Aplicación de un método in house para el estudio de la esclerosis múltiple / Application of an in-house method for the study of multiple sclerosis

Resumen El método de referencia para el estudio bioquímico de la esclerosis múltiple (EM) es el isoelectroenfoque (IEE) y la evaluación de las cadenas livianas libres (CLL) podría brindar una información adicional de relevancia. Por lo tanto, se propone evaluar la presencia de las CLL y la aplicabilidad de los estados de polimerización en el estudio de la EM. Se puso a punto un método compuesto por una separación electroforética en gel de poliacrilamida al 12,5% con posterior electrotransferencia (PAGE-WB) y se realizó la evaluación de 121 pacientes con sospecha de EM en simultáneo al IEE. Los patrones de PAGE-WB relacionados con la EM fueron el aumento de la concentración de monómeros Kappa o dímeros Lambda en líquido cefalorraquídeo (LCR) en comparación con el suero. Este método tuvo una muy alta significación de asociación con el IEE (p<0,0001) con sensibilidad del 95%, especificidad del 90%, VPP 83% y VPN 97%. La síntesis intratecal de CLL quedó evidenciada por el aumento de intensidad del monómero Kappa y/o el dímero Lambda observado en LCR. La técnica de PAGE-WB para CLL demostró ser un método alternativo para detectar la síntesis intratecal en pacientes con sospecha de EM.

Abstract The reference method for the biochemical study of multiple sclerosis (MS) is isoelectric focusing (IEF) and the evaluation of free light chains (FLC) could provide additional information of relevance. Therefore, it is proposed here to evaluate the presence of FLC and the applicability of the polymerisation states in the study of MS. A method consisting of a 12.5% polyacrylamide gel electrophoretic separation with a subsequent electrotransfer (PAGE-WB) was developed and the evaluation of 121 patients with suspected MS was carried out simultaneously with the IEF. MS-related PAGE-WB patterns were the increase in the concentration of Kappa monomers or Lambda dimers in CSF compared to serum. This method had a very high significance of association with the IEF (p<0.0001) with 95% sensitivity, 90% specificity, 83% PPV and 97% NPV. Intrathecal synthesis of FLC was evidenced by the increased intensity of the Kappa monomers and/or Lambda dimers observed in CSF. The PAGE-WB technique for FLC proved to be an alternative method to detect intrathecal synthesis in patients with suspected MS.

Resumo O método de referência para o estudo bioquímico da esclerose múltipla (EM) é a focalização isoelétrica (IEE) e a avaliação de cadeias leves livres (CLL) poderiam fornecer informações adicionais de relevância. Assim, propõe-se avaliar a presença das CLL e a aplicabilidade dos estados de polimerização no estudo de EM. Foi desenvolvido um método que consiste na separação eletroforética em gel de poliacrilamida a 12,5% com posterior eletrotranferência (PAGE-WB) e a avaliação de 121 pacientes com suspeita de EM foi realizada paralelamente à IEE. Os padrões de PAGE-WB relacionados com a EM foram o aumento na concentração de monômeros Kappa ou dímeros Lambda em líquido cefalorraquidiano (LCR) em comparação com o soro. Este método teve uma associação de significância muito alta com o IEE (p<0,0001) com sensibilidade de 95%, especificidade de 90%, VPP 83% e VPN 97%. A síntese intratecal de CLL foi evidenciada pelo aumento de intensidade do monômero Kappa e/ou dímero Lambda observado em LCR. A técnica PAGE-WB para CLL mostrou-se um método alternativo para detectar a síntese intratecal em pacientes com suspeita de EM.

Identification of major allergens from the house dust mites, Dermatophagoides farinae and Dermatophagoides pteronyssinus, by electroblotting

The allergens were separated from the extracts of house dust mites by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and identified by autoradiography. Over 30 protein bands of the whole body extract of Dermatophagoides farinae were apparent on 10-20% gradient SDS-PAGE, and 13 bands with MW between 93KD and 12KD bound with specific IgE antibodies in patients' sera sensitive to house dust mites. The major allergenic component of the whole body extract of D. farinae was the protein of MW 14-15KD, which was detected in 95.7% of 47 patients' sera sensitive to house dust mites. The extract of Dermatophagoides pteronyssinus supplied by Bencard Company, England was thought to contain feces enriched material as noted in a few broad protein bands on SDS-PAGE. Seven allergenic components were shown by autoradiography. The protein band of MW 14-15KD was one of the most frequently revealed allergens on autoradiography, which has appeared in 32.5% of 40 patients' sera sensitive to house dust mites. The electrobotting technique used in the present study was fast, convenient and highly useful for both the identification of allergen components and the screening of specific IgE antibody. The individual variations of IgE immune responses to the allergenic components of the two house dust mites were discussed.

Identification and partial purification of pollen allergens from Artemisia princeps

The pollen of Artemisia has been considered as the main late summer-autumn allergen source in this country. To identify its allergenic components, Artemisia princeps pollen extracts were separated by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membrane, where IgE binding components were detected by the reaction with sera of twenty Artemisia-allergic patients and 125I-anti-human IgE, sixteen components in the molecular range of 10,000 and 85,000 daltons were detected. Twelve bands bound to IgE from 50% of the sera tested, and two bands (37,000, 23,000 daltons) showed the highest (85%) frequency of IgE-binding in twenty sera tested. When the gel of SDS-PAGE with Artemisia pollen extracts was sliced into 11 allergenic groups (AG) and the protein of each AG was obtained by the gel elution method, the wormwool-RAST inhibition test showed that the AG 10 demonstrated to be the most potent, and the AG 7 was the next. Six AGs showed significant responses (more than 100% of wheal size to histamine, 1 mg/ml) on the skin prick test in more than 50% of the patients tested. It is suggested that electrophoretic transfer analysis with SDS-PAGE may be a valuable method for Artemisia allergen identification, and the possibility of partial purification of allergens by employing gel elution is discussed.

Identification and partial purification of pollen allergens from Artemisia princeps

The pollen of Artemisia has been considered as the main late summer-autumn allergen source in this country. To identify its allergenic components, Artemisia princeps pollen extracts were separated by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membrane, where IgE binding components were detected by the reaction with sera of twenty Artemisia-allergic patients and 125I-anti-human IgE, sixteen components in the molecular range of 10,000 and 85,000 daltons were detected. Twelve bands bound to IgE from 50% of the sera tested, and two bands (37,000, 23,000 daltons) showed the highest (85%) frequency of IgE-binding in twenty sera tested. When the gel of SDS-PAGE with Artemisia pollen extracts was sliced into 11 allergenic groups (AG) and the protein of each AG was obtained by the gel elution method, the wormwool-RAST inhibition test showed that the AG 10 demonstrated to be the most potent, and the AG 7 was the next. Six AGs showed significant responses (more than 100% of wheal size to histamine, 1 mg/ml) on the skin prick test in more than 50% of the patients tested. It is suggested that electrophoretic transfer analysis with SDS-PAGE may be a valuable method for Artemisia allergen identification, and the possibility of partial purification of allergens by employing gel elution is discussed.