Preparation of monoclonal antibodies against gamma-type phospholipase A₂ inhibitors and immunodetection of these proteins in snake blood

 

Jingjing Li¹, Ying Xiong², Shimin Sun¹, Lehan Yu¹, Chunhong Huang³

 

1 Department of Biochemistry, College of Basic Medical Science, Nanchang University, Nanchang 330006, China

2 Second Affiliated Hospital to Nanchang University, Nanchang University, Nanchang 330006, China

3 Jiangxi Province Key Laboratory of Tumor Pathogens and Molecular Pathology, Nanchang University, 461 Bayi Avenue, Nanchang 330006, China

 

ABSTRACT

Background:

The gamma-type phospholipase A₂ inhibitor (ΡLIγ) is a natural protein commonly found in snake serum, which can neutralize pathophysiological effects of snake venom phospholipases A₂. Therefore, this protein is a potential candidate to the development of a novel antivenom. To the best of our knowledge, there is no antibody currently available for PLIγ identification and characterization.

 

Methods:

Bioinformatics prediction of epitope using DNAStar software was performed based on the sequence of Sinonatrix annularis PLIγ (SaPLIγ). The best epitope ¹⁵¹CPVLRLSNRTHEANRNDLIKVA¹⁷² was chosen and synthesized, and then conjugated to keyhole limpet hemocyanin and bovine serum albumin for use as an immunogen and plate-coating antigen, respectively.

 

Results:

Eighteen IgG anti-PLIγ mAb hybridoma cell strains were obtained, and all the mAbs had positive interaction with recombinant His6-PLIγ and natural SaPLIγ. Moreover, the mAb from 10E9 strain was also successfully used for the immunodetection of other snake serum PLIγs. cDNA sequence alignment of those PLIγs from different snake species showed that their epitope segments were highly homologous.

 

Conclusions:

The successful preparation of anti-PLIγmAb is significant for further investigation on the relationship between the structure and function of PLIγs, as well as the interaction between PLIγs and PLA₂s.

 

Keywords: Monoclonal antibody; Phospholipase A₂ inhibitor; Epitope prediction

 

Received: March 31, 2017.

Accepted: July 25, 2017.

 

Correspondence: chhuang@ncu.edu.cn

 

Authors' contributions

JJ and XY contributed equally to his work. JJ performed the animal immunization and western blot. XY took part in hybridoma screening. SH collected the sera of snakes. LH was responsible for the PLIγ genes. CH conducted the experimental design and writing of the manuscript. All the authors read and approved the final manuscript.

 

Consent for publication

Not applicable.

 

Competing interests

The authors declare that they have no competing interests.