title: Production and characterization of specific monoclonal antibodies binding the Plasmodium falciparum diagnostic biomarker, histidine-rich protein 2.
abstract: BACKGROUND
  Early and accurate diagnosis of Plasmodium falciparum infection is important for providing appropriate treatment to patients with malaria. However, technical limitations of currently available diagnostic tests limit their use in control programs. One possible explanation for the vulnerability of current antibodies used in RDTs is their propensity to degrade at high ambient temperatures. Isolation of new antibodies with better thermal stability represents an appealing approach to improve the performance of RDTs.
  
  
  METHODS
  In this study, phage display technology was deployed to isolate novel binders by screening a human naïve scFv antibody library against recombinant Plasmodium falciparum histidine rich protein 2 (rPfHRP2). The isolated scFv clones were reformatted to whole IgG and the recombinant mAbs were produced in a mammalian CHO cell expression system. To verify the biological activity of these purified recombinant mAbs, range of functional assays were characterized.
  
  
  RESULTS
  Two unique clones (D2 and F9) were isolated after five rounds of biopanning. The reformatted and expressed antibodies demonstrated high binding specificity to malaria recombinant PfHRP2 and native proteins. When 5 μg/mL of mAbs applied, mAb C1-13 had the highest sensitivity, with an OD value of 1, the detection achieved 5 ng/mL of rPfHRP2, followed by mAbs D2 and F9 at 10 ng/mL and 100 ng/mL of rPfHRP2, respectively. Although the sensitivity of mAbs D2 and F9 was lower than the control, these recombinant human mAbs have shown better stability compared to mouse mAb C1-13 at various temperatures in DSC and blot assays. In view of epitope mapping, the predominant motif of rPfHRP2 recognized by mAb D2 was AHHAADAHHA, whereas mAb F9 was one amino acid shorter, resulting in AHHAADAHH. mAb F9 had the strongest binding affinity to rPfHRP2 protein, with a KD value of 4.27 × 10(-11) M, followed by control mAb C1-13 at 1.03 × 10(-10) M and mAb D2 at 3.05 × 10(-10) M.
  
  
  CONCLUSIONS
  Overall, the performance of these mAbs showed comparability to currently available PfHRP2-specific mouse mAb C1-13. The stability of these novel binders indicate that they merit further work to evaluate their utility in the development of new generation point of care diagnosis of malaria.
authors.affiliation: QIMR Berghofer Medical Research Institute, Brisbane, Australia. herng.leow@usm.my.
authors.forename: Chiuan Herng
authors.initials: CH
authors.lastname: Leow
authors.forename: Martina
authors.initials: M
authors.lastname: Jones
authors.forename: Qin
authors.initials: Q
authors.lastname: Cheng
authors.forename: Stephen
authors.initials: S
authors.lastname: Mahler
authors.forename: James
authors.initials: J
authors.lastname: McCarthy
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D000913:Antibodies, Protozoan / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D000953:Antigens, Protozoan / Q000276:immunology*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004356:Drug Storage; D042241:Early Diagnosis; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D016778:Malaria, Falciparum / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D051379:Mice; D019151:Peptide Library; D010963:Plasmodium falciparum / Q000276:immunology*; D055550:Protein Stability; D015800:Protozoan Proteins / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D012680:Sensitivity and Specificity; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology; D013696:Temperature
chemical_list: D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D000939:Epitopes; C514457:HRP-2 antigen, Plasmodium falciparum; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C055966:immunoglobulin Fv