| Objectives: It is very important for a patient to detect the blood platelets antibodies.The result is used for judgment the treatment effect in the disease. No a real clinical practical serological method appeared to be used by now.There are many differences between the results gained from the existing techniques. The methods of the molecular biology are not suitable for the clinical normal application. From the experiment principle analysis, the majority of serological methods are to adopt the blood platelets from the richly platelets plasma to wrap the micro-plate and further analyse the antibody with the direct or indirect methods. These techniques only detect whether the platelets surface adheaded Ig or the Ig in serum can integrate the platelets,can not distinguish that the antibody is the blood platelets particularity antibody or not, and exist some defects such as low particularity and/or the sensitivity,tedious operation,more blood volume etc.Therefore,it is necessary to found a set of more intelligent,accurate,practical and feasible ELISA technique to enhance the credibility of the experimentation.This research is to establish a capture-antigen-wrap ELISA method for detecting antibodies against blood platelets. This method also can filtrate the right donor at the same time.It serve for the clinical blood transfusion.Study design and methods: In order to establish a capture-antigen-wrap ELISA system,extraction and immunogenicity of platelets outer membrane protein complex (POMPC) are necessary, The admixture of O type blood platelets was constructed in order to the next experiment. Four methods were compared:SDS-PAGE electrophoresis, Rabbits test, Western-blotting and ELISA were used for testing the effect of dissolution and the immunogenicity.The Triton X-100 was of the best solubility among them. But the others are incomplete. The antigenicity of its deliquescence is strong and having the extensive cross reaction. The proteins of 50kD-160kD were the major besides some minor in the POMPC were determined. Through rabbit's immunity test, affinity purified, we gained highly reactive antibodies.Purified polyclonal antibody was immobilized for capture antigen.The donor's or the admixture of O type blood platelets was capture antigen.then reacted with the detected serum, second IgG-HRP as conjugate to report the ruselts.180 negative samples were detected with no false positive. When the method were used to detect 124 samples which had been reported anti-PLT negative by SEPSA, 13 samples showed positive. These 13 samples were tested with more specific methods such as FCM, and all of them remained positive. The results indicated that the capture-antigen-wrap ELISA we established showed high sensitivity and specificity.Results and Conclusion: False-positive results are due to some complications.Such as rheumatoid factor, hyperglobulinemia,non-specific antibodies to heterophilic antigens and fusion protein SOD in indirect ELISA.They are easy to mix the individual antigens.To resolve this problem, a more sensitive and specific capture-antigen-wrap ELISA may be helpful. the method of SEPSA and the microcolumn test are widely used to detect the antibody of platelet.These techniques cannot make sure the particularity of the antibody .The method we established can detect the PAIg and PBIg synchronously. The application of polyclonal antibody is more economical than the monoclonal and conquered the influence of the multiple-epitope.The period of diagnosis is shortened for those sufferers. The method is familiar and stable.Preliminary studies indicate that the new capture-antigen-wrap ELISA based on the purified polyclonal antibody is very sensitive and specific. Higher sensitivity may be obtained antibodies other than IgG (particularly IgM) could also be detected by this method, and this may replace the BPA. |
PDF Full Text Request