Specifications and quality control of a yeast-derived hepatitis B vaccine.

It is essential that the new generation of recombinant DNA yeast-derived hepatitis B vaccines be prepared according to techniques which are adequately controlled to ensure their safety and efficacy. Various national and supra-national authorities have already issued guidelines for specifications and standardization of such products. Based on these guidelines, the potential hazards, and the state-of-the-art technology, SmithKline Biologicals has developed specifications and analytical methods to cover all aspects of the quality assessment of its recombinant hepatitis B vaccine, Engerix-B. This includes adequate 'in-process' tests to guarantee the optimal reproducibility and standardization of the entire production process as well as methods to study the characteristics and identity of the expressed antigen protein. Finally, specifications and methods of analysis necessary to control the purified bulk product and vaccine in its final container ensure the purity, safety, identity, potency, and stability of each production lot.

Non-organ specific thermostable tissue antigens.

Thermostable ethanol insoluble antigens (BE antigens) were identified that occur in all human tissues and human dispersed cells, but which are absent from normal human serum. In contradistinction to previously described organ-specific BE antigens, these antigens were referred to as non-organ-specific tissue antigens (NOST). Antisera obtained by immunization of rabbits with BE preparations of human organs had been selected for being devoid of any organ specificity and had been absorbed by BE preparations of pooled human serum. Such antisera could be used as reagents for detection of NOST BE antigens in pathological human sera. Inhibition of enzyme immunoassay proved to be a convenient procedure for these studies. Inhibition of 35% or more was only exceptionally noted in studying sera of normal subjects 20-40 years old, but inhibition exceeding 35% (positive results) was noted in 48% of sera from subjects at the age of 70 years or more. A high incidence of positive results was also encountered in sera of patients with end-stage renal disease (30%), renal graft recipients (18%), and in patients with lymphoma or leukemia (44%), but interestingly enough, no positive tests were noted in patients with lepromatous leprosy.

A competitive ELISA technique for the measurement of von Willebrand factor antigen (vWF:Ag) using staphylococcal protein A peroxidase.

A competitive ELISA technique for measurement of von Willebrand factor antigen (vWF:Ag) using Staphylococcal Protein A peroxidase is described. The standard used in this assay is partially purified Factor VIII:C/vWF which has been standardized against a conventional method (electroimmunoassay). The results show a close correlation (correlation coefficient 0.956) as compared to the standard Laurell electroimmunoassay technique. Inter-assay and intra-assay coefficients of variation were less than 5%. The technique is simple to perform and results may be obtained within three hours of specimen collection.

Standardization of house dust mite extracts. Liberation of allergenic and antigenic components in relation to extraction conditions.

The spontaneous release of house dust mite components from cultures of Dermatophagoides pteronyssinus into slightly buffered water was studied against time, using both continuous and discontinuous extraction procedures. It was shown that proteins, carbohydrates, IgE binding components and precipitating antigenic components were rapidly released from the house dust mite cultures, reaching a maximal liberation within 1 h of extraction. Repeated extractions of house dust mite cultures (discontinuous extraction) showed an additional release of IgE components but the IgE binding potency declined after successive extractions, while showing increasing release of immunological inactive components. IgE binding to antigens immobilized to polystyrene surfaces (IgE-ELISA) appeared to be less sensitive compared with cyanogen-bromide activated discs (IgE-RAST). It was concluded that extraction procedures of house dust mite cultures with short incubation time of 1 h or less are to be preferred.

Successful immunotherapy for Triatoma protracta-induced anaphylaxis.

A successful program of immunotherapy for Triatoma protracta-induced anaphylaxis was developed. This program included a new passive extract-antigen preparation standardized by RAST inhibition. This antigen facilitated the development of a reliable skin test protocol for in vivo diagnosis of Triatoma protracta allergy. Five patients with T. protracta-induced anaphylaxis underwent a rapidly increasing dosage schedule of immunotherapy. The IgE- and IgG-antibody responses during immunotherapy were followed with solid-phase RIA. Protection against anaphylaxis was confirmed in all patients with a "bite challenge" by T. protracta. This is the first report of completely successful T. protracta immunotherapy.

Multitest CMI for standardized measurement of delayed cutaneous hypersensitivity and cell-mediated immunity. Normal values and proposed scoring system for healthy adults in the U.S.A.

The Multitest CMI system, a disposable device that simultaneously applies seven standardized preloaded antigens and diluent control, is a major advance for measurement of delayed type hypersensitivity (DTH) in assessment of cell-mediated immunity (CMI). The system was tested in 402 healthy adults, aged 17 to 92 years, to determine normal values for incidence and size of DTH responses. Incidence of positive responses to individual antigens varied from 85% to 46%, with great variability related to age and sex. To better assess CMI, a two-part score based on 48-hour readings was employed. The mean number of positive antigens ranged between four and five, and the mean sum of their mm induration ranged between 18 and 25, with both scores increasing with advancing age. A statistical zone of reduced DTH scores (hypoergy) was identified. The Multitest CMI system appears to be a practical means of reproducibly assessing CMI in subjects with immunologic, metabolic, infectious, or neoplastic disorders. The scores in our population may serve as reference values to which results from any tested adult can be compared.

[Development of a competitive enzyme immunoassay for factor VIII-related antigen]. / Entwicklung eines kompetitiven Enzymimmunoassay für das Faktor-VIII-assoziierte Antigen.

An enzyme immunoassay system basing on a competitive method has been developed to measure factor VIII related antigen (F. VIII R:Ag). A sufficient discrimination at low F. VIII R:Ag concentrations was gained. This method appears to be sensitive to 7,8 X 10(-3) U/ml F. VIII R:Ag showing an intraassay coefficient of variation (CV) of 0,11. In comparison to the commonly used Laurell electroimmunodiffusion assay for factor VIII significant less antisera per sample for the enzyme immunoassay technique is necessary.

Quantitation of a highly immunogenic leukocyte antigen (L1) by radioimmunoassay: methodological evaluation.

This paper describes a radioimmunoassay (RIA) for the quantitation of a major protein antigen (L1) released from human granulocytes. A protein A-carrying strain of Staphylococcus aureus was used as a solid phase IgG-binding reagent for the separation of antigen-antibody complexes from free antigen in the assay. A two-step version of the RIA may be completed in 2.5 h and has a detection limit of about 5 ng/ml. The sensitivity may be increased by minor modifications. A single step version takes 1.5 h and has adequate sensitivity to cover clinically important plasma levels. Selection and optimal use of reagents for the two-step procedure is described and their storage discussed. The simplicity of the procedure combined with high sensitivity and satisfactory accuracy, precision and reproducibility makes this assay suitable for quantitation of L1 in various body fluids.

IgE responses in human filariasis. II. Qualitative characterization of filaria-specific IgE.

Crossed radioimmunoelectrophoresis (CRIE) was used to characterize human IgE antibody responses to filarial parasites by using antigens derived from Brugia malayi (Bm) adult worms. A reference pool of patient sera was initially used to determine the sensitivity and specificity of CRIE. Because IgG-blocking antibodies interfered with IgE binding in certain sera, all sera were preabsorbed with protein A-Sepharose. As little as 50 ng of specific IgE antibody (determined by quantitative radioallergosorbent test [RAST]) in the reference pool bound to 20 of the 35 antigen precipitates in crossed immunoelectrophoresis. Increasing IgE antibody concentration did not increase the number of IgE-binding precipitates. Six patients from each of the three major clinical groups in lymphatic filariasis (i.e., tropical pulmonary eosinophilia [TE], chronic lymphatic pathology [CP], or circulating microfilaremia [MF]) were studied by CRIE with the use of a constant amount of IgE antibody (50 ng IgE anti-BmA). Distinct patterns of allergen recognition were observed among the groups. Individuals with TE recognized both anodic and cathodic antigens as allergens, whereas the other two groups recognized predominantly anodic antigens. The greatest number of allergens was recognized by patients with TE; this number ranged from nine to 18, whereas patients with CP or circulating MF recognized from six to 11 allergens. Although potentiated IgE responses at a quantitative level in parasitic helminth infections is a well-established phenomenon, our studies showing the diversity of antigens recognized as allergens indicate for the first time potentiated IgE responses at a qualitative level as well.