Characterization of a phase 1-d epitope on Salmonella typhi flagellin and its role in the serodiagnosis of typhoid fever.

A monoclonal antibody (MAb) directed against Salmonella typhi 52 kDa flagellin protein has been previously produced by our group. In this study, we have demonstrated that the epitope specific to the MAb is unique to phase 1-d. To map the epitope, plasmids encoding different regions of S. typhi flagellin gene were constructed. Analysis of protein produced from each recombinant plasmid indicated that the epitope specific to the MAb resided within amino acids 171-303 (region IV) of S. typhi flagellin protein. The recombinant region IV flagellin was used to develop an ELISA for the detection of IgM antibody to S. typhi in serum. In the hemoculture-positive typhoid group, the developed ELISA was positive in 77 of 92 cases. In patients with non-typhoidal Salmonella, gram-positive and gram-negative bacteria or dengue virus, the ELISA was negative in all 78 cases. Two from 116 healthy control subjects had positive reactions with the assay. The calculated sensitivity, specificity, positive and negative predictive values of the test were 83.7%, 99.0%, 97.5% and 92.8%, respectively. With such high validity together with the requirement of only a single serum specimen and one day for performing the test, the developed ELISA should become a valuable diagnostic test for typhoid fever.

A study of cell-mediated immune response to pancreatic antigens in patients with fibrocalculous pancreatic diabetes.

In order to investigate whether there was any association between autoimmunity to pancreatic antigens with FCPD as well as IDDM, cell-mediated immune response to pancreatic antigens was studied by lymphoproliferation assay in 7 FCPD, 17 IDDM, 33 NIDDM patients and 102 normal controls. Optimal pancreatic antigen concentrations used were 100, 150 and 200 micrograms/ml. Positive results were considered for each concentration of antigens tested, at stimulation index (SI) > (mean +/- 2 SD) SI obtained from normal age-matched controls with the use of the corresponding concentration of antigen. The one who gave positive result with any of these optimal antigen concentrations was considered to be the responder to pancreatic antigens. With this criterion, the responders were found to be 3/7 (42.9%) FCPD, 6/17 (35.3%) IDDM and 6/33 (18.2%) NIDDM patients; while there were 11 of all 102 (10.8%) normal controls.

Monoclonal antibodies against protein antigens of salmonellae causing paratyphoid fever and their diagnostic application.

Hybrid clones producing monoclonal antibodies (MAbs) specific for Salmonella paratyphi A (72 clones), S. paratyphi B (9 clones) and S. paratyphi C (8 clones) were produced by using the affinity purified Salmonella protein (Bp) as immunogens. MAbs to S. paratyphi A and S. paratyphi B reacted specifically with the 52 kDa homologous flagellin protein components while those to S. paratyphi C reacted with a 61 kDa flagellin protein component. The MAbs against S. paratyphi A and S. paratyphi B were used to establish a double antibody sandwich ELISA for detection of the 52 kDa flagellin antigens in serum samples from patients with acute paratyphoid A and paratyphoid B fever. With this assay system, 6.25 ng per ml of flagellin antigens of S. paratyphi A and S. paratyphi B could be detected. However, the assay system could not detect the flagellin antigens in patients' sera. The presence of IgM antibodies to the 52 kDa antigens of S. paratyphi A and S. paratyphi B in the acute sera from paratyphoid A or paratyphoid B patients suggested that the 52 kDa protein components of both salmonellae are good immunogens for human and might be used as antigens for early diagnosis of paratyphoid A and paratyphoid B fever.

Re-evaluation of ELISA and latex agglutination test for rheumatoid factor detection in the diagnosis of rheumatoid arthritis.

An indirect ELISA for the determination of each isotype (IgM, IgG, IgA, IgD, IgE) of rheumatoid factors (RF) was performed with sera obtained from 77 patients with either classical or definite rheumatoid arthritis (RA) and 319 controls, using rabbit IgG as the antigen. The results were compared with those of a commercial latex agglutination test, using denatured human gamma globulin as the antigen for rheumatoid factor determination. At the cut-off level at which positive results were found in less than 5% of normal controls, ELISA for IgM RF determination had sensitivity, specificity, efficiency, positive predictive value and negative predictive value of 46.75%, 98.12%, 88.13%, 85.71%, 88.41%, while those for IgA RF were 46.75%, 93.42%, 84.34%, 63.16%, 87.91% and for IgG RF were 59.74%, 92.16%, 85.86%, 64.78%, 90.46%, respectively. These indices by latex agglutination test were 83.11%, 93.73%, 91.67%, 76.19% and 95.83%, respectively. IgD RF titre greater than or equal to 1:5 was detected in 19/77 RA patients and 4/200 normal controls while IgE RF titre greater than or equal to 1:5 was detected only in 7/77 RA patients. Thus, ELISA did not appear to have any advantage over latex agglutination test for diagnosis of RA.

Dot-blot ELISA for the detection of IgM RF and IgA RF.

Dot-blot ELISA was developed for the detection of IgM RF and IgA RF. Normal rabbit IgG (NRIgG), concentration 100 micrograms/ml, was used as the antigen for dotting on the 0.45 microns pore size nitrocellulose membrane. Serum, conjugate and substrate incubation conditions were at room temperature for 1 hour, 1 hour and 3 minutes, respectively. The membrane with NRIgG dot could be sotred for 6 weeks before use in the assay. Positive results of IgM RF, at the serum dilution 1:800, were found in 31/51 patients with either classical or definite rheumatoid arthritis and 3/68 normal healthy individuals. Positive IgA RF, at the serum dilution 1:100, was found in 27/51 of the former and none of the latter. Significant concordance with high agreement index was found between the results of the dot-blot ELISA developed and those obtained from ELISA performed in microtitre plate (Kappa greater than or equal to 0.78 for IgM RF and 0.83 for IgA RF, p less than 0.001).

Value of an ELISA for mycobacterial antigen detection as a routine diagnostic test of pulmonary tuberculosis.

A double antibody sandwich ELISA was carried out with commercially available anti-BCG and peroxidase labeled anti-BCG, for the detection of mycobacterial antigens. By using purified protein derivative of tuberculin (PPD) as the antigen, the lowest detection limit of the assay was found to be 0.05 microgram/ml. At the cut off level of absorbance index (Al) greater than or equal to 5. positive results of ELISA were obtained from 24/25 sputum specimens which were positive for staining of acid fast bacilli (AFB), 5/16 specimens positive for culture of Mycobacterium tuberculosis and 67/69 specimens positive for both tests. The assay was positive in only 11/164 specimens negative for both staining of AFB and culture of M. tuberculosis. 4 of which were known to have tuberculosis. Thus, with sputum specimens, the sensitivity, specificity, efficiency, positive predictive value and negative predictive value of the ELISA were 87.27, 93.29, 90.88, 89.72 and 91.62 percent respectively. Positive results were also obtained in 2/111 sputum specimens which were positive for other bacteria but the presence of AFB in these specimens could not be ruled out. With pleural fluid specimens, positive ELISA with Al greater than 1 was found in 3/26 specimens of patients with tuberculous pleurisy and 0/11 of those with malignancy. Twenty-six sera and urine specimens of tuberculous patients and also all control specimens (138 sera and 86 urine specimens) assayed, gave negative ELISA results (Al less than 1).

The detection of antigen and antibody in cerebrospinal fluid of patients with tuberculous meningitis.

The ultimate diagnosis of tuberculous meningitis can be convincingly made through a positive culture of Mycobacterium tuberculosis from the cerebrospinal fluid (CSF). However, this process requires at least 6-8 weeks. Early diagnosis, preferably at time of presentation, is preferable to avoid the unfavorable sequelae. The detection of tuberculous antigen in the CSF of children with tuberculous meningitis was carried out by an ELISA technique. Anti-PPD in the CSF was detected by latex agglutination or measured by ELISA. CSF anti-PPD was not able to be detected by latex agglutination but was demonstrated by ELISA (titre > 1: 5). The sensitivity of test was 50 percent, with specificity of 94.7 percent and accuracy of 77.4 percent. Tuberculous antigen in the CSF, however, could not be detected by an ELISA technique used in this study (< 0.1 mcg/ml). We concluded that anti-PPD antibody by ELISA could be demonstrated in these children with tuberculous meningitis and this test could potentially be utilized in a clinical situation to aid in the rapid diagnosis of tuberculous meningitis patients.

Diagnostic value of carcinoembryonic antigen in pleural fluid.

A double antibody sandwich ELISA was used for the determination of carcinoembryonic antigen levels in sera and pleural fluid samples of 25 cancer and 16 tuberculous patients. It was found that while cancer patients had pleural fluid CEA levels significantly higher than those in sera (p < 0.01), this was not true for tuberculous patients. When the CEA level at 20 ng/ml was used as the cut-off value for the diagnosis of malignancies, 8 of 25 (32%) cancer patients could be diagnosed from serum CEA level while 16 of them (64%) could be diagnosed from CEA level in the pleural fluid. In addition, none of tuberculous patients had CEA above the cut-off level. Based on the results of cytologic examination, only 11 of the 25 cancer patients (44%) could be diagnosed. However, if either positive cytologic examination or pleural fluid CEA level higher than 20 ng/ml was used as a criterion for diagnosis, then 20 of them (80%) could be diagnosed.

Detection of Salmonella typhi protein antigen in serum and urine: a value for diagnosis of typhoid fever in an endemic area.

Using haemoculture as the gold standard, a double antibody sandwich ELISA for the detection of Salmonella typhi Barber protein antigen (BP) was compared with the Widal test. Specimens used were serum and urine obtained from normal healthy individuals and from patients with typhoid fever, paratyphoid fever, pyrexia caused by other bacteria and pyrexia with negative haemoculture. The ELISA for antigenuria gave a significantly higher sensitivity, specificity, accuracy and positive predictive value than the Widal test (p less than 0.05). The ELISA for antigenaemia gave a significantly higher sensitivity and positive predictive value only. All other values were not significantly different. The timing of specimen collection was critical for sensitivity in the ELISA for antigenaemia and antigenuria, and the best results could be obtained by carrying out both assays simultaneously. The clearance of BP from serum into urine occurred around 16 days after the onset of fever in one patient. In two patients, BP could be detected in sera up to 3 weeks after the onset of fever. In two patients, serum BP could still be detected although haemoculture was negative.

Systemic and intestinal immunities after different typhoid vaccinations.

The comparative studies of systemic and intestinal immunities to S. typhi were performed in 29 healthy volunteers during 2 years after receiving oral vaccination with attenuated S. typhi Ty21a in gelatin capsule, parenteral vaccination with acetone inactivated or heat inactivated-phenol preserved S. typhi Ty2. The methods used were immunobead ELISA for total secretory IgA and indirect ELISA for specific secretory IgA in the intestinal lavage fluid. The specific systemic IgG, IgM and anti-O, anti-H agglutinins were measured by indirect ELISA and Widal test respectively. The leukocyte migration inhibition test was used for the measurement of systemic cell mediated immunity. The results indicate that the oral S. typhi Ty21a stimulated intestinal immunity better than both parenteral vaccines but evoked less systemic antibody response. The stimulation of systemic cell-mediated immunity by the live attenuated and acetone inactivated vaccine was comparable while stimulation by heat inactivated-phenol preserved vaccine was less pronounced. The same studies were performed in 26 healthy volunteers during 6 months following different doses of oral vaccination with S. typhi Ty21a in enteric-coated capsule. The results suggest that the stimulation of intestinal and systemic immunities by this vaccine is dosage dependent. Three doses of vaccine provide better stimulation than two doses and one dose, respectively.