The Preparation Of Monoclonal Antibodies Against CMV Pp65Antigen And Establishment Of Methodology For The Detection Of The Cytomegalovirus Antigenemia As Well As Clinical Comparison Test

[BACKGROUND]Human cytomegalovirus was one of the most common recessive infection pathogens in human, and the serum cytomegalovirus antibody positive rate was very high in common population. CMV infection generally didn’t cause serious clinical consequences in people with normal immune function, but the virus would been dormant in vivo for life. The dormant cytomegalovirus in vivo would turn to active infection and caused serious clinical consequences in patients with impaired immune systems. Cytomegalovirus low phosphorus matrix phosphoprotein (CMVpp65antigen) was coded by CMV UL83gene. It was a kind of tegument proteins about65KD and was the most viral protein under the active replication. Currently, the CMVpp65antigenemia was accepted as the early and rapid indicator for CMV active infection. However, the diagnostic kits for CMV pp65antigenemia detection in domestic market were almost imported.[OBJECTIVE]The purpose of this study was to prepare monoclonal antibodies against CMV pp65antigen, and an indirect immunofluorescent method based on the monoclonal antibody was set up to detect peripheral blood CMV pp65antigenemia, which would led to development of domestic kits.[MATERIALS AND MATHODES]Cytomegalovirus inclusion antigens were prepared from human embryonic lung fibroblast (MRC-5) infected with CMV AD169for24,48and72hours and were inactivated by formaldehyde. BALB/C mice were immunonized with the CMV inclusion antigens as well as CMV recombinant antigen for preparation of monoclonal antibodies against CMV pp65antigen with a standard methodology. An indirect immunofluorescent method based on the monoclonal antibody was set up to detect peripheral blood CMV pp65antigenemia. The clinical comparison tests were carried out between the self-bult method and the imported kit for CMV pp65antigenemia detection. Then we analyzed the consistency of the two methods by using the SPSS17.0statistical software.[RESULTS]1. Two strains of hybridoma which produce anti-CMV pp65McAbs were cloned and named13D1-3,29F1-1respectively. The immunogloblin subclasses of13D1-3and29F1-1were determined as IgG1and lgG2a. There were no cross reactions between the two McAbs with other virus or cells antigen such as HSV-1/2, MRC-5, BGMK, VZV. The titers of the mouse ascites produced by hybridomas of13D1-3and29F1-1was1:5000and1:2000respectively in the indirect immunofluorescent assay. The ascites antibodies still had the immunological characteristics after protein G affinity chromatography purification.2. The method to detect peripheral blood CMV pp65antigenemia based on13D1-3McAb was established. The peripheral blood leukocytes were fixed with3.7%formaldehyde, permeabilized by IGEPAL CA-630, blocked with0.5%BSA in0.01M PBS. The13D1-3McAb and FITC labeled sheep against rat IgG were used for the detection of CMV pp65antigen in peripheral blood leukocytes. Good consistency between the imported kit and selt-built method were found, positive coincidence rate was88.8%, negative coincidence rate was98.9%, the total coincidence rate was97.3%, and kappa value was0.895.[CONCLUSION]Monoclonal antibodies anti-cytomegalovirus pp65protein were prepared successfully, and the indirect immunofluorescent method based on13D1-3McAb for detecting peripheral blood CMV antigeneima was found to have good compliance with CMV BriteTM kit. [BACKGROUND]Syphilis is a sexually transmitted disease caused by infection with the Treponema pallidum, blood transfusion or wound also can spread. Now, syphilis is still the important public health problem worldwide. There was nearly12million syphilis infections were reported every year. Traditional screening algorithm which used the nontreponemal assay as the initial screening assay and followed by treponemal assay for sure was recommended by The Centers for Disease Control and Prevention (CDC), had high missed diagnosis rate and low sensitivity. The reverse algorithm that began with treponemal assay and followed by nontreponemal assay was gradually widely accepted, but it recommended by the western countries where pregnant women or other populations who had risk sexual behavior were recommended to take the syphilis serological screening. However, in China, because of special medical environment, routine screening tests for syphilis were taken in all patients who would have invasive intervention such as endoscopy or transfusion or surgery, as well as pregnant women at first antenatal visits. So these screening modes may not be suitable for the Chinese population, especially in comprehensive hospital patients with low risk patients.[OBJECTIVE] The goal of the present study was to analyze the reverse screening algorithm for syphilis and improve the screening procedure in order to reduce false positive rate by establishment of a new serologic screening model which will be applied to low-risk population in China.[METHODS]The treponema pallidum chemiluminescence micro particle immunoassay (CMIA) was used for initial screening for syphilis in Peking Union Medical College Hospital, China between August2013and January2014. The serum specimens with positive reaction on CMIA were re-tested by treponema pallidum particle agglutination assay (TP-PA) and syphilis rapid plasma regain test (RPR). FTA-ABS was used for confirmation in specimens with discordant results (CMIA-positive but TPPA-negative).[RESULTS] There were41275assays for syphilis serology screening in Peking Union Medical College Hospital between August2013and January2014. Most of the samples came from the departments which would take invasive measures for diagnosis or treatment, such surgery departments (30%), gynecology (17%), internal medicine (14%), or gastroenterology (13%). While the sample proportion from the department which was most likely associated with syphilis infection such as dermatology, infectious department or neurology department only had three percent totally. Totally608assays (M/F264/344, average age of50.87, age rangel-100) were positive reactive on CMIA tests with the average S/CO was13.18C1-54.92), so the initial positive rate were1.47%(608/41275). Of the608CMIA-positive samples, there were only369assays (60.69%) with positive reaction on TPPA test and182assays (29.93%) with positive reaction on RPR test.239samples with discordant results (CMIA-positive and TPPA-negative) were all negative on FTA-ABS IgM test. But there were19samples with positive reactive and220assays with negative reactive on FTA-ABS IgG test[CONCLUSION]False positive results were found in initial screening of treponemal test with CMIA. The improved screening algorithm is designed as that treponemal test (CMIA) is performed as the initial screening test, if CMIA result is positive; TPPA test is carried out for confirmatory. The positivity is reported if the confirmatory test (TPPA) is positive and the RPR is also tested in order to know more about the disease activity or monitor the treatment efficacy. The negativity is reported if the confirmatory test (TPPA) is negative.