Cross-reaction Of Broad Spectrum Monoclonal Antibodies To Lipopolysaccharide And Establishment Of Endotoxin Double Antibodies Sandwich ELISA

Lipopolysaccharide (LPS) is the main part of envelop of Gram-negative bacteria. Low dose of LPS could induce broad biological reaction. Clinically, 65% hematosepsis is caused by Gram-negative bacteria infection. The morality accounts for 22.7%---40.0%. Over a long period of time, endotoxemia have been diagnosed by LT ( limulus Test, LT) clinically. However, due to nonspecificity of the regents and the influence of multifactors in plasma, the diagnosis of endotoxemia has not been specific and accurate. The ~~~successful preparation of McAb to LPS provides important experimental tool~ for diagnosis of LPS. Aided with the cross-reaction of 2 broad spectra monoclonal antibodies, this study firstly established the endotoxin double anti-bodies sandwich ELISA, which provides a prompt, easily-performed, highly-specific, and highly-sensitive detective method. This study consists of three major parts: 1. Observation of associated factors in the ascites production of hybridoma cell lines. In order to obtain a great amount of hybridoma cell lines ascites, we observed the influence of ascites production caused by such factors as adjuvant of pretreatment mice, types of mice,and hybridoma cell lines. Results: the result caused by pretreatment mice treated with pristane and Freund's incomplete adjuvant was more dramatic than that caused by pretreatment mice treated with liquid paraffin. The ascites production of the former was quicker and more excessive than that of the latter (P<0.5). The production of ascites was much more in female mice having ever given birth to a baby mice than in the mice having never given birth to a baby mice; the ascites of C3A2 cell line was more than that of H3 D3 cell line. But the difference was not significant (p>O.O5). 2. Confirmation of cross reaction spectra of different species bacteria's endotoxin caused by 2 monoclonal antibodies. It is the focus for the establishment of endotoxin ELISA whether endotoxin monoclonal antibodies can cause broad cross-reaction in the endotoxin of different bacteria species. Therefore, 21 genera, say, 48 species of bacteria were collected, and ELISA and PHA were respectively applied to the 2 monoclonal antibodies. Results: the cross-reaction was strongest with such Enterobacteriaceace as Escherichia, Enterobacter, Klebsiella, Proteus, and was comparatively weak with such Glueose nonfermeuters as Acinetobacter, Pseumonas, Alcaigenes, as well a with Candida. No cross-reaction was observed with Gram-positive bacteria. As far as cross-reaction was concerned, no different significance was observed between ELISA and PHA (passive hemolysis assay). The cross-reaction of C3A2 was stronger than that of H3D3. But their broad spectra were almost the same. Thus, 2 broad spectra LPS McAb can be used as antibodies for LPS detection. 3. Establishment and initial application of endotoxin double antibodies sandwich ELISA. Under both the guide of the associated literature and our experiment, endotoxin double antibodies sandwich ELISA was established by us. The method was experimented with sensitivity, specificity, accuracy and repetition, and 40 clinic samples were detected too. Results: the sensitivity of double antibodies sandwich ELISA (DAS ELISA) was 50 pg/mi, higher than that of LT, lower than that of quantitative LT instrument turbidimetric method (over lpgIml). But the accuracy of DAS ELISA was higher...