Abstract:

Objective To establish a novel colloidal gold immunochromatography assay for the specific detection of Legionella pneumophila（Lp）. Methods Lp-PAL recombinant protein was prepared and purified by genetic engineering,and hybridoma cell lines secreting anti-Lp-peptidoglycan-associated lipoprotein（PAL） recombinant protein monoclonal antibody were screened. The indirect enzyme-linked immunosorbent assay（ELISA） and immunoblotting were used to screen and identify the specificities of Lp-1 and Lp-2 monoclonal antibodies. The specificity of antigen-antibody response was identified using bio-layer interferometry（BLI） technology. A colloidal gold immunochromatography test strip was prepared based on double antibody sandwich principle,and its detection performance（specificity,sensitivity and stability） was initially evaluated. Results Totally,2 hybridoma cell lines that secreted anti-Lp-PAL protein antibodies were screened out,and 2 monoclonal antibodies,Lp-1 and Lp-2,were purified. Indirect ELISA showed that Lp-1 and Lp-2 monoclonal antibodies only positively reacted with Lp,and reacted with 10 other common respiratory pathogens（Streptococcus pneumoniae,Moraxella catarrhalis,Haemophilus influenzae,Mycoplasma pneumoniae,Staphylococus aureus,Klebsiella pneumoniae,Klebsiella oxytoca,Acinetobacter baumannii,Pseudomonas aeruginosa and Proteus mirabilis） had no cross-reactivity. The results of western blotting showed that Lp-1 and Lp-2 monoclonal antibodies had strong reactions with natural PAL protein and Lp-PAL recombinant protein,and had no purposeful reaction bands with Streptococcus pneumoniae,Haemophilus influenzae and Moraxella catarrhalis. The verification results of BLI technology showed that the antigen-antibody interactions were strong,and the dissociation rate was slow. Lp-1 and Lp-2 monoclonal antibodies recognized different epitopes of the same antigen,and only had a strong reaction with Lp. They had no specific binding reaction with 10 other pathogens. The minimum detection limit of the prepared colloidal gold immunochromatography test strip was 1×10⁷ CFU/mL,which specifically reacted with the 4 common serotypes of Lp（Lp14,Lp12,Lp9 and Lp6） and did not react with 10 other pathogens such as Streptococcus pneumoniae and Moraxella catarrhalis. The strips could be stored at 25 ℃ for at least 6 months. Conclusions Lp outer membrane protein PAL,which possesses a high degree of surface antigen accessibility and antigenicity,could be used as a molecular marker for Lp infection. The colloidal gold immunochromatography test strip with the features of rapidity,convenience and sensitivity provides a unique tool for the on-site surveillance and diagnosis of Lp infection.

Key words: Legionella pneumophila, Monoclonal antibody, Peptidoglycan-associated lipoprotein, Bio-layer interferometry technology, Colloidal gold immunochromatography assay