Production and characterization of monoclonal antibodies against substrate specific loop region of Plasmodium falciparum lactate dehydrogenase.
Plasmodial lactate dehydrogenase, terminal enzyme of the glycolytic pathway, has been shown to be biochemically, immunologically and structurally different from the mammalian enzyme. The substrate specific loop region of plasmodial lactate dehydrogenase (pLDH) has 5 amino acids insert (DKEWN) important for anti-malarial drug targeting. In the present study, we have produced six monoclonal antibodies, which are against three different epitopes of Plasmodium falciparum LDH (PfLDH). Two of these monoclonal antibodies (10C4D5 and 10D3G2) are against the substrate specific loop region of PfLDH (residues 98-109, AGFTKAPGKSDKEWNR). The 10C4D5 and 10D3G2 monoclonals bind to substrate specific loop region resulting in inhibition of PfLDH activity. A Microplate Sandwich ELISA was developed employing high affinity non-inhibitory (10A5H5, Kaff 1.272 ± 0.057 nM) and inhibitory (10C4D5, Kaff 0.306 ± 0.011 nM) monoclonal antibodies and evaluated using gossypol, a well known inhibitor of pLDH. The binding of gossypol to substrate specific loop region resulted in inhibition of binding of 10C4D5 monoclonal. This Microplate Sandwich ELISA can be utilized for identification of compounds inhibitory to PfLDH (binding to substrate specific loop region of parasite LDH) from combinatory chemical libraries or medicinal plants extracts. The Microplate Sandwich ELISA has also shown potential for specific diagnosis of malaria using finger prick blood samples.
Division of Parasitology, CSIR-Central Drug Research Institute , Lucknow 226001 , India and.
Nuzhat A
NA
Kaushal
Deep C
DC
Kaushal
D000911:Antibodies, Monoclonal / Q000302:isolation & purification / Q000494:pharmacology*; D000953:Antigens, Protozoan / Q000276:immunology; D002478:Cells, Cultured; D020650:Combinatorial Chemistry Techniques; D006072:Gossypol / Q000494:pharmacology; D006801:Humans; D007167:Immunotherapy / Q000379:methods*; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000276:immunology; D016778:Malaria, Falciparum / Q000276:immunology / Q000628:therapy*; D010936:Plant Extracts; D010963:Plasmodium falciparum / Q000276:immunology*; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan; D010936:Plant Extracts; D007770:L-Lactate Dehydrogenase; D006072:Gossypol
Lactate dehydrogenase; Plasmodium falciparum; malaria parasite; monoclonal antibodies; substrate specific loop region of PfLDH