Estimation of vacuolating cytotoxin secreted by different strains of Helicobacter pylori using bead enzyme-linked immunosorbent assay & its correlation with bacterial genotype.
OBJECTIVE
A highly sensitive bead enzyme-linked immunosorbent assay was applied for the quantitative determination of vacuolating cytotoxin (VacA) released in the culture supernatant of 40 well characterized Helicobacter pylori strains in order to clarify the significance of allelic combination of the vacA gene as the predictor of the level of toxin secretion and also to determine the most appropriate genotype of H. pylori associated with high VacA release. Attempts were also made for the detection of VacA in the gastric juice of patients for the rapid diagnosis of H. pylori infection.


METHODS
The genotypes of 40 H. pylori strains cultured from the gastric biopsy samples were determined by specific PCRs. The cell-free culture supernatant of the strains as well as the gastric juice of the patients were used for bead-ELISA and the purified VacA from the H. pylori strain ATCC49503 was used as positive control.


RESULTS
Ninety per cent of the strains with vacAs1m1 allele combination secrete on an average 146.4 ng/ml of VacA while the corresponding value was 19.1 ng/ml for s1m2 strains. None of the s2m2 as well as the ice negative H. pylori strains produced detectable VacA in the medium while strains expressed the toxin irrespective of the presence or absence of cagA gene. Fifteen of 22 gastric juice samples yielded positive bead-ELISA results.


CONCLUSIONS
vacAs1, vacAm1 and iceA1 could be considered as the determinants of high VacA secretion. Also, the detection of VacA by bead-ELISA in the gastric juice could be considered as an alternative approach in the diagnosis of H. pylori infection.
Datta
S
S
National Institute of Cholera & Enteric Diseases (ICMR), Kolkata, India.
Kurazono
H
H
Chattopadhyay
S
S
Chowdhury
A
A
Chaudhuri
S
S
Bhattacharya
S K
SK
Mukhopadhyay
A K
AK
Berg
D E
DE
Hirayama
T
T
Nair
G B
GB
D000328:Adult; D000368:Aged; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D005838:Genotype; D016480:Helicobacter pylori / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; C074689:VacA protein, Helicobacter pylori