Immunochemical characterization of a high molecular weight basic allergen (HMBA) of rye grass (Lolium perenne) pollen

A high molecular weight basic allergen (HMBA) was isolated from the mixture of non-dialysable components of the aqueous extract of defatted rye grass pollen by a combination of gel filtration and isoelectrofocussing. HMBA, a glycoprotein of mol. wt 56,800 (17% carbohydrate) contained all naturally occurring amino acids. A hyperimmune rabbit anti-HMBA serum gave only a single precipitin band with the crude extract of the rye grass pollen in crossed immunoelectrophoresis. Thus. it was concluded that HMBA was a unique and highly purified antigen. The allergenicity of HMBA was revealed by its ability to elicit immediate skin reactions in grass allergic patients. Moreover, all patients' sera tested had IgE antibodies to HMBA detectable by direct RAST with HMBA allergosorbent discs, These observations indicated that HMBA was a major allergenic constituent of rye grass pollen. Treatment of HMBA by 6 M guanidine HC1 led to a significant reduction in its ability to combine with human IgE antibodies. The treatment also resulted in the complete loss of allergenicity (i.e. inability to elicit PCA reactions with a murine reaginic antiserum to HMBA) and antigenicity (inability to form precipitins with rabbit anti-HMBA): hence, it would appear that the allergenic and antigenic determinants of HMBA are ‘conformational’.     

Pharmacokinetic investigation of oral and IV dihydroergotamine in healthy subjects

Summary A new radioimmunoassay (RIA) for the specific measurement of dihydroergotamine (DHE), sufficiently sensitive for the determination of low plasma concentrations, has been used to investigate the pharmacokinetics of unchanged DHE. In a randomized crossover trial, eight healthy male volunteers received single doses of DHE 5 mg, 10 mg and 20 mg orally and 0.1 mg and 0.5 mg intravenously. It was possible to determine plasma concentrations and urinary excretion of DHE over the following 48 h. A long terminal plasma elimination phase of unchanged DHE (half-life 15 h) was found. A similar terminal elimination half-life was also calculated from urine data. The multi-exponential decline in plasma DHE with a long terminal half-life suggests that distribution into a deep compartment contributes to the long-lasting effect of the drug. Plasma protein binding was 93%. Despite extensive tissue distribution (V_z=33 l/kg) and a high plasma clearance (CL_P=2l/min), dose-independent linear pharmacokinetics was observed. The present assay was at least 20-times more specific than the polyvalent RIA used previously and appears suitable to explore the pharmacokinetics of unchanged DHE in patients on low-dose therapy. The long terminal elimination half-life of DHE only reported previously in studies using ³H-labelled drug, and considered to be due to metabolites, was also true for the parent compound. This, in addition to the sustained pharmacological activity of the 8-hydroxy metabolite already shown, provides a further explanation for the long duration of action of DHE in animals and man.     

Detection of specific IgE in isocyanate and phthalic anhydride exposed workers: comparison of RAST RIA, Immuno CAP System FEIA, and Magic Lite SQ

Sera from 94 workers occupationally exposed to isocyanate were tested by RAST RIA, Immuno CAP FEIA, and Magic Lite SQ (ML) against the allergens HDI, MDI, TDI, and phthalic anhydride. Twenty sera showed increased levels of diisocyanate-specific IgE antibodies, and five sera were phthalic anhydride positive. High total IgE titer was not correlated with positive specific IgE titer ( r = 0.47), showing that nonspecific IgE binding was low. The results of ML and CAP correlated well ( r = 0.91), but tended to be slightly higher than the results obtained with the RAST isotope test. CAP and RAST data also correlated significantly ( r = 0.90), but the correlation between ML and RAST data was lower ( r = 0.82). The results of the new in vitro tests, CAP and ML, showed good reproducibility (5% CV for CAP and 8% CV for ML). In summary, the CAP and ML methods were found to be appropriate for routine diagnosis of specific IgE antibodies against the allergens HDI, MDI, TDI, and phthalic anhydride.     

Enzyme-linked immunosorbent assay to monitor colorectal carcinoma patients treated with a monoclonal antibody (17-1A)

An enzyme-linked immunosorbent assay (ELISA) was compared to a radioimmunoassay (RIA) for the detection and quantification of mouse monoclonal antibody MoAb 17-1A and for measurement of the host response (i.e. anti-mouse immunoglobulin in sera from patients receiving immunotherapy with MoAb 17-1A. Comparable sensitivity and reproducibility were noted with RIA and ELISA but ELISA was more rapid to perform than RIA. Thus quantitative ELISA compared favorably with the RIA for MoAb detection.     

A simple computer programme for standard curve tracing and calculations in RIA following sigmoid patterns

A computer programme is described for the analysis of radioimmunoassays assuming a general sigmoid shape. The mathematical treatment uses the results obtained from a sigmoid equation to perform a limited operational search in order to optimize the fitness of the curve. The programme can be installed in most desk top microcomputers and has been tested for the calculation of Ig concentrations in biological fluids.     

Cryptic association of e antigen with different morphologic forms of hepatitis B surface antigen

When highly purified HBsAg particles, separated by rate zonal centrifugation into populations differing in predominant size, were tested for HBeAg, the e1 specificity was detected preferentially in association with particle fractions containing large filaments and Dane particles. These results were obtained both by agar gel diffusion and by radioimmunoassay for e antigen. The e antigen activity present in these fractions was potentiated by prior treatment of particles with Tween 80, suggesting cryptic localization of e1 specificity within or under the outer membrane. The HBeAg released by detergent treatment from a purified preparation composed predominantly of small-particle forms of HBsAg was separated by electrofocusing into a peak of nonparticulate e antigen in the pH range of 5.7--6.0. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed three major polypeptides in this preparation with approximate molecular weights of 25,000, 55,000, and 70,000. Furthermore, two additional peaks of e antigen activity were detected which migrated in association with HBsAg particles at isoelectric points of 4.4 and 5.5--5.6. The major portion of e antigen remained in association with particles after further purification by rate zonal centrifugation.     

Monoclonal antibodies to human growth hormone can distinguish between pituitary and genetically engineered forms

Monoclonal antibodies (MABs) prepared against human pituitary growth hormone (hGH) have been compared for their binding to pituitary-derived and genetically engineered methionyl growth hormone (met-hGH). The antibodies bind to four non-overlapping epitopes of which two are completely shared with human choronic somatomammotropin (hCS). The determinant defined by MAB NA27 was expressed on met-hGH to a lesser degree than on hGH of pituitary origin. However, another antibody, QA68, which binds to a determinant closely related to NA27, failed to discriminate between hGH and met-hGH. A further two MABs (EB1 and NA71) were similarly ineffective in distinguishing between the two forms of the hormone. The determinant recognized by antibody EB2 was equally represented on hGH and met-hGH when assessed by a liquid-phase radioimmunoassay: however, measurement of the binding in a solid-phase assay resulted in a two-four-fold lower binding to met-hGH. Bioactivity assessed by both an in vitro cell proliferation assay and an in vivo cartilage sulphation bioassay failed to distinguish between the two hormones. It is therefore concluded that the NH2-terminal methionine on bacterially derived growth hormone results in altered antigenicity of the hormone without any measurable effect on bioactivity.     

Oyster-associated gastroenteritis in Australia: the detection of Norwalk virus and its antibody by immune electron microscopy and radioimmunoassay

Following widespread outbreaks of oyster-associated gastroenteritis in Australia during 1978 in which Norwalk virus was implicated as the causative agent, collaborative studies were undertaken between laboratories in Australia and the United States to confirm the etiology. Immune electron microscopy (IEM) techniques were used in Australia and radioimmunoassay (RIA) methods in the United States. Norwalk virus was detected by IEM in seven of 15 faecal samples, and four were positive by RIA. A much better correlation was found with antibody determinations. Both methods demonstrated significant increases in antibody to Norwalk virus in 22 of 30 sets (73%) of "acute" and "convalescent" sera, confirming that Norwalk virus was responsible for the majority of cases. It is significant that the RIA serology was determined using Norwalk antigen originating in the United States and the IEM serology was determined using 27--30-nm particles originating in Australia.     

Quantitation of specific antibodies bound to feline leukemia virus in the plasma of pet cats

A method is described for determining levels of circulating immune complexes (CIC) composed of feline leukemia virus (FeLV) antigens and corresponding antibodies in plasma of persistently-infected pet cats. The procedure is based on the ability of high-titered heterologous anti-FeLV serum to chase cat anti-FeLV IgG from dissociated CIC by successfully competing for binding of free antigen. The eluted cat antibody is then collected and quantitated. In a study of cats in the process of clearing persistent FeLV infections, measured levels of FeLV-specific CIC correlated well with fluctuating levels of free FeLV antigen and antibody. The Raji cell assay for CIC in those cats was of comparatively little value in following the clearance of the virus, presumably because that assay does not distinguish between CIC containing viral and those containing non-viral antigens. The method described can be adapted to studies of specific immune complexes associated with a variety of syndromes, provided that the antigen eliciting the immune response is known.     

Dot-based ELISA and RIA: Two rapid assays that screen hybridoma supernatants against whole live cells

Two assays are described that are suitable for the screening of large numbers of hybridoma supernatants as well as for the screening of a wide range of tissues for the distribution of a given cell surface antigen. These assays use whole live cells as targets, avoiding fixation or extraction, which could alter the antigenic structural profile. The assays are rapid, simple and inexpensive. Their advantages and applications are discussed.