu³⁺-labelled toxoids and to 0.0035 IU/ml using Sm³⁺-labelled toxoids. The assay was fast with a high capacity making it a suitable method for serological surveillance studies.     

Endocrinology: Comparative pharmacokinetics of two urinary human follicle stimulating hormone preparations in healthy female and male volunteers

These studies were designed to compare the pharmacokinetic characteristicsof a very highly purified urinary human follicle stimulatinghormone (FSH-HP) preparation (sp. act. 9000 IU FSH/mg of protein),Metrodin HP^®, with a standard urinary FSH preparation Metrodin^®(FSH). The two preparations were administered in a balanced,random-order, cross-over sequence as single doses of 150IU,separated by 1 week of washout to 12 female volunteers by i.v.injection and to 12 male volunteers by i.m. and s.c. routes.FSH concentrations were measured by immunoradiometric assayand by an in-vitro rat granulosa cell aromatase bioassay. Afteran i.v. bolus, the pharmacokinetics of the two FSH preparationswere identical. Total clearance was 0.5 and 0.15 1/h respectivelyfor immunoassay and bioassay data. Immunoassay showed that thetwo preparations were similar for renal clearance (0.1 1/h),volumes of distribution at steady state (9 1), distributionand terminal half-lives (2 and 17 h, respectively). After parenteraladministrations, the absorption half-life of FSH was 3 h andthe apparent terminal half-life was 1.5 days. Both preparationshad relative bio-availabilities close to 100% for i.m. and s.c.administrations. Immunopurification, which results in a veryhighly purified FSH-HP, does not modify the pharmacokineticproperties of FSH. This study also confirmed that s.c. and i.m.doses of FSH-HP are equivalent from the pharmacokinetic andpharmacodynamic points of view.     

Use of multivariate statistical methods to identify immunochemical cross‐reactants

Quantitative competition immunoassays with appropriate combinations of antibodies give consistent dose‐response patterns which may be used to identify and estimate amounts of cross‐reacting compounds. Previously reported methods of analyzing cross‐reaction patterns include multiple regression, principal components analysis and minimum estimates of variance (MEV). Four other techniques which are preferable in theory have been surveyed: discriminant analysis (DA), maximum likelihood estimates (MLE), classification and regression trees (CART), and computational neural networks (NN). MLE and simple back‐propagation neural networks can estimate the concentration, as well as the identity, of individual compounds. These four methods worked well with unfitted, unscaled data from monoclonal assays of triazines, phenylureas and avermectins. Immunoassays must be properly designed to provide adequate data for pattern recognition. Cross‐reactivity pattern analysis will make multi‐analyte, multi‐antibody immunoassays feasible for many applications in toxicology and hazard assessment.     

Detection of Mycoplasma hominis antigen in clinical specimens by using a four-layer modification of enzyme immunoassay (EIA)

A 4-layer modification of enzyme immunoassay (EIA) was developed for the detection of Mycoplasma hominis antigen in clinical specimens. Microtiter plates were sensitized with rabbit anti-mycoplasma immunoglobulin, guinea pig anti-mycoplasma immunoglobulin was used as the secondary antibody, and horseradish peroxidase-conjugated anti-guinea pig immunoglobulin was used as the indicator antibody. The specificity of the assay was confirmed by using guinea pig immunoglobulins from preimmunization sera. The sensitivity of the assay is down to 10 ng/ml of antigen protein. Marked cross-reactivity was demonstrated for different strains within the species M. hominis, whereas the other genital mycoplasma species tested showed no reactivity in the assay. A comparison was made of EIA and conventional culture of vaginal specimens from 24 women. All 6 specimens positive by culture were also positive for M. hominis antigen by EIA. Antigen detection by EIA is a sensitive, rapid and simple method for the detection of M. hominis in clinical specimens.     

A computer-interfaced photometer and systematic spacing of duplicates to control within-plate enzyme-immunoassay variation

A Multiskan photometer for reading microtiter plate enzyme immunoassays was linked with a time sharing computer to facilitate control of assay variation and analysis of results. The interface that converted photometer output to RS-232-C format required changes to divide the output into segments short enough for input to the computer. To measure within-plate variation and investigate how the method of allocating sample duplicates to plate wells may affect the estimation of sample variance, uniformity tests were conducted with 47 plates. Coefficients of variation (CV) among wells within-plates ranged from 4.6 to 20.7% and in two-thirds of the plates exceeded 10%. Duplicates allocated to adjacent wells (method 1) gave consistently higher CV for sample means than duplicates allocated to opposite plate quadrants (method 2). In general, the CV by method 2 was about 30% smaller than that by method 1. Analysis of variance confirmed the effectiveness of the quadrant pattern of duplicate allocation as a method of controlling variation that arises from well position effects.     

High rate of false positives in blood donor screening for antibodies to hepatitis C virus

Summary Anti-hepatitis C virus antibody screening of blood donors in different countries revealed prevalences ranging from 0,4–1,4%. These results were obtained with an enzyme immunoassay based on a recombinant hepatitis C virus antigen. We applied a specific inhibition assay (neutralization assay) and a recombinant immunoblot assay to determine the specificity of positive reactions in the enzyme immunoassay.Of 2836 blood donor sera tested, 10 (0,35%) were reactive in the enzyme immunoassay, however, only 3 sera (0,1%) proved to be specifically anti-HCV positive in the inhibition assay. The recombinant immunoblot assay gave similar results. The prevalence of anti-hepatitis C virus antibodies among blood donors has been overestimated in recent publications. Furthermore the high rate of false positives in the enzyme immunoassay may explain reports claiming that only a minor part of EIA positive blood units transmitted the hepatitis C virus to recipients.The inhibition assay was also applied to sera of haemophiliacs and of patients with hepatopathy which had reacted positively in the anti-hepatitis C virus antibody enzyme immunoassay. The antihepatitis C virus specificity was confirmed for all sera from the haemophiliacs group (100%) and for 77% of the hepatopathy patients group. Thus, the anti-hepatitis C virus enzyme immunoassay has a high predictive value when it is used to screen groups with high risks of parenteral hepatitis C virus infection, however, its predictive value is very low when it is used for blood donor screening.Abbreviations EIA enzyme immunoassay - HCV hepatitis C virus - RIBA recombinant immunoblot assay - SOD superoxide dismutase     

C3b-Mediated chemiluminescence of human lymphocytes

Department of Microbiology, S. M. Kirov Medical Institute, Gor'kii. (Presented by Academician of the Academy of Medical Sciences of the USSR Yu. A. Vladimirov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 108, No. 11, pp. 587–589, November, 1989.