Colloidal gold immunochromatographic strip for rapid detection of Haemophilus influenzae / 生物工程学报

To establish a novel colloidal gold immunochromatography assay (GICA) for rapid, sensitive and accurate detection of Haemophilus influenzae infection by using the outer membrane protein P6 as detection target. First, the linear antigen epitope located in the extracellular domain of the P6 protein (GenBank accession number: AGH02799) was predicted by bioinformatics analysis. The region (62-75 aa of the protein) with strong antigen specificity was chosen and synthesized. Two rabbits were then immunized by the polypeptides (14 aa) for production of polyclonal antibodies. Then, the recombinant P6 proteins were also obtained to produce polyclonal antibodies. Finally, based on the two antibodies, a novel colloidal GICA for detection of Haemophilus influenzae infection was established and the specificity, sensitivity, repeatability and stability of this method were evaluated. At the same time, the method was tested in clinical simulation, and the plate culture method was used to verify its accuracy. The test strip for Haemophilus influenzae infection was successfully prepared. The detection limit of the test strip was as low as 1×105 CFU/mL and the whole process can be completed within 15 minutes. The strip specifically recognized Haemophilus influenzae and did not react with nine of other common respiratory pathogens such as Streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumonia, and Legionella pneumophila. And the strips could be stored at 25 °C for at least 6 months without losing sensitivity or specificity. The coincidence rate between the results of 200 clinical samples and the plate culture method was 90.5%. Haemophilus influenzae protein P6, which possessed a high degree of surface antigen accessibility and antigencity, could be used as a marker for Haemophilus influenzae detection. The immunochromatographic colloidal gold test strip which bears the features of rapidity, convenience and sensitivity provides a unique tool for the on-site surveillance and diagnosis of Haemophilus influenzae infection in clinical test.

Serum concentrations of antibodies against outer membrane protein P6 and its T-and B-combined antigenic epitopes of nontypeable Haemophilus influenzae in children and adults of different ages / 中华微生物学和免疫学杂志

Objective To measure the concentrations of antibodies against outer membrane protein P6 and its T-and B-combined antigenic epitopes of nontypeable Haemophilus influenzae ( NTHi) in children and adults of different ages and to evaluate the differences among different subjects for further investigation on NTHi multiple antigenic peptide vaccine .Methods A prokaryotic expression system was established to ex-press the recombinant outer membrane protein P 6 of NTHi.The expressed protein was purified by using Ni-NTA affinity chromatography .T-and B-cell epitopes in protein P6 were predicted with Epitope prediction software 1.0 and ANTIGENIC program and were used to synthesize T-and B-combined antigenic epitopes .A total of 605 subjects aged from 1 day to 103 years old were recruited from October 2013 to March 2014 .Ser-um concentrations of antibodies against protein P 6 and its T-and B-combined antigenic epitopes were meas-ured by using ELISA .Mann-Whitney U test was used to analyze the differences between groups .Pearson product-moment correlation coefficient was used for correlation analysis .Results Four T-and B-combined antigenic epitopes including P 6-2, P6-61, P6-95 and P6-122 were predicted and synthesized .The levels of antibodies against NTHi P6 and P6-2, P6-61, P6-95 and P6-122 were significant lower in the <1 months group than those in the 1-6 months group (all P<0.001) and 7 months-3 years group (all P<0.001).Three groups including 7 months-3 years group , 4-6 years group and 7-14 years group showed significant differ-ences regarding to the antibodies levels , among the 7 months-3 years group showed the highest levels , fol-lowed by the 4-6 years group and the 7-14 years group.However, no significant difference was found be-tween other adjacent groups .Concentrations of antibodies against P 6-2, P6-61, P6-95 and P6-122 were pos-itively correlated with the level of antibody against P 6 (P<0.0001).Conclusion The distribution of anti-bodies against T-and B-combined antigenic epitopes in P6 was highly in accord with those against P6, which indicated good immunogenicity of those epitopes .The highest antibodies levels were found in subjects aged 7 months to 3 years old , which might correlate with the high risk of NTHi infection at that stage .