[The results of international comparative testing of reagents to swine serum protein allotypes conducted in 1987-1988]. / O rezul'tatakh mezhdunarodnogo sravnitel'nogo ispytaniia reagentov k allotipam syvorotochnykh belkov svin'i, provedennogo v 1987-1988 godakh.

Antisera to 15 allotypes of pig serum protein were raised and compared with those prepared elsewhere, within the framework of International Comparison Test of pig blood groups and polymorphic proteins and enzymes in 1987-1988. Of 15 allotypes tested 8 were found to coincide with the known GpB, GpD, GpA, Gpa, IGH3 C1, Lpb3, Lpb12, Lbr1. For 7 allotypes left, no analogues were found out. They seem very likely to be new allotypes of pig serum proteins.

Effects of analytical method and lyophilized sera on measurements of apolipoproteins A-I and B: an international survey.

In 1987 a collaborative study was initiated with 140 laboratories worldwide to evaluate the effects of analytical method and lyophilization on the measurement of different concentrations of apolipoproteins (apo) A-I and B in four lyophilized serum pool samples. This survey confirmed that the lyophilized apo Reference Material of the International Union of Immunological Societies (IUIS) is useful for apo A-I assays as an international serum-based reference material, because among-method variation is negligible. The apo A-I concentration value of 1.24 g/L is now assigned to the IUIS Reference Material (CDC 1883) by a Centers for Disease Control RIA in-house reference method. Use of lyophilized serum preparation as a reference material for some modes of apo B measurement is questionable because of lyophilization and matrix effects. Both radial immunodiffusion and liquid immunoprecipitin methods demonstrated bias in measured apo B concentrations, compared with overall method-weighted means values on the IUIS Reference Material. Because of the uncertainty associated with LDL primary standard, protein analysis, and concentration differences among analytical methods, assigning a single apo B concentration value to the IUIS Reference Material appears inadvisable at present.

An international collaborative study of an anti-infectious bursal disease virus reference serum.

Fourteen laboratories participated in a collaborative study of a freeze-dried preparation of anti-infectious bursal disease virus serum to assess the suitability of the serum as a standard for use in the infectious bursal disease virus neutralization test. Ten laboratories carried out micro-virus neutralization tests and six carried out plaque reduction tests, two laboratories carrying out both tests. When titres were expressed as a proportion of that obtained for a reference preparation there was a marked reduction in variation between results from different laboratories. The use of a reference preparation was therefore of value when comparing results from different laboratories. It is proposed that the reference preparation used in this study be used as a standard to facilitate the comparison of results from different laboratories. The proposed standard contains by definition 10,000 UK units.

Development of quality control procedures for the human immunodeficiency virus type 1 antibody enzyme-linked immunosorbent assay.

A standardized pool of human sera that was positive for human immunodeficiency virus type 1 (HIV-1) antibody was developed. This positive control serum was used to analyze test differences among eight laboratories, among the HIV-1 antibody test kits of three different manufacturers, among different lots of the same test kit, and among pipetting devices and techniques. The standardized pool of human sera was tested 327 times by the different laboratories. In terms of positive tests, a reproducibility of 99.69% was achieved; however, significant test variance among laboratories, among test kit lots, and among pipetting devices and techniques could be demonstrated if the tests were compared on the basis of the net positive optical density (OD) value. This value was calculated by subtracting the cutoff OD value (i.e., the value below which an OD value was considered negative for HIV-1 antibody) from the observed OD value of the standardized pool of human sera. The results obtained suggest that this strategy can be used for proficiency testing, for monitoring the quality of HIV-1 antibody enzyme-linked immunosorbent assay reagents, and for evaluating pipetting devices and techniques.

[Antitoxoplasma serum--derivation of the national standard (ELISA)]. / Antitoxoplazmické sérum--odvození národního standardu (ELISA).

When assessing the content of specific antibodies in serum there is tendency to abandon the specification expressed as dilution and use more reproducible data, i.e. international units. To this end it is necessary to derive from the international standard the national standard according to which positive control sera in diagnostic sets, produced by SEVAC can be described in international units. In pooled positive human serum recommended as the national standard and in the international standard first the parallel course of the dose/response curve was tested. Then both sera were repeatedly compared: into a close dilution series of the national standard one dilution of the international standard was included and in both sera the dilution with same response was found (by optic density) and thus the activity of the suggested national standard was assessed as 2400 units/ml. The author discusses also the recommended routine procedure for expressing the activity of the examined sera in international units.

Indirect double sandwich ELISA for the specific and quantitative measurement of mouse IgM, IgA and IgG subclasses.

We have developed sensitive enzyme-linked immunosorbent assays (ELISA) which measure mouse serum heavy chain immunoglobulin isotypes in nanograms per milliliter. In each case the specific isotypic Ig is sandwiched between an isotype-specific antibody used for coating and another isotype-specific antibody coupled to biotin for detection (with alkaline phosphatase coupled to avidin). These methods are simple to perform, specific for each isotype, reproducible with an average coefficient of variation of 5% for IgG1, 3% for IgG2a, 7% for IgG2b, 10% for IgG3, 3% for IgA and 7% for IgM, and at least 100 times more sensitive than radial immunodiffusion. The assays have been used to determine the absolute concentrations of mouse serum heavy chain Ig isotypes.

Quantification of IgG and IgG4 antibodies to bee venom phospholipase A2 by competitive inhibition in ELISA.

Phospholipase A2 (PLA) is the major antigen of bee venom. Individuals often stung by bees, such as bee keepers, show a restricted immune response mainly of anti-PLA IgG4 antibodies. In contrast, patients allergic to bee venom produce high levels of PLA-specific IgE. This isotype restriction, the clinical relevance and the well defined structure of the PLA antigen, provide a useful model for the study of the principles regulating isotype expression in the human antibody response. A fundamental requirement for such studies is the availability of quantitative and sensitive assays to measure PLA-specific antibodies. Here we describe an ELISA method for direct isotype-specific quantification of anti-PLA IgG and IgG4 antibodies. Serum containing anti-PLA IgG antibodies was added at a predetermined dilution to PLA coated microtiter plates. Then mouse monoclonal antibodies to human IgG or IgG4 and different concentrations of purified human IgG and IgG4, respectively, were added simultaneously. The concentration of anti-PLA IgG and IgG4 antibodies in the serum was calculated from the resulting inhibition curve. Additionally, an analytical method to compare unknown antibody samples with a standard in ELISA - avoiding problems of different affinities - is described. Using the technique described here, isotype-specific quantification of anti-PLA antibodies can be performed at a sensitivity of approximately 70 pg/ml with a reproducibility range of 10-15%.

[The influence of thermal load on the immunochemical detection of dried blood plasma supplements in meat mixtures]. / Einfluss der thermischen Belastung auf den immunchemischen Nachweis von Trockenblutplasma-Zusätzen in Fleischgemengen.

Addition of blood plasma to meat products is not permitted in the FRG unless these products are heat processed using an internal temperature of 80 degrees C (German regulation of meat and meat products: "Verordnung für Fleisch und Fleischerzeugnisse"). Such heat process may have an unfavourable effect on the detectability of blood plasma. Since blood plasma or dried plasma may originate from different animal species (porcine or bovine) two different anti dried blood plasma-sera (porcine and bovine) are required for immunochemical analysis. The varying quality of these sera has to be considered when interpreting the results. Seven M urea extract turned out to be suitable for detection of dried plasma additives and proved to be highly effective particularly when examining heated samples. Both the gel-diffusion and the electro immuno assay proved useful for the detection of dried blood plasma, provided the examined extracts had been adequately diluted. Immunochemical reactivity was hampered by the heat process which was given to the sample. Accordingly, the concentration of the plasma in a particular sample cannot be determined unless the time/temperature data of the process applied to the sample were given and model samples were tested for comparison.

[Diagnostic meningococcal sera and their quality]. / Diagnosticheskie meningokokkovye syvorotki i ikh kachestvo.

The findings evidence that preparation of highly active specific meningococcal sera depends on the selection of production strains and reference cultures for the assessment of their conformity to technological requirements. Meningococcal cultures selection conditions may be unified if standard immune sera are developed, characterized by stable titres of antibodies to group capsule antigens. Employment of such sera will improve the quality of commercial agglutinating sera.

[The use of immunoenzyme assay for the control of specific activity of antisera against vasopressin]. / Primenenie immunofermentnogo analiza dlia kontrolia spetsificheskoi aktivnosti antisyvorotok k vazopressinu.

A procedure is described for production of antiserum against peptide hormone Arg-vasopressin. A sensitive immunoenzyme assay is developed for estimation of the antiserum activity and specificity.

Studies and observations on Lewis grouping of body fluids and stains.

Leb positive individuals may phenotypically express both Lea and Leb in their secreted body fluids. Therefore, the interpretation of a Le(a + ,b-), non-secretor result is dependent on the absence of Leb. This study emphasises the importance of accurate procedure and biased selection of antisera such that Leb is preferentially detected in comparison with Lea. The relationship of the ABO group to the expression of Le is discussed in conjunction with the selection of samples for testing antisera and inclusion as control standards.

Quality of commercially produced Shigella serogrouping and serotyping antisera.

Shigella grouping antisera from five manufacturers and typing antisera from two were purchased and evaluated with homologous and heterologous Shigella strains in the slide agglutination test. Only 31 of 73 (42%) antisera were satisfactory. In many instances, the antisera gave negative, as opposed to weak, reactions when they should have given strong positive reactions. Four reagents cross-reacted with Shigella strains. Of the 19 polyvalent grouping antisera to subgroups Shigella dysenteriae serotypes 1 through 7, S. flexneri serotypes 1 through 6, S. boydii serotypes 1 through 7, and S. sonnei forms I, II, only one S. sonnei reagent and five S. flexneri reagents were satisfactory with greater than or equal to 90% of the homologous strains. The reagent of poorest quality was satisfactory with only 18% of the homologous strains. There were three polyvalent antisera to the higher types of S. dysenteriae and S. boydii, which were available from only one company, that adequately identified 80, 63, and 65% of the homologous strains. Typing antisera were available from only two companies, and 30 of 51 (59%) were satisfactory. Commercially available Shigella antisera are inadequate for the laboratory testing required for planning the development of and evaluating Shigella vaccines.

Panel of reference strains for evaluating serologic reagents used to identify gonococci.

A panel of strains for evaluating Neisseria gonorrhoeae serologic reagents was developed. The strains selected for the panel were antigenically diverse and representative of strains isolated worldwide and had been isolated from a variety of anatomic sites. A few strains with characteristics that can cause problems in serologic tests were included. The panel of 52 gonococcal and 20 nongonococcal strains was used to evaluate two commercially produced kits with monoclonal antibody reagents, GonoGen and Phadebact, and one Phadebact kit with absorbed rabbit antiserum. The GonoGen reagent correctly identified all gonococcal strains and did not react with any of the nongonococcal strains. The Phadebact absorbed antiserum reagent correctly identified 47 of 48 gonococcal strains but reacted with 2 of the 20 nongonococcal strains. The Phadebact monoclonal antibody reagent correctly identified all the gonococcal strains; however, it gave positive reactions with 8 and trace reactions with 4 of the 20 nongonococcal strains.

The effect of storage temperature and physical state on the performance of antisera in radioimmunoassay after long-term storage.

The performance in radioimmunoassay of four antisera after storage at temperatures ranging from -40 degrees C to room temperature, in three physical states (frozen, liquid or freeze dried) was investigated over a 3-year period. No deterioration in antiserum performance in terms of precision and accuracy of quality control serum measurement or recovery of ligand was apparent under any of the storage conditions studied. Some lowering of titre became apparent in two of the antisera over the study period. Deterioration was most marked when antiserum was stored lyophilised at room temperature. Storage of antiserum frozen confers no advantage over storage at 4 degrees C provided precautions are taken to minimise bacterial contamination when storing antiserum in liquid form.

Antigenic studies on Rhizopus microsporus, Rh. rhizopodiformis, progeny and intermediates (Rh. chinensis).

Fourteen isolates belonging to Rhizopus microsporus, Rh. rhizopodiformis, the progeny of Rh. microsporus x Rh. rhizopodiformis and an intermediate species, Rh. chinensis, were serologically tested either by a modification of the exoantigen technique of Kaufman & Standard (1978) or by the more analytical cross immunoelectrophoresis technique with intermediate gel. Common antigenic determinants in the isolates studied indicated that the species, their progeny and the intermediate species Rh. chinensis, are antigenically very closely related. Through absorptions, a specific reference antiserum for Rh. microsporus was obtained. With this adsorbed monospecific antiserum, using the exoantigen test, the identity of the isolates in study, determined after their general morphology as Rh. microsporus, was confirmed. For comparative purposes, the use of reversed phase high performance liquid chromatography for the investigation of fungal isolates is also reported.