ABSTRACT
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.
Subject(s)
Antigens, CD/metabolism , Leukocyte Common Antigens/metabolism , Erythrocytes , Female , Fetus , Flow Cytometry , Glycophorins/metabolism , Humans , Immunohistochemistry , Immunomagnetic Separation , Leukocyte Reduction Procedures , Polymerase Chain Reaction , Pregnancy , Prenatal Diagnosis/methods , Receptors, Transferrin/metabolism , Sensitivity and Specificity , Sex Determination Analysis/methodsABSTRACT
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.
Subject(s)
Bacteremia/blood , Burkholderia pseudomallei/isolation & purification , Enzyme-Linked Immunosorbent Assay , False Positive Reactions , Hemagglutination Tests , Humans , Immunoblotting , Immunoglobulin G/blood , Melioidosis/blood , Predictive Value of Tests , Retrospective Studies , Sensitivity and Specificity , ThailandABSTRACT
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.
Subject(s)
Antigens, Bacterial/immunology , Case-Control Studies , Enzyme-Linked Immunosorbent Assay , Hemagglutination Tests , Humans , Immunoblotting , Melioidosis/diagnosis , Sensitivity and Specificity , ThailandABSTRACT
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.