Establishment Of System For Detection Of SEC In Staphylococcal Enterotoxin C Injection And Preliminary Bioactivity Analysis Of Staphylococcal Enterotoxins

Superantigen is a kind of proteins,which can stimulate non-specific T-cell proliferation based on their V_βreceptor usage.Their abilities to stimulate lymphocyte proliferation and lymphokine production at concentrations as low as 10^-13-10^-16M makes them among the most potent T cell activators known.As superantigens,SEs have profound effects on the immune system,both acute and long-term.Acute effects include food poisoning and toxic shock syndrome.Long-term effects include autoimmune diseases and immunodeficiency such as atopic allergy,Kawasaki's disease and periodontal disease.Further,superantigens can cause deregulation of immune responses,resulting in autoimmune diseases of immunodeficiency.However, SEs can induce enhancement of desirable immune responses if used correctly,and their ability of inducing apoptotic death of tumor cells is of particular interest.The filtrate of Staphylococcus aureus culture has been used as ampul named Staphylococcal Enterotoxin C Injection for cancer therapy in clinic for ten years in China and proved to be effective,such as Gaojushen(also named HAS),Sifusheng and Engefei.The active constituent of these three injections is claimed to be staphylococcal enterotoxin C(SEC),and the content of SEC is used to be the quality control index.However,the correct content of SEC was not known and the relative amount of SEC was very low because of the complicated components of the filtrate. Further more,there was no certifiable evidence to illustrate which were the active components actually.Therefore,firstly,a rapid,reliable,and sensitive immunological assay system is eagerly required,which is used to be a quality control of the content of the SEs for Staphylococcal Enterotoxin C Injection.Secondly,SEs are a family of many major serological types of heat stable enterotoxins(SEA through SEE,SEG through SEQ, TTST-1,TTST-2 and SEC including subtypes of SEC₁,SEC₂ and SEC₃)produced by Staphylococcus aureus and share a number of genetic and biochemical characteristics with similar functions,but their working sites and mechanisms are different.Though Staphylococcal Enterotoxin C Injection proved to be safe and effective and there was no evidence showing organ damage or decreases in white cell counts over the years of clinical treatment,the complicated components make it difficult to control the inevitable side effects and quality of the drugs.It is possible to find one or several pure SEs and use the ideal combination which has the lowest side effects and best treatments in clinic,and that will be helpful to the improvements of effects, controllable side effects and quality of these drugs.In this research,we designed schemes in following studies.First,we established a proper ELISA system for the detection of SEC for Staphylococcal Enterotoxin C Injection used in clinc,which will improve the quality control of Staphylococcal Enterotoxin C injuction.Second,we established a proper bioactivity analysis system of SEs in vitro,for further investigations of the active components of these drugs and establishment of foundations for development of novel anti-cancer drugs.A.Establishment of BA-ELISA system for detection of SEC of Staphylococcal Enterotoxin C injection1.Production and identification of polyclonal and monoelonal antibodies of Staphylococcal enterotoxin C₂(SEC₂)1.1 Preparation and identification of polyclonal antibodies of SEC₂Three rabbits were hyperimmunized by injection of an emulsion of SEC₂-His in complete Freund adjuvant.After four times of immunization,we used double-gel immunodiffusion assay and indirect ELISA to test the titer of the serum.Then its specificity was identified with Western blotting.Finally,rabbit blood was collected from its heart and the polyclonal antibodies were in the collected serum.1.2 Purification of polyclonal antibodiesThe polyclonal antibodies were purified from the serum by two-step method using caprylic acid and amine sulphate.The caprylic acid was applied to eliminate the albumin and the solid amine sulphate was applied to deposit IgG from the serum. Then the deposition was dissolved into PBS buffer and dialyzed in PBS buffer for 48h.1.3 Selection of McAbAfter several times of selections and limiting dilutions,the 202 hybridoma cell strain was selected with supreme potency.The potency of McAb was detected by indirect ELISA and the potency of 202 strain is 1.9×10⁵.1.4 Purification of the McAbThe protein A can bind with the Fc region of IgG and it was used to purify the McAb from the ascites.The ascites was purified by the protein A affinity column.The McAb was eluted by the elution buffer,after washing the column with binding buffer. The result of SDS-PAGE indicated that pure McAb was obtained.2.Establishment of the immunoassay system for SEC₂2.1 Establishment of sandwich ELISA methodThe purified McAb was coated onto a solid-phase support to capture the antigen SEC₂ from the test material.The SEC₂-specific polyclonal antibodies were used to detect the content of the captured SEC₂.A third anti-rabbits IgG antibodies conjugated to HRP enzyme were used to detect the content of SEC₂-specific polyclonal antibodies which had been binded with the captured antigen.The content of the protein SEC was detected with the subsequent reaction of the enzyme and the substrate,based on a quantitative colorimetry.2.2 Establishment of BA-ELISA methodBased on the method of sandwich ELISA,we used biotin-streptavidin system to enlarge the reaction signal.The purified McAb was coated onto a solid-phase support to capture the antigen SEC₂ from the test materials.The SEC₂-specific polyclonal antibodies were used to test the content of the captured SEC₂.Biotin labeled anti-rabbits IgG antibodies and streptavidin which were conjugated to HRP enzyme were used to detect the content of SEC₂-specific polyclonal antibodies.The content of the protein SEC was detected with the subsequent reaction of the enzyme and the substrate,based on a quantitative colorimetry.B.Comparative studies on bioactivity analysis of two kinds of Staphylococcal Enterotoxin C injection and several kinds of SEs1.Establishment of a bioactivity analysis system for SEs in vitroWe used MTT assay to establish a proper system to examine the bioactivity of SEC₂,both wild type and recombinant type.The MTT assay on the purified nSEC₂ and rSEC₂ was applied to investigate their abilities of stimulating T cells and inhibiting the proliferation of tumor cells in vitro.Three kinds of tumor cells were investigated in this assay.2.Abilities of stimulating T cells of two kinds of Staphylococcal Enterotoxin C injection and eight kinds of SEsComparative studies on the abilities of stimulating splenic lymphocyte of ICR mice of Gaojusheng,Engefei and rSEA/rSEO/rSEM/rSEN/rSEG/rSEC₂/nSEC₂/rSEI were investigated,based on the established model for bioactivity analysis of SEs.3.Abilities of stimulating T cells of seven kinds of SEs working together mutuallyComparative studies on the abilities of stimulating splenic lymphocyte of ICR mice of rSEA/rSEO/rSEM/rSEN/rSEG/rSEC₂/rSEI mixed mutually with equal quality were observed,based on the established model for bioactivity analysis of SEs.It is an initial exploration for the synergistic effects which may exist in SEs.As a conclusion,First,we established a proper BA-ELISA system for the quality control of Staphylococcal Enterotoxin C injection.This system was simple,rapid with high sensitivity and stablity.The results of the detections of three different kinds of Staphylococcal Enterotoxin C injection indicated that two of them were unqualified and over 99.9%contents of proteins of Staphylococcal Enterotoxin C injection were unknown.In addition,it also established foundations of highly acute quantitative detections for other SEs.Second,we established a proper bioactivity analysis system for SEs in vitro. While used separately,several SEs,such as SEI,SEM and SEN showed greater bioactivities than other SEs,including two kinds of Staphylococcal Enterotoxin C injection and SEC₂.The results of the bioactivity of seven SEs mixed with equal quality mutually indicated the possibility of synergistic effects within SEs.This research was an initial exploration for the selection,combination and investigation of SEs and provided evidences for the further researches and investigations of novel potentially therapeutically important protein drugs of SEs.