The Detection And Research Of Anti-antibiotic Antibody And Screen Of Rare Blood Group By Moleculer Method

Not even the body patient of the drugs, but also the production of anti-drug antibodies would cause of the use of medicine especially the antibiotics during the clinical therapy. After the antibodies produced, drugs, anti-drug antibodies and red blood cells will form an immune complex, resulting in the destruction of red blood cells and removing from the body. This may cause immune hemolytic anemia. Drugs used invalidly would endanger the patients. The incidence of anti-drugs antibody produceed is very low, but more than 100 medicines that can cause drug-resistant antibodies have been reported. With advances in pharmaceutical industries, more and more drugs will be put into use. Therefore, establishing an effective method to detect anti-drug antibodies is widely used in drug-induced immune hemolytic anemia detection and the guidance of medication. The serological method reacted of drugs, common RBC and plasma or RBC elution of the patients is the classic method of anti-drug antibodies detection. But the method can't fully explain the anti-drugs antibodies in patients against with the complex of RBC membrane and the drugs, or the modifid membrane structure by drugs. Therefore, understanding the mechanism of antibody production is profoundly significant for the definition of anti-drugs antibody. In this study, penicillin, azithromycin, levofloxacin, cefuroxime, cefepime, cefoperazone, Cephradine, Ceftazidime and Cefaclor 9 antibiotics widely used in Shanghai were selected, and prepared of drug-coated RBC in special buffer systems. We used the drug-coated RBCs to detect the anti-drug abtibodies. The coated cells mainly against the antibodies produced by hapten mechanism and the autoantibodies against the new antigen site exposed after the reaction of drug and RBC membrane protein. Samples of the 181 cases (61 cases of blood match difficultly,51 cases of antibody screening and Coombs'test in the Shanghai Blood Center blood group reference laboratory, and 70 cases of bone marrow transplantation patients in Ruijin Hospital) were detected. Using of fluorescent-labeled antibodies against CD55 and CD59 detecte the change of the two CD molecular in cephalosporin treated RBC. And penicillin as the represente drugs, react with the RBC membrane protein and then detect the bingding site by Far-Western-Blotting method.By serological methods,181 samples were detected of drug-coated RBCs.4 cases (6.56%) were found containing anti-penicillin antibodies,1 case (0.16%) has specific anti-cephalosporin antibody and 2 cases (0.33%) of non-specific anti-drug antibodies in 61 samples of blood match difficultly; 4 cases (8%) found containing specific anti-penicillin antibody in 50 samples of antibody screening and Coombs'tests; and only 1 case (1.43%) found containing specific antibodies against penicillin in 70 samples of bone marrow transplantation. From the perspect of targeted drugs, the samples containing anti-drug antibody against penicillin is most of 9 cases (4.97%), the sample containing antibody against cephalosporins is only 1 case (0.55%), rather than the sample contaning nonspecific anti-drug antibody of 2 cases (1.1%). Flow cytometry showed that the CD55 and CD59 on the membrane of RBC treated by cephalosporin were decreased. Far-Western-Blotting results show the RBC membrane protein binging with penicillin have the molecular weight over 130KD.There is a very low probability for the produce of anti-drug antibodies.The results of penicillin reacting and the RBC membrane proteins suggest that drugs may act on blood group antigen proteins. Certain protein on RBC membrane which drugs will be combined is unknown.Another study of this thesis is to analyze the frequency of CO, YT, LU, K, DI 5 rare blood group alleles in the Chinese population. There are about 300 antigen substances on human red blood cell, belonging to 30 blood group systems. Each blood type may lack certain blood group antigens in a very low chance. These RBC will show a rare blood group. It is difficult to detect the presence of rare blood group by the conventional serological, clinical transfusion reactions may lead to the occurrence. For the difficulty of serological methods for rare blood proup individual, we can establish a stable molecular biological method, to detect rare blood group system. Rare blood group test to the donor and the accepter will reduce the possible of blood transfusion reaction in the the maximum rare.According to the SNP sites of CO, YT, LU, K, DI and other blood group gene, we designed sequence-specific primers to establish a stable amplification system, and screened a variety of rare blood group antigen in a system. A total of 438 cases of samples (random sample of 321 cases in Shanghai, the 61 samples of Tu minority,28 samples of Dong minority and 28 samples of Lisu minority) were screened 8 allele sites of Coa, Kpb, Yta, Lub, k, Dib, Jsb1910 and Jsb2019 in 5 blood groups. All the samples were detected by multi-PCR-SSP. And there are O cases of Coa-, Kpb-, Yta-, Lub-, k-, Dib-, Jsb1910-and Jsb2019-samples.In this study, based on the conventional screening of anti-drug antibodis, we also have maken a preliminary study of further mechanism in anti-drug antibody production. Instead of conventional marked proteins, using the drugs as the bridge molecule, study the drug target sites on RBC membrane proteins by Far-Western-Blotting method. Though detection of the anti-drug antibodies by the establishment of drug-coated RBC and the study of the mechanism of anti-drug antibodies production, we found that anti-drug antibodis have some chance of appearance in population used antibiotics, there will be some changes of the membrane proteins after combining with cephaliosporins. On the other hand, screening of the blood group antigen alleles by multi-PCR-SSP make up the small number of rare blood group antigens detection and the affection about lack of antibodies application or the restriction under the lowest efficiency of current serological tests. And this also expands the rare blood group antigen detection and detection in clinical specimens. Although a false positive results shows in the research, coupled with serological technical and other further tests, detecting the rare blood group antigens before transfusion, will help screening of suited blood targeted, reducing blood transfusion reactions, and improving transfusion efficacy.