Anticorpos policlonais para o imunodiagnóstico de vírus bovino / Polyclonal antibodies for the immunodiagnostic of bovine viruses

O objetivo do estudo foi produzir anticorpos policlonais para vírus bovino e testar a reatividade em imunoensaios como imunofluorescência, imunoperoxidase e slot blot. Como imunógeno utilizou-se cepas e/ou isolados dos herpesvírus bovino tipo 1, 2 e 5 (BoHV-1, BoHV-2 e BoHV-5), vírus da diarreia viral bovina (BVDV), vírus respiratório sincicial bovino (BRSV), vírus da língua azul (BTV) e vírus vaccina (VACV) amplificados em cultivo celular. Os coelhos foram imunizados em intervalos regulares e o soro hiperimune produzido foi utilizado como anticorpo primário nos imunoensaios. A diluição de trabalho para cada antissoro foi determinada por diluição seriada limitante e variaram entre 1:800 e 1:51.200. As maiores diluições foram observadas para imunoperoxidase, quando comparadas com a imunofluorescência e slot blot. Diluições menores que 1:800 foram associadas com aumento de reações inespecíficas. Os antissoros anti-BoHV-1, -BoHV-5, -BVDV e -BRSV reagiram frente a isolados heterólogos em ensaios de imunofluorescência e imunoperoxidase. Conclui-se que os antissoros produzidos possuem elevadas concentrações de anticorpos específicos para os vírus e é uma alternativa para a utilização em imunoensaios.(AU)

The objective of the study was to produce polyclonal antibodies to bovine viruses and to test the reactivity in immunoassays such as immunofluorescence, immunoperoxidase and slot blot. Bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine herpesvirus type 1, 2 and 5 (BoHV-1, BoHV-2 and BoHV-5) blue tongue virus (BTV) and vaccinia virus (VACV) were amplified in cell culture and used to immunize rabbits. Animals were immunized at regular intervals and the hyper immune serum produced was used as primary antibody in the immunoassays. The working dilution for each antiserum was determined by limiting serial dilution and varied from 1: 800 to 1: 51,200. The highest dilutions were observed for immunoperoxidase, when compared with immunofluorescence and slot blot. Dilutions lower than 1:800 were associated with the presence of nonspecific reactions. Anti-BoHV-1, -BoHV-5, -BVDV and -BRSV antisera reacted against heterologous isolates in immunofluorescence and immunoperoxidase assays. It is concluded that the produced polyclonal antibodies have high concentrations of virus-specific antibodies and are an alternative for use in immunoassays.(AU)

Anticorpos policlonais para o imunodiagnóstico de vírus bovino / Polyclonal antibodies for the immunodiagnostic of bovine viruses

O objetivo do estudo foi produzir anticorpos policlonais para vírus bovino e testar a reatividade em imunoensaios como imunofluorescência, imunoperoxidase e slot blot. Como imunógeno utilizou-se cepas e/ou isolados dos herpesvírus bovino tipo 1, 2 e 5 (BoHV-1, BoHV-2 e BoHV-5), vírus da diarreia viral bovina (BVDV), vírus respiratório sincicial bovino (BRSV), vírus da língua azul (BTV) e vírus vaccina (VACV) amplificados em cultivo celular. Os coelhos foram imunizados em intervalos regulares e o soro hiperimune produzido foi utilizado como anticorpo primário nos imunoensaios. A diluição de trabalho para cada antissoro foi determinada por diluição seriada limitante e variaram entre 1:800 e 1:51.200. As maiores diluições foram observadas para imunoperoxidase, quando comparadas com a imunofluorescência e slot blot. Diluições menores que 1:800 foram associadas com aumento de reações inespecíficas. Os antissoros anti-BoHV-1, -BoHV-5, -BVDV e -BRSV reagiram frente a isolados heterólogos em ensaios de imunofluorescência e imunoperoxidase. Conclui-se que os antissoros produzidos possuem elevadas concentrações de anticorpos específicos para os vírus e é uma alternativa para a utilização em imunoensaios.

The objective of the study was to produce polyclonal antibodies to bovine viruses and to test the reactivity in immunoassays such as immunofluorescence, immunoperoxidase and slot blot. Bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine herpesvirus type 1, 2 and 5 (BoHV-1, BoHV-2 and BoHV-5) blue tongue virus (BTV) and vaccinia virus (VACV) were amplified in cell culture and used to immunize rabbits. Animals were immunized at regular intervals and the hyper immune serum produced was used as primary antibody in the immunoassays. The working dilution for each antiserum was determined by limiting serial dilution and varied from 1: 800 to 1: 51,200. The highest dilutions were observed for immunoperoxidase, when compared with immunofluorescence and slot blot. Dilutions lower than 1:800 were associated with the presence of nonspecific reactions. Anti-BoHV-1, -BoHV-5, -BVDV and -BRSV antisera reacted against heterologous isolates in immunofluorescence and immunoperoxidase assays. It is concluded that the produced polyclonal antibodies have high concentrations of virus-specific antibodies and are an alternative for use in immunoassays.

Estudo do perfil microbiológico da Seção de Envasamento e Acondicionamento de Soros e Vacinas desenvolvidos no Instituto Butantan

Diversos são os controles que devem ser realizados pela indústria farmacêutica para garantir a qualidade do ambiente onde são fabricados os medicamentos. Este trabalho contemplou alguns pontos do controle ambiental da Seção de Envasamento e Acondicionamento de soros e vacinas desenvolvidos no Instituto Butantan, como os pontos críticos de amostragem, isolamento dos microrganismos presentes nesses pontos, a identificação microbiológica por métodos macro e microscópicos e técnicas de biologia molecular e as possíveis fontes contaminação, de acordo com as características do microrganismo. Os gêneros de bactérias mais frequentes associados à amostragem das áreas de produção foram Staphylococcus spp., Micrococcus luteus e Bacillus spp. Os pontos de amostragem com maior frequência de microrganismos isolados foram nos monitoramentos de operadores e de partículas viáveis em operação. A sanitização das áreas limpas é um aspecto importante na fabricação de produtos estéreis. Assim, a implantação de um rodízio de sanitizantes pode ser mais efetiva por ampliar o espectro de ação destes em diferentes espécies de microrganismos.

Estudo do perfil microbiológico e monitoramento ambiental da Seção de Plasmas Hiperimunes e do Serviço de Formulação de Soros e Vacinas do Instituto Butantan

Diversos são os controles que devem ser realizados pela indústria farmacêuticapara garantir a qualidade do ambiente onde são fabricados os medicamentos.Este trabalho contemplou alguns pontos do controle ambiental da Seção dePlasmas Hiperimunes e do Serviço de Formulação de soros e vacinas, comoos pontos críticos de amostragem, isolamento dos microrganismos presentesnesses pontos e a identificação desta microbiota por métodos macro emicroscópicos e técnicas de biologia molecular. O gênero de bactéria maisfrequente associado à amostragem das áreas de produção foi Staphylococcusspp., seguido de Bacillus spp. e Micrococcus luteus. Os pontos de amostragemcom maior frequência de microrganismos isolados foram nos monitoramentosde operadores e de partículas viáveis em operação.

Intralaboratory validation of kinetic chromogenic Limulus amebocyte lysate assay for bacterial endotoxin determination in anti-bothropic serum.

Over the years, substituting in vitro biological methods for in vivo tests has posed an ever increasing challenge for researchers, including those who study the applications for snake antivenom. In the quality control of antivenons, the only official test recommended by pharmacopoeias for detecting pyrogenicity is the rabbit pyrogen test. In the present study, we propose intralaboratory validation of a method to replace the rabbit pyrogen test: in vitro determination of bacterial endotoxin in anti-bothropic serum (ABS) with quantitative kinetic chromogenic limulus amebocyte lysate (LAL) assay. The kinetic chromogenic LAL assay is specific to the detection of gram-negative bacterial endotoxin. The validation of the test involved the determination of performance parameters required by the Agência Nacional de Vigilância Sanitária Brasileira (ANVISA, the Brazilian National Health Surveillance Agency), the United States Food and Drug Administration (FDA) and the United States Pharmacopeia (USP) 35. In all experiments, the correlation coefficient of the curve obtained with the control standard endotoxin (CSE; Escherichia coli 055:B5 strain, range, 0.005-50 EU/mL) was between -0.998 and -1.000; and the recovery of endotoxin added to the sample of ABS (0.5 EU/mL) at the working dilution (1:10) followed the recuperation criteria (i.e., 50-200%). We performed six determinations, in each of which the coefficient of variation for the intermediate precision was between 5.6% and 13.8% (below the 15% threshold) and the accuracy was between 90.7% and 114.3% (within the acceptable range of 80-120%). The endotoxin concentration limit for the ABS was determined to be ≤ 2.9 EU/mL. The intralaboratory validation of the methodology was considered to have been successful because it met the criteria for all of the performance parameters.

Paenibacillus larvae enolase as a virulence factor in honeybee larvae infection.

Paenibacillus larvae is a gram-positive spore-forming bacteria, causative agent of American Foulbrood (AFB), a severe disease affecting larvae of the honeybee Apis mellifera. In an attempt to detect potential virulence factors secreted by P. larvae, we identified an enolase among different secreted proteins. Although this protein is a cytosolic enzyme involved in glycolytic pathways, it has been related to virulence. The aim of the present work was to evaluate its role during the infection of honeybee larvae. Toxicity assays showed that enolase was highly toxic and immunogenic to honeybee larvae. Its production was detected inside P. larvae vegetative cells, on the surface of P. larvae spores and secreted to the external growth medium. P. larvae enolase production was also confirmed in vivo, during the infection of honeybee larvae. This protein was able to hydrolyze milk proteins as described for P. larvae, suggesting that could be involved in larval degradation, maybe through the plasmin(ogen) system. These results suggest that P. larvae enolase may have a role in virulence and could contribute to a general insight about insect-pathogen interaction mechanisms.

Transcriptomic basis for an antiserum against Micrurus corallinus (coral snake) venom.

BACKGROUND: Micrurus corallinus (coral snake) is a tropical forest snake belonging to the family Elapidae. Its venom shows a high neurotoxicity associated with pre- and post-synaptic toxins, causing diaphragm paralysis, which may result in death. In spite of a relatively small incidence of accidents, serum therapy is crucial for those bitten. However, the adequate production of antiserum is hampered by the difficulty in obtaining sufficient amounts of venom from a small snake with demanding breeding conditions. In order to elucidate the molecular basis of this venom and to uncover possible immunogens for an antiserum, we generated expressed sequences tags (ESTs) from its venom glands and analyzed the transcriptomic profile. In addition, their immunogenicity was tested using DNA immunization. RESULTS: A total of 1438 ESTs were generated and grouped into 611 clusters. Toxin transcripts represented 46% of the total ESTs. The two main toxin classes consisted of three-finger toxins (3FTx) (24%) and phospholipases A(2) (PLA(2)s) (15%). However, 8 other classes of toxins were present, including C-type lectins, natriuretic peptide precursors and even high-molecular mass components such as metalloproteases and L-amino acid oxidases. Each class included an assortment of isoforms, some showing evidence of alternative splicing and domain deletions. Five antigenic candidates were selected (four 3FTx and one PLA(2)) and used for a preliminary study of DNA immunization. The immunological response showed that the sera from the immunized animals were able to recognize the recombinant antigens. CONCLUSION: Besides an improvement in our knowledge of the composition of coral snake venoms, which are very poorly known when compared to Old World elapids, the expression profile suggests abundant and diversified components that may be used in future antiserum formulation. As recombinant production of venom antigens frequently fails due to complex disulfide arrangements, DNA immunization may be a viable alternative. In fact, the selected candidates provided an initial evidence of the feasibility of this approach, which is less costly and not dependent on the availability of the venom.

Detecção de anticorpos antinucleossomos em soros com diferentes padrões de imunofluorescência em células HEp2 / Detection of antinucleosome antibodies in sera with different patterns of immunofluorecence in HEp 2 cells

Objetivo: Este trabalho tem como objetivo a detecçãode anticorpos antinucleossomos em soros positivoscom diferentes padrões de fluorescência para anticorposantinucleares. Métodos: Até recentemente, não se dispunha de metodologia para a detecção destes anticorpos. Atravésda técnica de imunofl uorescência indireta (IFI) emcélulas HEp2 para a análise de fatores antinucleares(FAN) e ensaios de ELISA (ORGENTEC diagnostika GmbH, Mainz, Alemanha) para detecção de ANucls, realizou-se pesquisa em 61 soros obtidos na rotina de laboratório clínico. A detecção de anticorpos anti-DNAn foi realizado por IFI no tripanossomo Crithidia luciliae (DTS, Randburg, R.S.A) e anti-ENA por ELISA (Hemagen, São Paulo, Brasil). Resultados: Do total de soros pesquisados, 80,3 foram positivos e 19,7 negativos para o FAN, com diferentes padrões de fluorescência. Todos os FANs negativos não apresentaram ANucls. Das amostras positivas, 57 foram também positivas para ANucls. Com relação aos padrões de fluorescência o padrão homogêneo mostrou 91,6 de positividade, pontilhado grosso 91, pontilhado fi no denso 50, centromérico e nucleolar 0. A concomitância de ANucls e anti-DNAn ocorreu em 8 e anti-ENA com ANucls em 28,5. Conclusão: Pôde-se concluir que ANucls detectados em 57 dos casos de FAN positivo ocorreram de forma isolada ou concomitante com outros auto anticorpos antinucleares (DNAn e ENA). Finalmente, este trabalho demonstra que o conceito prático de que ANucls ocorre apenas com padrão homogêneo não corresponde à realidade

Variant surface glycoprotein from Trypanosoma evansi is partially responsible for the cross-reaction between Trypanosoma evansi and Trypanosoma vivax.

Salivarian trypanosomes use antigenic variation of their variant-specific surface glycoprotein (VSG) coat as a defense against the host immune system. Although about 1000 VSG and pseudo-VSG genes are scattered throughout the trypanosome genome, each trypanosome expresses only one VSG, while the rest of the genes are transcriptionally silent. A 64-kDa glycosylated cross-reacting antigen between Trypanosoma evansi and Trypanosoma vivax (p64), which was purified from the TEVA1 T. evansi Venezuelan isolate, was proven here to represent the soluble form of a VSG. Initially, a biochemical characterization of p64 was carried out. Gel filtration chromatography, sedimentation, and chemical cross-linking provided evidences of the dimeric nature of p64. The hydrodynamic parameters indicated that p64 is asymmetrical with a frictional ratio f/fo = 1.57. Isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis revealed that p64 contained two isoforms with isoelectric points of 6.8-6.9 and 7.1-7.2. When p64 and three p64 Staphylococcus aureus V8 proteolytic fragments were sequenced, the same N-termini sequence was obtained: Ala-Pro-Ile-Thr-Asp-Ala-Asp-Leu-Gly-Pro-Ala-Gln-Ile-Ala-Asp, which displayed a significant homology with a putative Trypanosoma brucei VSG gene located on chromosome 4. Additionally, immunofluorescence microscopy on T. evansi and T. vivax established that p64 and its T. vivax homologue were confined to the surface of both parasites. An immunological characterization of this antigen was also carried out using several Venezuelan T. evansi isolates expressing different VSGs, which were obtained from naturally infected animals. Although sera from animals infected with the various T. evansi isolates recognized p64, only one isolate, besides TEVA1, contained polypeptides that were recognized by anti-p64 antibodies. All these results together with prior evidences [Uzcanga, G. et al. (2002) Parasitology 124, 287-299] confirmed that p64 is the soluble form of a T. evansi VSG, containing common epitopes recognized by sera from animals infected with T. evansi or T. vivax. Despite the huge repertoire of VSG genes existing on bloodstream trypanosomes, our data also demonstrated the potential use of a VSG variant from the TEVA1 T. evansi isolate as a diagnostic reagent.

[The production of a latex immunoglobulin conjugate for the diagnosis of Gardnerella vaginalis]. / Obtención de un conjugado látex inmunoglobulina para el diagnóstico de Gardnerella vaginalis.

To obtain an anti-Gadnerella vaginalis latex globulin, specific immunosera to reference strain (ATCC 14018) and to a clinical isolation strain pool were produced. These immunosera were characterized using PAGE-SDS and immunoblotting where a close antigenic relation between the clinical isolation strains and the reference strain was observed. The latex globulin conjugates obtained from these 2 immunosera were evaluated in vitro with a resulting level of detection of 10 ufc/mL of Gardnerella vaginalis. These two conjugates were also evaluated in clinical samples and compared with the 6 vaginalis culture and the criteria considered for diagnosing bacterial vaginosis. The anti-strain pool (II) latex globulin conjugate turned out to be more sensitive and specific than anti-G. vaginalis ATCC strain (I) latex globulin.

Impacto de la administración y calidad del calostro sobre los niveles de inmunoglobulinas séricas en becerro / Impact of colostrum management and quality over serum immunoglobulin levels in claves

La hipogamaglobulinemia se presenta muy frecuentemente en becerros, y es una de las causas más importantes de la morbilidad y mortalidad en estos animales. Cuando no existe una transferencia de inmunidad calostral apropiada se presentan diferentes grados de hipogamaglobulinemias. Se determinaron la densidad y la concentración de proteínas en el suero del calostro del primer ordeño de 305 vacas. Se registró el volumen de calostro consumido por parte de los becerros nacidos de esas vacas y se tomó el tiempo posparto del consumo. Las concentraciones séricas de proteínas totales se determinaron por método de Biurete e inmunoglobulinas (Ig) con la técnica de turbidez con sulfato de zinc. Se determinó una relación directa entre la densidad del calostro y las proteínas en suero de calostro, así como de proteínas de suero de calostro con las proteínas e Ig séricas del becerro. Los nivel de Ig en el suero sanguíneo de los becerros a los 2 a 4 días de edad dependen del volumen, tiempo y también significativamente de la calidad del calostro del primer consumo. Los niveles séricos de Ig fueron de 0-5 unidades de turbidez de sulfato de zinc (UTSZ) en 10.8 por ciento; de 6-10 UTSZ en el 21.3 por ciento, de 11-17 UTSZ en 33.5 por ciento y de 18 UTSZ en 34.4 por ciento, siendo esto becerros normoglobulinémicos. Cuando los animales consumen 2 litros de calostro de buena calidad (densidad >1.050) antes de 3 h y otra dosis dentro de las primeras 10 h posparto, alcanzan niveles séricos de 18 UTSZ. En la práctica diaria es muy importante evaluar la calidad del calostro que consumen los becerros, así como los niveles de Ig séricas a los 2 a 4 días, para garantizar una buena protección contra septicemias e infecciones

Pre-treatment with Concanavalin-A increases resistance of mice to peritoneal infection by Serratia marcescens.

Mice pre-treated with Concanavalin-A largely survived an intra-peritoneal inoculum of 2 x 10(7) Serratia marcescens, whereas all control mice died within 15 h of inoculation. A subpopulation of peritoneal macrophages from Con-A pre-treated mice was able to phagocytose the bacteria in vitro (6.7 SEM 1.2% phagocytosing cells) and in vivo (16.9 SEM 2.1%), whereas control phagocytes did not phagocytose S. marcescens. The survival of Con-A pre-treated mice allowed their immunisation with living bacteria, and the antiserum thus produced increased the phagocytosis of S. marcescens in vitro. Control mice largely survived an inoculum of S. marcescens suspended in 50% immune serum, although the bacteria were resistant to the bactericidal activity of that serum. These results suggest that, in contrast to the delayed humoral protection afforded by immunisation, phagocytosis by phagocytes activated by Con-A conferred early protection to mice against experimental infection by S. marcescens.

Meningoencefalitis por Enterovirus en los últimos 5 años / Meningoencephalitis due to Enterovirus in the last five years

Se describen los resultados del estudio de Enterovirus como agentes productores de meningoencefalitis viral, desde 1990 hasta 1994. Fueron estudiados en este período 546 muestras de heces fecales, 95 líquidos cefalorraquídeos y 1 058 sueros pareados, obtenidos de 1 388 pacientes diagnosticados clínicamente con esta enfermedad. Las muestras para aislamiento viral se inocularon en 2 sistemas celulares diferentes, el mayor número de aislamiento se encontró en células diploides de fibroblasto humano. Las determinaciones de anticuerpos se realizaron por prueba de neutralización (micrométodo) con 11 antígenos de Enterovirus (Echo 4,6,9,11 y 30 y Coxsackie B1,2,3,4,5 y 6) y en períodos epidémicos con el virus aislado. En los años estudiados se produjeron 2 brotes epidémicos; uno por Coxsackie A9 (1990-1991) y otro por Echo 30 (1994). En los sueros pareados se encontró mayor positividad a los Echo 6 y 11(AU)

Meningoencefalitis por Enterovirus en los ultimos 5 anos / Meningoencephalitis due to Enterovirus in the last five years

Se describen los resultados del estudio de Enterovirus como agentes productores de meningoencefalitis viral, desde 1990 hasta 1994. Fueron estudiados en este periodo 546 muestras de heces fecales, 95 liquidos cefalorraquideos y 1 058 sueros pareados, obtenidos de 1 388 pacientes diagnosticados clinicamente con esta enfermedad. Las muestras para aislamiento viral se inocularon en 2 sistemas celulares diferentes, el mayor numero de aislamiento se encontro en celulas diploides de fibroblasto humano. Las determinaciones de anticuerpos se realizaron por prueba de neutralizacion (micrometodo) con 11 antigenos de Enterovirus (Echo 4,6,9,11 y 30 y Coxsackie B1,2,3,4,5 y 6) y en periodos epidemicos con el virus aislado. En los anos estudiados se produjeron 2 brotes epidemicos; uno por Coxsackie A9 (1990-1991) y otro por Echo 30 (1994). En los sueros pareados se encontro mayor positividad a los Echo 6 y 11

Respuesta anómala de anticuerpos de infecciones virales productoras de rash / Anomalous response of antibodies in rash producing viral infections

Se estudiaron 20 pares de sueros provenientes del Sistema de Vigilancia Seroepidemiológica Nacional de la vacuna triple viral que llegaron al laboratorio con el diagnóstico de rash febril. Mediante la técnica de inhibición de la hemaglutinación se observó una respuesta anormal de anticuerpos, tanto a rubéola como a sarampión, manifiesta por una caída del título de anticuerpos a una o ambas entidades o a una de ellas con seroconversión a la otra. Con el objetivo de definir la respuesta de anticuerpos a la familia Herpesviridae (HSV, EBV, CMV, VZV), se encontró el 80 por ciento de la respuesta a estos virus. Los resultados se presentan y se discuten(AU)

Respuesta anomala de anticuerpos de infecciones virales productoras de rash / Anomalous response of antibodies in rash producing viral infections

Se estudiaron 20 pares de sueros provenientes del Sistema de Vigilancia Seroepidemiologica Nacional de la vacuna triple viral que llegaron al laboratorio con el diagnostico de rash febril. Mediante la tecnica de inhibicion de la hemaglutinacion se observo una respuesta anormal de anticuerpos, tanto a rubeola como a sarampion, manifiesta por una caida del titulo de anticuerpos a una o ambas entidades o a una de ellas con seroconversion a la otra. Con el objetivo de definir la respuesta de anticuerpos a la familia Herpesviridae (HSV, EBV, CMV, VZV), se encontro el 80 por ciento de la respuesta a estos virus. Los resultados se presentan y se discuten

A rapid method to evaluate the bactericidal activity of hiperimmune serum against Vibrio cholerae 01 using oxidase test

Desenvolveu-se ensaio rapido para medir o efeito de soros imunes contra o Vibrio cholerae 01, tendo-se por base a atividade de oxidase de culturas de bacterias em placas de ELISA

Hyperimmune antirabies sera titration by standard mouse neutralization and counterimmunoelectrophoresis tests, comparing results of different laboratories.

To determine the rabies antibody level of twenty-four hyperimmune equine sera, Standard Mouse Neutralization (SMN) and Couterimmunoelectrophoresis (CIE) tests were carried out, both at the Instituto Butantan (IB) and Instituto Panamericano de Proteccíon de Alimentos y Zoonosis (INPPAZ). Statistical analysis has shown a correlation (r) of 0.9317 between the SMN and CIE performed at the IB, while at the INPPAZ it scored 0.974. Comparison of CIE data of both laboratories yielded a correlation of 0.845. The CIE technique has shown to be a sensitive and efficient as the SMN in titrating antirabies hyperimmune equine sera. Based on CIE results, a simple, rapid and inexpensive technique, titers of sera antibody can be rellably estimated in SMN test.

Valores de anticuerpos anticardiolipina como referencia para una población pediátrica / Reference values for anti-cardiolipin antibodies (aAC) in a pediatric population

Se determinaron anticuerpos anticardiolipina (aAC) por el método de ELISA en 112 sueros de pacientes en edad pediátrica (un mes a diez y siete años), de ambos sexos, que acudieron a su toma de muestras de los servicios de ortopedia, oftalmología y cirugía del Instituto Nacional de Pediatría, para someterse a una cirugía menor, en quienes se descartó algún problema autoinmune o infeccioso. También se incluyeron 13 sueros de pacientes positivos para aAC que padecían el síndrome antifosfolípido, sueros controles positivos y negativos valorados en el Instituto Nacional de la Nutrición y un pool de sueros preparados con 50 muestras de los 112 sujetos antes mencionados. El valor de corte o negatividad para los sueros normales se obtuvo conforme la indica Loizou y col. aumentando cinco desviaciones estándares (1 DS = 0.1890, 5 DS = 0.9450) al valor promedio de las absorbencias (X = 0.4049) obtenidas de los 112 sujetos sanos, quedando un valor de 1.3499 (0.4049 + 0.9450)