Isolation of fetal nucleated red blood cell from maternal blood using immunomagnetic beads for prenatal diagnosis.

The immunomagnetic beads method for isolation of fetal nucleated red blood cells (FNRBCs) from peripheral blood of 78 pregnant women for prenatal diagnosis was developed. The study subjects were classified into 8-10 and 11-14 weeks of gestation (n = 39 each). Peripheral blood cells were divided into two for the FNRBCs isolation using two protocols, one with anti-CD45 depletion followed by anti-CD71 and anti-GPA monoclonal antibodies and another without CD45 depletion. The use of CD45 depletion gave a slightly higher number of sorted cells but not significantly different (p > 0.05). The percentage of CD71+ and GPA+ cells obtained from 8-10 weeks and 11-14 weeks of gestation was not different (p > 0.05). The sensitivity in determining the sorted FNRBCs for male fetal sex by PCR using 8-10 and 11-14 weeks of gestation was generally 50 and 69%, respectively. The method so developed is simple and cost effective and may thus be applied for prenatal diagnosis.

Retrospective study on the diagnostic value of IgG ELISA, dot immunoassay and indirect hemagglutination in septicemic melioidosis.

Three serological methods for diagnosis of melioidosis were compared with the culture method currently used as the "gold standard". The diagnostic values of the serological methods were evaluated retrospectively in 306 patients residing in an endemic area. The enzyme-linked immunosorbent assay (ELISA), using affinity purified antigen for detecting specific IgG antibody, showed a slightly higher specificity (86.0%) than the dot immunoassay (DOT) (84.0%) and both were superior to indirect hemagglutination (IHA) (72.0%). The sensitivity of DOT (96.4%) and ELISA (85.7%) were considerably higher than that of IHA (50.0%). The primary benefit of the high negative predictive value of both ELISA (96.4%) and DOT (99.0%) in an area of high prevalence is the ability to rule out most of the non-melioidosis patients.

Use of culture-filtrated antigen in an ELISA and a dot immunoassay for the diagnosis of melioidosis.

An enzyme-linked immunosorbent assay (ELISA) and a dot immunoassay with culture-filtrated antigen were developed for detection of Burkholderia pseudomallei specific antibodies in melioidosis patients. Sixty-eight sera of bacteriologically confirmed melioidosis patients, 45 sera of other bacterial infected patients and 80 sera of healthy blood donors from endemic area were investigated. The samples were subjected to those assays im comparison with indirect hemagglutination (IHA). The sensitivity, specificity, positive and negative predictive values in this dot immunoassay were 94.1%, 99.2%, 98.5% and 96.9%, respectively, with cut-off dilution at 1:4,000, whereas those in ELISA were 92.6%, 96.8%, 94.0% and 96.0%, respectively, with cut-off value of OD = 0.47 at 490 nm. Meanwhile, those in IHA were 64.7%, 93.6%, 84.6%, 83.0% respectively, with a cut-off value of > or = 1:80. The results in this study demonstrated that the dot immunoassay was more reliable and rapid than ELISA as the serological test for diagnosis of melioidosis.

Characterization of Pseudomonas pseudomallei antigens by SDS-polyacrylamide gel electrophoresis and western blot.

Immunological characterization of various Pseudomonas pseudomallei preparations was carried out by SDS-PAGE and Western blot using sera from infected humans and from patients with other bacterial infections. Somatic (SOM) and partially purified cell extracts (PCE) gave more complex SDS-PAGE patterns: M(r) ranged from 86 to 12.7 and 48 to 10 kDa, respectively. The culture-filtrated antigens (CF) from 3 different kinds of synthetic media consisted of fairly simple profiles with common bands M(r) of 40, 26 and 16 kDa. PCE and CF reacted specifically with infected human sera; SOM did not. The components with M(r) of 40 kDa in CF reacted consistently with all infected sera but failed to react with sera infected with Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, Proteus mirabilis, Salmonella spp., Staphylococcus aureus, Streptococcus spp., Pseudomonas aeruginosa and P. stutzeri. This peptide was demonstrated to be a major component in CF thus suggesting its potential for development of immunodiagnostic methods for melioidosis.

Immunodiagnosis of opisthorchiasis.

Monoclonal antibody-based enzyme-linked immunosorbent assay and DNA dot blot hybridization techniques were developed and evaluated for their potential in the detection of Opisthorchis viverrini. A mixture of IgG monoclonal antibodies specific for the 89 kDa metabolic product of O. viverrini was captured on a microtiter plate by rabbit anti-mouse IgG and used in a sandwich ELISA for the detection of soluble parasite antigen in the feces of patients with opisthorchiasis. As little as 0.1 ng of the antigen could be detected. A specific O. viverrini DNA probe was used in a dot blot hybridization of parasite DNA. The labeled probe could detect DNA released from as few as five O. viverrini eggs. Both approaches were highly specific for O. viverrini and their sensitivity appeared to be comparable with that of the classical parasitological method. Preliminary data obtained from a field trial showed that these two methods have potential in the diagnosis of opisthorchiasis. Moreover, the limited data currently available showed that it is possible to use these methods to detect the presence of O. viverrini metacercariae in naturally infected fish.