Development of assay systems for canine pancreatic dieaseses using anti-trypsin monoclonal antibodies

取得学位：博士(薬学)，学位授与番号：博乙第245号，学位授与年月日：平成14年3月22日,学位授与年：200

An ELISA protocol to improve the accuracy and reliability of serological antibody assays

To assay serum antibodies by indirect ELISA, it is critical to eliminate a variety of false positive and negative reactions attributed to the principle. These include 1) the background (BG) noise reaction caused by hydrophobic binding of immunoglobulin components in sample specimens to solid surfaces, 2) false positive reaction caused by non-specific binding of immunoglobulins to target-antigens by protein-protein interactions, and 3) other false positive and negative reactions caused by buffer components. No current blocking agents can prevent these false positive and negative reactions, and antibody assay results vary significantly depending on the buffer system used. To address these fundamental problems, we investigated all types of non-specific reactions involved in indirect ELISAs, and the blocking efficacy of current buffer systems and a newly developed ELISA buffer, ChonBlock™. The accuracy and reliability of these assay results were examined in detail by inhibition tests in individual buffer systems. Based on these studies, we are providing a definitive ELISA protocol for all users to improve ELISA technique and obtain accurate, reliable, and reproducible assay data against a variety of antigens

Development and evaluation of mouse anti-Ara h 1 and Ara h 3 IgE monoclonal antibodies for advancing peanut allergy research

Immediate hypersensitivity reactions to peanuts are a considerable public health concern due to the acute and severe IgE mediated reactions. To conduct research on the pathogenesis and therapeutics of peanut allergies, it is imperative to have mouse anti-crude peanut extract (CPE) IgE monoclonal antibodies (mAbs) for both in-vitro and in-vivo assays. Without these tools, it is difficult to advance research in this field.In this study, four hybridomas producing anti-CPE IgE mAbs were developed and the IgE mAbs were validated using immune-blot analysis, Sandwich ELISA, Indirect ELISA, a cell-based assay using RBL-2H3 cells, and footpad type I hypersensitivity reaction studies in mice.The results indicate that two of the four mAbs can be effectively used for both in-vitro and in-vivo peanut allergy studies, as they induce allergic reactions with sensitization alone in mice. These novel anti-Ara h1 and Ara h 3 IgE mAbs, in combination with the detailed protocols outlined in this article, offer valuable guidance for studying acute allergic reactions involving mast cells across various platforms. With some considerations, the IgE mAbs can significantly advance peanut allergy research

New Studies of Pathogenesis of Rheumatoid Arthritis with Collagen-Induced and Collagen Antibody-Induced Arthritis Models: New Insight Involving Bacteria Flora

Much public research suggests that autoimmune diseases such as rheumatoid arthritis (RA) are induced by aberrant “self” immune responses attacking autologous tissues and organ components. However, recent studies have reported that autoimmune diseases may be triggered by dysbiotic composition changes of the intestinal bacteria and an imbalance between these bacteria and intestinal immune systems. However, there are a few solid concepts or methods to study the putative involvement and relationship of these inner environmental factors in RA pathogenesis. Fortunately, Collagen-Induced Arthritis (CIA) and Collagen Antibody-Induced Arthritis (CAIA) models have been widely used as animal models for studying the pathogenesis of RA. In addition to RA, these models can be extensively used as animal models for studying complicated hypotheses in many diseases. In this review, we introduce some basic information about the CIA and CAIA models as well as how to apply these models effectively to investigate relationships between the pathogenesis of autoimmune diseases, especially RA, and the dysbiosis of intestinal bacterial flora

Linkage of RF with IgG and IgA antibody responses to bacterial pathogens in RA patients.

<p>IgG and IgA antibody levels against individual pathogens and their IgA/IgG antibody ratio were analyzed for possible correlation with RF levels in 54 patients with RRP (a) and 101 patients with non-RRP (b) by Spearman’s rank correlation coefficient analysis. NOTE: Pink: significant correlation at p<0.05, Blue: trending toward correlation at 0.05≤p<0.15, No color: no correlation.</p

Comparison of antibody responses to potential pathogenic environmental agents between RA and NL controls.

<p>IgG and IgA antibody levels against <i>E</i>. <i>coli</i>-LPS, Pg-LPS and PG-PS were determined in sera from 38 NL controls, 54 patients with RRP and 101 patients with non-RRP (a). IgG and IgA antibody levels of individual patients were divided by the average values of NL controls, and shown as IgG and IgA index values (b). Data are shown as median and interquartile range (IQR). <i>E</i>. <i>coli</i>: <i>Escherichia coli</i>, Pg: <i>Porphyromonas gingivalis</i>, LPS: lipopolysaccharide, PG-PS: peptidoglycan polysaccharide from <i>Streptococcus pyogenes</i>, RRP: rapid radiographic progression, NOTE: Index 1: sum of anti-<i>E</i>. <i>coli</i>-LPS + anti-Pg-LPS, Index 2: sum of anti-<i>E</i>. <i>coli</i>-LPS + anti-PG-PS, Index 3: sum of anti<i>-E</i>. <i>coli</i>-LPS + anti-Pg-LPS + anti-PG-PS.</p