Quantification of high mobility group protein (HMGB1) by enzyme linked immunosorbent assay (ELISA)

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Quantification of high mobility group protein (HMGB1) by enzyme linked immunosorbent assay (ELISA)


Author: Sin, Hon-wa
Title: Quantification of high mobility group protein (HMGB1) by enzyme linked immunosorbent assay (ELISA)
Degree: M.Sc.
Year: 2006
Subject: Hong Kong Polytechnic University -- Dissertations
Protein binding
Protein microarrays
Enzyme-linked immunosorbent assay
Department: Dept. of Applied Biology and Chemical Technology
Pages: viii, 56 leaves : ill. ; 30 cm.
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b1893943
URI: http://theses.lib.polyu.edu.hk/handle/200/1420
Abstract: High mobility group protein (HMGBl) was discovered and purified in 1973. It was discovered as a nuclear protein and considered as a structural protein. HMGBl is member of the HMGB protein family. HMGBl was believed to involve in some vital functions, in which HMGBl deficient mice died within the first day of life due to hypoglycaemia. HMGBl consists of 259 amino acids, and with molecular weight of approximately 25000. The human HMGBl gene is located on chromosome 13. The protein is highly conserved among different mammals, which Chinese hamster, pig, bovine, rat, canine HMGBl has more than 97% homology to human. HMGBl is highly charged with about 50% amino acid in the chain are charged. It has a tripartite structure that is consisted of two repeated domains, domain A and domain B, and an acidic tail. Binding of HMGBl to DNA chain will cause bending of the DNA chain. It is believed that the binding is not sequence specific. DNA bending has been proposed to facilitate or enhance the effect of other transcription factors. In 1999, HMGBl has been found to be a pro-inflammation mediator. HMGBl have been found to be involved in inflammation reaction of different organs, such as lung and liver. It was believed that HMGBl have an important role in inflammation reaction. Besides, some studies showed that anti-HMGBl antibodies have some protective effect in mice that has induced systemic sepsis. Due to its inflammation mediator characteristics, there were a lot of studies on the relationship between HMGB1 and inflammation has been performed. The current method for quantifying HMGB1 is Western blot technique. This is a complicated and time consuming technique. Therefore, a sensitive, fast and easy technique will be desired. In this study, a competitive enzyme linked immunosorbent assay (ELISA) of HMGB1 was developed by using commercially available monoclonal anti-HMGBl antibody. The competitive ELISA has the characteristic of being sensitive, fast and easy to perform. The ELISA is able to produce reproducible result on aqueous samples. However, the performance is heavily dependent on the quality of the purify HMGB 1 protein. The result of this study shows the possibility of quantify HMGB1 by competitive ELISA. The time needed for HMGB1 research may be shortening by using ELISA in quantifying HMGB 1. The high sensitivity of ELISA may also allow exploration into more secret about HMGB1 which have not been discovered in past studies.

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