Production and characterization of monoclonal antibodies against protein antigens of Pseudomonas pseudomallei

Abstract

Melioidosis is a disease caused by Pseudomonas pseudomallei. In Thailand, most of the patients lived in the northeast. At present, melioidosis oases were increasingly reported. The clinical manifestations are variable tanging from chronic to aoute fatal septicemia which the mortality rate is 80-90 %. Moreover, signs and symptoms of melioidosis can mimio many diseases. Isolation and identification of cultures from patients specimens is the only available confirmatory method for melioidosis but it is time consuming. Serological tests have been developed and modified for the rapid diagnosis. Most of them are concerned with the detection of antibody. However, these tests are of limited value because of the high background antibody titer in normal individuals, particularly in those resided in the endemic areas of infection Thus, the antigen detection approach should be the method of choice for a rapid diagnosis. In the present study, the monoclonal antibodies specific to the protein antigen of Pseudomonas pseudomallei (prepared by extracting with veronal buffer and followed by precipitation with trichloroacetic acid) were produced and characterized by indirect ELISA, immunoblotting and radioimmunoprecipitation. Ten specific clones were obtained. They did not cross react with the protein antigens prepared from Ps. aeruginosa, Ps. cepacia, Ps. stuzeri, K. pneumoniae, 5. typhi, E. coli and L. pneumophila. Clone 7B6 was the only one that gave strong reactivity by all three tests in this study and was evaluated for its potential in detecting the Ps. pseudomallei antigen. It was found that this clone could detect the protein antigen of Ps. pseudomallei in aqueous solution at a concentration as low as 15 ng/ml by B-SA ELISA. However , this clone failed to detect the antigen in the serum specimens from melioidosis patients. This failure may be due to the presence of immune complexes in the patients sera. Therefore, if the antigen was present in any of the specimen, it must be present at a concentration below 15 ng/ml which was the limit of the sensitivity of the test. However, it is possible to use MoAb for the detection of antigen in the patients specimens if the MoAb used has specificity that is different from the antibody found in the patient serum. Further studies on the epitope mapping are needed if one wants to overcome the interference by immune complexes.