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Production and characterization of specific monoclonal antibodies binding the Plasmodium falciparum diagnostic biomarker, histidine-rich protein 2.

Leow CH, Jones M, Cheng Q, Mahler S, McCarthy J - Malar. J. (2014)

Bottom Line: 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.Overall, the performance of these mAbs showed comparability to currently available PfHRP2-specific mouse mAb C1-13.

View Article: PubMed Central - HTML - PubMed

Affiliation: QIMR Berghofer Medical Research Institute, Brisbane, Australia. herng.leow@usm.my.

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.

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Related in: MedlinePlus

Analysis of purified recombinant mAbs D2 and F9 on 4-12% SDS-PAGE. The gel was stained with Coomassie Blue R250 (A). Lane 1: SeeBlue Plus2 Pre-Stained Standard (Invitrogen); Lanes 2 and 3: non-reduced mAb D2; Lanes 4 and 5: non-reduced mAb F9; Lanes 6 and 7: reduced mAb D2; Lanes 8 and 9: reduced mAb F9, at neat, and 1 in 10 dilutions. Analytical size exclusion chromatography of protein-A purified recombinant mAbs D2 and F9 at 280 nm are shown in panels B and C, respectively.
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Figure 2: Analysis of purified recombinant mAbs D2 and F9 on 4-12% SDS-PAGE. The gel was stained with Coomassie Blue R250 (A). Lane 1: SeeBlue Plus2 Pre-Stained Standard (Invitrogen); Lanes 2 and 3: non-reduced mAb D2; Lanes 4 and 5: non-reduced mAb F9; Lanes 6 and 7: reduced mAb D2; Lanes 8 and 9: reduced mAb F9, at neat, and 1 in 10 dilutions. Analytical size exclusion chromatography of protein-A purified recombinant mAbs D2 and F9 at 280 nm are shown in panels B and C, respectively.

Mentions: The D2 and F9 scFv fragments were reformatted to whole mAbs, expressed in CHO cells and purified by Protein A chromatography [26]. SDS-PAGE analysis of the purified mAbs showed the whole intact mAb in non-reducing conditions (~150 kDa), and a 50 kDa heavy chain and 25 kDa light chain in reducing conditions. Size exclusion HPLC showed no significant aggregation or degradation (Figure 2).


Production and characterization of specific monoclonal antibodies binding the Plasmodium falciparum diagnostic biomarker, histidine-rich protein 2.

Leow CH, Jones M, Cheng Q, Mahler S, McCarthy J - Malar. J. (2014)

Analysis of purified recombinant mAbs D2 and F9 on 4-12% SDS-PAGE. The gel was stained with Coomassie Blue R250 (A). Lane 1: SeeBlue Plus2 Pre-Stained Standard (Invitrogen); Lanes 2 and 3: non-reduced mAb D2; Lanes 4 and 5: non-reduced mAb F9; Lanes 6 and 7: reduced mAb D2; Lanes 8 and 9: reduced mAb F9, at neat, and 1 in 10 dilutions. Analytical size exclusion chromatography of protein-A purified recombinant mAbs D2 and F9 at 280 nm are shown in panels B and C, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4120728&req=5

Figure 2: Analysis of purified recombinant mAbs D2 and F9 on 4-12% SDS-PAGE. The gel was stained with Coomassie Blue R250 (A). Lane 1: SeeBlue Plus2 Pre-Stained Standard (Invitrogen); Lanes 2 and 3: non-reduced mAb D2; Lanes 4 and 5: non-reduced mAb F9; Lanes 6 and 7: reduced mAb D2; Lanes 8 and 9: reduced mAb F9, at neat, and 1 in 10 dilutions. Analytical size exclusion chromatography of protein-A purified recombinant mAbs D2 and F9 at 280 nm are shown in panels B and C, respectively.
Mentions: The D2 and F9 scFv fragments were reformatted to whole mAbs, expressed in CHO cells and purified by Protein A chromatography [26]. SDS-PAGE analysis of the purified mAbs showed the whole intact mAb in non-reducing conditions (~150 kDa), and a 50 kDa heavy chain and 25 kDa light chain in reducing conditions. Size exclusion HPLC showed no significant aggregation or degradation (Figure 2).

Bottom Line: 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.Overall, the performance of these mAbs showed comparability to currently available PfHRP2-specific mouse mAb C1-13.

View Article: PubMed Central - HTML - PubMed

Affiliation: QIMR Berghofer Medical Research Institute, Brisbane, Australia. herng.leow@usm.my.

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.

Show MeSH
Related in: MedlinePlus