A multi-laboratory evaluation of an enzyme-linked immunoassay quantitating human antibodies to Streptococcus pneumoniae polysaccharides.

An enzyme-linked immunoassay (EIA) is described and evaluated which quantitates human antibodies to serotype specific S. pneumoniae polysaccharide (PnPs) in human sera. Based on the observations previously described by Koskela (1), native PnPs are used as coating antigens and sera are absorbed with a soluble pneumococcal absorbant material containing C-polysaccharide (CPs) to ensure measurement of serotype specific anti-PnPs antibodies. The robustness of this method was evaluated by ten laboratories using the same reagents, protocol, and five human serum samples. Reproducible antibody values were obtained for IgM, IgG, and IgA antibodies to five different PnPs serotypes, 3, 6B, 14, 19F, and 23F. The overall mean percent coefficients of variation in this interlaboratory study for all five selotype specific anti-PnPs determinations with the five coded sera were 30% for IgG, 3/% for IgM, and 36% for IgA. This assay can be standardized for quantitation of serotype specific anti-PnPs antibodies, allowing comparison of antibody values in vaccine trials evaluating pneumococcal vaccines.

Estudio comparativo de las técnicas de Machado Guerreiro y Elisa en el diagnóstico de infección chagásica / Compartive assay of Machado Guerreiro and Elisa techniques in diagnosis of chagas disease

Se procesaron 241 muestras de donantes voluntarios del banco de Sangre del Estado Zulia de sexo masculino y femenino, en edades comprendidas entre 18 y 30 años; a cada una de las cuales se le practicaron las pruebas de Elisa realizadas en el IVIC utilizando cepas puras del antígeno de T.cruzi, ELISA utilizando un kit comercial y Machado Guerreiro. Ambas técnicas realizadas en el Banco de Sangre del Estado Zulia. De los resultados obtenidos, con la técnica de Machado Guerreiro, 7 sueros resultaron positivos y 234 negativos, con ELISA (Banco de Sangre) 21 positivos y 220 negativos; con ELISA (IVIC) 32 positivos y 209 negativos. La comparación de Machado Guerreiro con ambas ELISA empleando el método de análisis Ji-Cuadrado modificado por Brand-Snedecor, reveló que las diferencias observadas entre los resultados de las técnicas fueron significativas. Evaluando los resultados ELISA (Banco de Sangre) y ELISA (IVIC) se demostró que no hubo diferencias significativas entre ambas pruebas. Los resultados de este estudio demostraron que ELISA es más sensible que Machado Guerreiro ya que detectó cantidades mínimas de anticuerpos específicos para antígenos chagásicos que no fueron detectados por Machado Guerreiro. El uso de la técnica ELISA amplía el rango de seguridad para las transfunsiones sanguíneas en relación a pacientes con infección chagásica

Infección por virus de inmunodeficiencia humana (VIH-1) en donantes de sangre: Maracaibo, Edo. Zulia-Venezuela / Infection by human immunodeficiency virus (HIV-1) in blood donors: Maracaibo State Zulia-Venezuela

Fueron procesados 16.489 sueros sanguíneos por los Métodos de Inmunoanálisis Enzimático ELISA Abbot Laboratorio y Western Blot, para la investigación de anticuerpos contra el Virus de Inmunodeficiencia Humana (VIH-1) en donantes de sangre del Instituto Hematólogico de Occidente (IHO) Maracaibo Edo. Zulia, Venezuela durante los meses de enero-diciembre 1993; obteniéndose 16 casos positivos (0.097 por ciento)

Immunohistochemical study of hemangioblastoma with special reference to its cytogenesis.

The cytogenesis of so-called hemangioblastoma arising in the central nervous system was investigated using the immunoreactivity to various proteins in cerebellar hemangioblastomas, extracerebellar hemangioblastomas, cerebral hemangiopericytomas, and hemangioendotheliomas, as well as a variety of meningiomas. The antisera used included vimentin, glial fibrillary acidic protein, epithelial membrane antigen, factor VIII-related antigen, and cytokeratin. Lipid-laden cells often encountered in partially degenerated meningiomas revealed almost identical reactivity patterns as the lipid-laden cells, i.e., so-called stroma cells, of hemangioblastomas. The results indicate that the so-called hemangioblastoma of the central nervous system is derived from arachnoid cells in meningotheliomatous meningiomas by way of cellular degeneration.

Desenvolvimento de laboratórios para fagotipagem de Staphylococcus aureus e para classificaçäo de estreptococos beta-hemolíticos na disciplina de microbiologia da FMRP-USP / Development of laboratories for phage-typing of Staphylococcus aureus and for classification of beta-hemolytic streptococci in the division of microbiology, Ribeiräo Preto Medical School, University of Säo Paulo, Brazil

Os reagentes biológicos dos laboratórios de Staphylococcus (fagotipagem) e de Streptococcus (grupagem e tipagem T) da FMRP-USP, foram produzidos e avaliados na Disciplina de Microbiologia no Departamento de Parasitologia, Microbiologia e Imunologia dessa Faculdade com sementes-padräo doadas por organismos científicos especializados de idoneidade internacional e reavaliados, aprovados e controlados por esses mesmos organismos, respectivamente, o "Central Public Health Laboratory", Colindale, Londres, (Elizabeth Asheshov, Maureen de Saxe) e os "Centers for Disease Control", Atlanta, GA, EEUU (Elaine L. Updyke, Max D. Moody, Richard R. Facklam e Peter Byrd Smith)

Estudio comparativo entre el uso del método de Spicer-Edwards y la relación de coaglutinación para identificación de antígenos de Salmonella / Comparative study between Spicer-Edwards method and a coagglutination method to detect Salmonella antigen

Se realizó un estudio comparativo para la identificación de antígenos flagelares de Salmonella empleando el método de aglutinación en tubo con sueros de Spicer-Edwards y la reacción de coaglutinación utilizando proteína A de Staphylococcus aureus Cowan 1 (NCTC 8530). Se tipificaron por ambos métodos 39 serotipos de Salmonella pertenecientes a ocho serogrupos del esquema antigénico de Kauffman-White. Cada uno de los serogrupos incluía salmonelas de los diferentes serotipos monofásicos y bifásicos. El análisis estadístico de los resultados demostró que la reacción de coaglutinación tuvo mayor sensibilidad y especificidad para la detección de antígenos flagelares que la reacción clásica de aglutinación en tubo. El uso de menor cantidad de antisueros para detectar antígenos de Salmonella sin pérdida de sensibilidad y/o especificidad en la prueba de coaglutinación representa un ahorro importante para laboratorios clínios y de investigación epidemiológica que requieren de la identificación de estos microorganismos

Use of immunoblotting to characterize the mitochondrial antigens recognized by anti-mitochondrial autoantibodies.

Sera with anti-mitochondrial autoantibodies detected by indirect immunofluorescence and/or enzyme-linked immunosorbent assay (ELISA) were examined by immunoblotting against pig heart mitochondria. Seven types of reactions were defined, according to the pattern of the labelled bands. Type I sera reacted with 12 bands located within four zones. The most intensively labelled bands were located at 70, 67, 58, 63 and 43 kDa. Other types gave decreasing band numbers. When beef heart mitochondria were used, sera belonging to each of the above types had a profile of labelled bands which sometimes differed from those obtained with pig heart mitochondria. When the chloroform extracted F1-ATPase from beef heart mitochondria was used to prepare the immunoblots, primary biliary cirrhosis (PBC) sera with anti-mitochondria antibodies reacted with all the bands although zone A bands were less labelled. Rat liver mitochondria gave seven bands with type I sera among which the 57 and 35 kDa bands were specific for rat liver mitochondria, as shown by absorption tests. Sera of PBC patients were also tested in immunoblotting against rat liver subcellular fractions including mitoplasts, submitochondrial particles, inner membrane, outer membrane, matrix proteins and inter-membrane proteins. Antigenic bands of A and B zones were localized in the inner membrane and/or in the matrix proteins and the 35 kDa band in inter-membrane proteins. The outer membrane gave no reaction. The most frequent anti-mitochondrial autoantibody types in PBC were type II, then I, whilst for chronic active hepatitis type III was the most common. Type V was only seen in a patient suffering from a typical PBC. Some sera from patients with syphilis, collagenous colitis or progressive systemic sclerosis labelled one or two bands distinct from those labelled by the PBC sera. Sera from patients with drug-induced hepatitis with endoplasmic reticulum antibodies and with systemic lupus erythematosus were generally found negative by immunoblotting.

Neoantigens appear in human IgG upon antigen binding: detection by antibodies that react specifically with antigen-bound IgG.

We raised rabbit antibodies that specifically recognize antigen-bound but not monomeric human IgG. These rabbit IgG antibodies (RAb) were induced in rabbits that were made tolerant to monomeric human IgG. They bound to immune complexes (IC) made with human IgG and various antigens including tetanus toxoid, sheep erythrocytes (E), rabbit E, or human Rh(D) + E, and were very poorly inhibitable with monomeric IgG compared to conventional rabbit anti-human IgG. RAb did not recognize complement components bound to the IgG containing IC. Cleavage of the Fc domain from human IgG markedly decreased binding of RAb to IC. Surprisingly, RAb did not bind to heat-aggregated human IgG (agg-IgG) better than to monomeric IgG. We conclude that human IgG expresses an Fc neoantigen when it binds its own antigen, and that this determinant is not expressed by agg-IgG. The implications of these findings for the biologic functions of antigen-complexed IgG are discussed.

The biochemical genetics of TL antigens on mouse thymocytes.

Molecules bearing thymus leukemia (TL) alloantigen were isolated by immunoprecipitation from detergent-solubilized thymocyte lysates. Antisera used included monoclonal antibodies (anti-TL.m1, anti-TL.m2, anti-TL.m3), monospecific anti-TL.5 alloantisera and multispecific anti-TL.1,2,3,5 antiserum. Apparently, each of these reagents immunoprecipitates the same single 45,000 molecular weight Tla gene product as shown by identity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing and by sequential precipitation studies. Allelic TL molecules, coded by the Tlaa and Tlad genes, were shown to be distinguishable by SDS-PAGE, and tryptic peptide mapping experiments. Both allelic TL molecules could be isolated from thymocytes of (Tlaa x Tlad)F1 mice. These results suggest that, at least for the Tlaa-Tlad allelic differences, the polymorphism and antigenicity of TL is determined by variation in amino acid composition.

Immunoregulatory circuits which modulate responsiveness to suppressor cell signals: characterization of an effector cell in the contrasuppressor circuit.

Spleen cells from neonatal animals, placed in culture for 6 days spontaneously develop the ability to block the activity of suppressor T cells, a phenomenon that is referred to as contrasuppression. The effector cell which is derived from the interactions among the cells which comprise a contrasuppressor "circuit" is an Ly-1 T cell. It can be separated from Ly-1 helper cells by three criteria other than function: its generation is dependent on Ly-2+ cells, it is I-J+, and it sticks to the Vicia villosa lectin. Those cells which deliver help to B cells under the experimental conditions studied are not dependent on Ly-2+ cells for generation and neither express determinants that our anti-I-J antisera recognize nor stick to V. villosa. The mechanism by which these Ly-1 contrasuppressor cells function was elucidated by adding them to "'intermediate cultures" containing activated Ly-2 suppressor cells and in vivo immunized Ly-1.1-congenic helper cells. After 48 h in these intermediate cultures, the neonatal Ly-1.2 contrasuppressor cells and the Ly-2 suppressor cells were removed by treatment with the appropriate antiserum plus complement. The remaining activity of the in vivo generated Ly-1.1 helper cells was assayed in fresh cultures of B cells. The contrasuppressor cells not only diminished suppression of the Ly-1 helper cells by the Ly-2 suppressor cells in the intermediate culture, but actually conferred a state of relative resistance to suppression upon the helper cells. This state persisted after the contrasuppressor cells were removed. Why such a cellular circuit, which confers resistance to suppression, might be beneficial to neonatal mice and how considering its attributes might help explain some immunological paradoxes is the subject of discussion.

Use of antisera for identification of enterotoxigenic Escherichia coli.

The usual methods for identifying enterotoxigenic Escherichia coli (ETEC) strains involve testing for production of heat-labile enterotoxins. To simplify the identification of ETEC, antisera against common ETEC O serogropus were used to identify ETEC in the stools from 618 patients with acute diarrhoea and dehydration (greater than or equal to 5% loss of body-weight) receiving treatment at a hospital in Dacca, Bangladesh. Compared with enterotoxin testing the antisera had a sensitivity of 64%, a specificity of 96%, and a predictive accuracy of 89%. These antisera may be useful in the identification ETEC in clinical laboratories which are unable to perform toxin testing and should be evaluated in other geographical areas.

HLA serum screening based on an heuristic solution of the set cover problem.

A computer program initially written by the Milwaukee Blood Bank has been modified to use a new algorithm for the assignment of HLA specificities to antisera. The assignment is based on the reactions of cells with known specificities. Specificities which are present only on cells which do not react are first ruled out. This step is followed by one or more steps in which the 'least reactive' specificities are ruled out. The rationale for the algorithm is discussed and an example is presented.

Indirect 125I-labeled protein A assay for monoclonal antibodies to cell surface antigens.

An assay for detection of monoclonal hybridoma antibodies against cell surface antigens is described. Samples of spent medium from the hybridoma cultures are incubated in microtest wells with cells, either as adherent monolayers or in suspension. Antibodies bound to surface antigens are detected by successive incubations with rabbit anti-immunoglobulin serum and 125I-labeled protein A from Staphylococcus aureus, followed by autoradiography of the microtest plate or scintillation counting of the individual wells. Particular advantages of this assay for screening hybridomas are: (1) commerically available reagents are used, (2) antibodies of any species and of any immunoglobulin class or subclass can be detected, and (3) large numbers of samples can be screened rapidly and inexpensively. We have used the assay to select hybridomas producing monoclonal antibodies to surface antigens of human melanomas and mouse sarcomas.

Immunochemical studies on blood groups. LIV. Classification of anti-I and anti-i sera into groups based on reactivity patterns with various antigens related to the blood group A,B,H, Le a, Le b and precursor substances.

Quantitative precipitin assays were carried out with eleven anti-I and five anti-i cold agglutinin sera. Their reaction patterns with precursor substances and with certain antigens related to the A, B, H, Le(a), and Le(b) substances revealed at least six types of I and four types of i specificity. All of the I and i determinants appear to be represented in varying amounts on that fraction of the ovarian cyst precursor substance OG which was precipitated by 20% ethanol (OG 20% 2X). Only some of these determinants can be detected in the first periodate oxidation stages of A, B, and H substances, in cow substances, and in hydatid cyst fluid.