In order to obtain high amounts of specific antibodies against porcine enteropathogenic E . coliThe course of antibody concentrations in serum and egg yolk was detected by a specific ELISA Advantages of egg yolk antibodies and potential therapeutic applications in human and veterin-strains laying hens were immunized with a vaccine for pregnant sows.system. ary medicine are discussed.
This paper describes a specific and highly sensitive ELISA system using monoclonal antibodies in order to assay an organophosphorus compound. The soman derivative methyl phosphonic acid, p-aminophenyl 1,2,2,-trimethyl-propyl diester (MATP) served as model substance. In order to obtain antibody-producing hybridomas BALB/c mice were immunized with MATP linked onto human serum albumin (HSA). The spleen cells of immunized mice were fused with syngenic plasmacytomas of the non-producer-line X63Ag8.653 with the aid of polyethylene glycol. To eliminate undesirable cross-reaction, common screening procedures were modified by directly coating the ELISA plates with hapten. Five out of 15 positive cell-lines were cloned by limiting dilution and further propagated. The respective immunoglobulin class and subclass of the obtained monoclonal antibodies was determined. Four of which were identified as IgG1, the other as IgG2a. After enrichment of antibodies in ascites and their isolation by protein A-sepharose, the affinity of various monoclonal antibodies was estimated in competitive inhibition enzyme immunoassay (CIEIA) by measuring the IC50 rates of free MATP. The rates were found to lie between 2.5 x 10(-6) mol/l and 4.3 x 10(-4) mol/l MATP. The IC10 rate for detectable MATP concentration was 5.4 x 10(-7) mol/l MATP. Test duration was 280 min. The reactivity of the monoclonal antibodies with structurally related substances was used to check their specificity. Cross-reaction turned out to be negative. In order to develop a direct competitive ELISA, MATP was linked to horse radish peroxidase (HRPO) by adding a spacer. This helped to reduce total duration to 40 min.(ABSTRACT TRUNCATED AT 250 WORDS)
The aim of the present study was the development of a sensitive and specific ELISA system for the quantitative and qualitative assay of chicken Ig Isotypes G, M, and A using monoclonal antibodies. Five hybridoma cell lines were developed that synthesized specific antibodies against chicken IgG and three lines each producing specific antibodies against IgM or IgA. Using an immunodiffusion test, the subclasses were determined. Isolation of monoclonal antibodies from ascites was carried out by way of affinity chromatography with protein G sepharose. The purity of the eluates were determined by both SDS-PAGE and HPLC. A Sandwich ELISA was found to be the most suitable technique for the assay. Specificity testing was carried out by Western blotting. An epitope analysis was also carried out. By variation of the single steps concerning incubation times, quantities, and concentrations of the substances to be applied, the whole procedure was optimized. Assay limits for individual Ig isotypes were determined. The limits were 20 ng/mL for IgG, 80 ng/mL for IgM, and 160 ng/mL for IgA.
An alpha-N-acetylgalactosaminidase IV able to remove blood type specificity of human A(II)-erythrocytes and not effecting B(III)-erythrocytes was isolated from the marine bacterium Arenibacter latericius KMM 426T. The alpha-N-acetylgalactosaminidase IV preparation exhibits high activity during inhibition of hemagglutination with blood group substance A containing determinants analogous to A-erythrocytes. The enzyme has a pH optimum from 7.0 to 8.0 and completely retains its activity during 30-min heating at 50 degrees C and for a week at 20 degrees C. The enzyme can be stored under the sterile conditions for any length of time at 4 degrees C, but it does not withstand freezing. The alpha-N-acetylgalactosaminidase is resistant to NaCl; for p-nitrophenyl-alpha-N-acetyl-D-galactosaminide, the Km is 0.38 mM. The molecular mass of the enzyme determined by gel filtration is 84 kD.
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