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Foi desenvolvido e validado um ELISA tipo sanduíche "in house" para a quantificação de anticorpos (Ac) anti-anexina V em plasma. Os parâmetros de validação estudados foram: (i) precisão, expressado como coeficiente de variação (CV) inter-e intra-ensaio, (ii) exatidão, expressado como porcentagem de desvio entre o valor obtido e o valor real, (iii) limite de detecção (LOD), avaliado a partir do branco de reagentes e (iv) robusteza, obtida através da introdução propositada de pequenas variações em diferentes parâmetros. Além disso, a técnica "in house" foi comparada com um método comercial. Encontrou-se que ambos CV foram < 20%, a exatidão foi de 100 ± 20%, o limite de detecção foi menor que 1 U mL -1 e as pequenas variações na técnica não produziram variações significativas nos resultados. A comparação com o método comercial mostrou uma correlação aceitável. Concluiu-se que o método desenvolvido cumpre satisfatoriamente com os parâmetros de padronização e validação para imuno-análise.An "in house" sandwich ELISA for the quantification of plasma anti-annexin V antibodies was developed and validated. The validation parameters studied were: (i) precision, expressed either as the intra-or the inter-assay coefficient of variation (CV), (ii) exactitude, expressed as the percentage deviation between the obtained value and the real value, (iii) limit of detection (LOD), evaluated from the reagents blank and (iv) robustness, obtained by deliberately introducing slight variations in different parameters. Also, a comparison between the "in house" technique and a commercial method was performed. The research revealed that both CV were < 20%, exactitude was within the 100 ± 20% range, limit of detection was below 1 U mL -1 and that slight variations in the technique did not produce any significant variations in the results. Comparison with the commercial method showed an acceptable correlation. It was concluded that the method developed here satisfactorily accomplishes the parameters of standardization and validation for an immunoassay.Keywords: ELISA, annexin V, antibodies, anti-phospholipid syndrome, foetal losses IntroductionAnnexins belong to a family of proteins that are able to bind to negatively charged phospholipids and membrane bilayers through calcium dependent interactions. Though their fine structure has been well described, their functions have not been clearly identified yet.1 Like others, annexins constitute a group of ubiquitous cytoplasmic proteins involved in signal transduction. 2Annexin V is a 320-amino acid-residue, 36-kDa-protein that is folded into a planar cyclic arrangement of four repeats with each repeat composed of five alpha-helical segments. 3,4 It is expressed in various cell types, including placental trophoblasts and vascular endothelial cells. This protein is highly expressed in an apparently constitutive manner by placental trophoblasts and is displayed Quantification of Autoantibodies to Annexin V in Plasma by an "In House" Sandwich ELISA J. Braz. Chem. Soc. 454 on the apical membrane o...