Enzyme-linked immunosorbent assay (ELISA) using a glycolipid antigen for the serodiagnosis of melioidosis

Abstract

The serodiagnosis of melioidosis is commonly performed with tests using protein or polysaccharide as antigen. However, due to the low sensitivity, specificity and difficulty in the preparation of the antigens, more simple, precise and reproducible diagnostic tests were required. A purified glycolipid antigen (GL) which is a specific lipid component of Burkholderia pseudomallei has been used in an ELISA. With this antigen, specific immunoglobulin G (IgG) was detected in 49 out of 50 melioidosis sera. IgG was also detected in 2 out of 185 (Japanese) and 16 out of 181 (Vietnamese) control sera. Thus, the sensitivity was 98.0%, and specificity was 98.9% and 91.1% in the Japanese and Vietnamese sera, respectively. When the ELISA and indirect haemagglutination (IHA) tests were combined, a sensitivity of 100% and specificity of 97.8% were achieved. The advantages of the glycolipid antigen are ease of preparation, stability, high sensitivity and specificity.

Bioterrorism and Infectious Agents: A New Dilemma for the 21st Century

“…Various cut-off titers for regarding the test as "positive" have been suggested (Leelarasamee, 1997;Sirisinha, 1991), but most workers in endemic areas have found that it lacks both sensitivity and, more importantly, specificity (Khupulsup and Petchclai, 1986), probably due to recurrent exposure to the ubiquitous Burkholderia spp. Potential candidates included a 45 kDa protein (Lertmemongkolchai et al, 1991), LPS (Petkanjanapong et al, 1992), a 40 kDa protein (Wongratanacheewin et al, 1993), a 19.5 kDa antigen (Anuntagool et al, 1993), exotoxin (Embi et al, 1993), a glycolipid antigen (Phung et al, 1995), a monoclonal antibody-affinity purified nonprotein surface antigen (Dharakul et al, 1997), and a recombinant 18.7 kDa antigen (Wongprompitak et al, 2001). Early attempts to improve the serological diagnosis of melioidosis focused on the differentiation of IgG and IgM responses, using techniques such as immunofluorescence (Ashdown, 1981;Khupulsup and Petchclai, 1986;Vadivelu et al, 1995;Vadivelu and Putucheary, 2000), enzyme-linked immunosorbent assay (ELISA) (Ashdown et al, 1989;Chenthamarakshan et al, 2001;Kunakorn et al, 1990;Vadivelu et al, 1995) or gold blot (Kunakorn et al, 1991b).…”

Procedurally Similar Competitive Immunoassay Systems for the Serodiagnosis ofBabesia Equi, Babesia Caballi, Trypanosoma Equiperdum, andBurkholderia MalleiInfection in Horses

“…5,11,12,15,17 Patterns of CF response have also been investigated after experimental infections of horses with T. equiperdum and B. mallei. 1,6,7,13 Although similar patterns of cELISA response have not been as extensively investigated for the latter 2 agents, the CF responses to them have also been shown to often wane or fluctuate erratically within a relatively short period after infection. Theoretically, cELISA procedures might be expected to be less immunoglobulin class and subclass dependent than CF procedures, and the c-ELISA procedures for dourine and glanders serodiagnosis would be expected to be superior to CF methods in detecting long-standing infections with those diseases as well.…”

“…Mallein, a heat-killed, primarily lipopolysaccharide (LPS) extract of B. mallei, was used as the glanders cELISA antigen. 7,13 Both piroplasmosis antigens, the dourine antigen, and the glanders antigen were stored at Ϫ70 C until used.…”

“…2,3 The OIE has also recognized alternative serodiagnostic methods for each etiologic agent, including separate indirect fluorescent antibody tests (IFATs) for B. equi and B. caballi, enzyme-linked immunosorbent assay (ELISA) for B. mallei, and ELISA, IFAT, or agar gel immunodiffusion procedures for T. equiperdum serodiagnosis. 4,6,7,12,13,17 In practice, the battery of 4 CF procedures has been the most convenient, rapid, single test system for accomplishing the required piroplasmosis, dourine, and glanders serodiagnostic testing. Unfortunately, the CF tests require careful continuous titration of numerous labile reagents and, for piroplasmosis, the laborious expensive preparation of antigen from infected splenectomized horses.…”

Bioterrorism and Infectious Agents: A New Dilemma for the 21st Century

“…Then, during the 1990s, several groups attempted to identify individual antigens that might be purified and used in tests to give greater sensitivity and specificity than the mixtures used in the earlier assays. Potential candidates included a 45 kDa protein (Lertmemongkolchai et al, 1991), LPS (Petkanjanapong et al, 1992), a 40 kDa protein (Wongratanacheewin et al, 1993), a 19.5 kDa antigen (Anuntagool et al, 1993), exotoxin (Embi et al, 1993), a glycolipid antigen (Phung et al, 1995), a monoclonal antibody-affinity purified nonprotein surface antigen (Dharakul et al, 1997), and a recombinant 18.7 kDa antigen (Wongprompitak et al, 2001). Some impressive sensitivities, specificities, and predictive values have been found in studies of relatively small numbers of sera, and there is little doubt that some of these newer assays are an improvement on the IHA test Wongratanacheewin et al, 2001), but none of these tests has ever been subjected to a large scale, multicenter analysis.…”