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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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Sensitivity of mAbs D2, F9 and C1-13 against rPfHRP2. The binding efficacy of mAbs against serially diluted recombinant PfHRP2 is determined by ELISA (Left panel) and dot blot (Right panel). A: 5 μg/mL of mAbs; B: 1 μg/mL of mAbs. Dot line represents cut-off OD absorbance at 1.0.
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Figure 5: Sensitivity of mAbs D2, F9 and C1-13 against rPfHRP2. The binding efficacy of mAbs against serially diluted recombinant PfHRP2 is determined by ELISA (Left panel) and dot blot (Right panel). A: 5 μg/mL of mAbs; B: 1 μg/mL of mAbs. Dot line represents cut-off OD absorbance at 1.0.

Mentions: The specificity of mAbs against recombinant and native malaria proteins was evaluated by Western blot analysis (Figure 4). Both mAbs D2 and F9 reacted to rPfHRP2, showing a single band at 82 kDa, the correct size for the thioredoxin-fused protein. For detection of native parasite proteins from blood stage infection, P. falciparum-infected red blood cells were tested in Western blot. The results show that all mAbs were capable of recognizing native PfHRP2 protein at ~50 kDa, with such band was not detected from uninfected RBC protein lane. No cross-reactivity with the other two malaria biomarkers, namely P. falciparum lactate dehydrogenase (PfLDH) and Plasmodium vivax aldolase (PvAldolase) was observed on Western blot (Additional file 1) or ELISA (Additional file 2).The sensitivity of detecting rPfHRP2 protein by mAbs was investigated by ELISA and dot blot analysis (Figure 5). The ELISA data indicated a difference in sensitivity between three mAbs when comparing the detection limit against serial diluted rPfHRP2. While 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 (Figure 5A). However, sensitivity fell when less concentrated antibodies were applied (1 μg/mL) (Figure 5B). These results were consistent with results of the semi-quantitative studies carried out by dot blot. Compared to the commercial antibody C1-13, the sensitivity of both antibodies D2 and F9 was lower. Detection limits were ten times (D2) and 100 times (F9) lower than for C1-13.To determine the accuracy and linearity of rPfHRP2 detection, mAbs of D2 and F9 were each designed as capture or detection antibodies in sandwich ELISA, in combination with mAb C1-13. The mean reactivity for duplicate measures of serially diluted rPfHRP2 protein from 1 μg/mL to 0.01 fg/mL detected by mAbs D2 and F9 are represented in Figure 6. From the results, both antibodies D2 and F9 showed binding to rPfHRP2 protein in heterologous configurations with mAb C1-13. However, the sensitivity of heterologous antibodies varied despite the use of the same mAbs D2 or F9 as either capture or detection antibodies. When using an OD cut-off value of 0.25, the detection limit for D2-Cap/C1-13-Detection was at 50 pg/mL, whereas C1-13-Cap/D2-Detection was at 1 pg/mL (Figure 6A). The configuration of mAbs F9 and C1-13 influenced different sensitivity. The lowest detection limit for F9-Cap/C1-13-Detection was 5 ng/mL, and C1-13-Cap/F9-Detection was ~0.5 ng/mL at the OD cut-off value of 0.25 (Figure 6B). Overall, the configuration of C1-13-Cap/D2-Detection was the most sensitive for detection of rPfHRP2 protein by ELISA. Neither heterologous mAbs D2 nor F9 reacted to 1% BSA protein.


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)

Sensitivity of mAbs D2, F9 and C1-13 against rPfHRP2. The binding efficacy of mAbs against serially diluted recombinant PfHRP2 is determined by ELISA (Left panel) and dot blot (Right panel). A: 5 μg/mL of mAbs; B: 1 μg/mL of mAbs. Dot line represents cut-off OD absorbance at 1.0.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4120728&req=5

Figure 5: Sensitivity of mAbs D2, F9 and C1-13 against rPfHRP2. The binding efficacy of mAbs against serially diluted recombinant PfHRP2 is determined by ELISA (Left panel) and dot blot (Right panel). A: 5 μg/mL of mAbs; B: 1 μg/mL of mAbs. Dot line represents cut-off OD absorbance at 1.0.
Mentions: The specificity of mAbs against recombinant and native malaria proteins was evaluated by Western blot analysis (Figure 4). Both mAbs D2 and F9 reacted to rPfHRP2, showing a single band at 82 kDa, the correct size for the thioredoxin-fused protein. For detection of native parasite proteins from blood stage infection, P. falciparum-infected red blood cells were tested in Western blot. The results show that all mAbs were capable of recognizing native PfHRP2 protein at ~50 kDa, with such band was not detected from uninfected RBC protein lane. No cross-reactivity with the other two malaria biomarkers, namely P. falciparum lactate dehydrogenase (PfLDH) and Plasmodium vivax aldolase (PvAldolase) was observed on Western blot (Additional file 1) or ELISA (Additional file 2).The sensitivity of detecting rPfHRP2 protein by mAbs was investigated by ELISA and dot blot analysis (Figure 5). The ELISA data indicated a difference in sensitivity between three mAbs when comparing the detection limit against serial diluted rPfHRP2. While 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 (Figure 5A). However, sensitivity fell when less concentrated antibodies were applied (1 μg/mL) (Figure 5B). These results were consistent with results of the semi-quantitative studies carried out by dot blot. Compared to the commercial antibody C1-13, the sensitivity of both antibodies D2 and F9 was lower. Detection limits were ten times (D2) and 100 times (F9) lower than for C1-13.To determine the accuracy and linearity of rPfHRP2 detection, mAbs of D2 and F9 were each designed as capture or detection antibodies in sandwich ELISA, in combination with mAb C1-13. The mean reactivity for duplicate measures of serially diluted rPfHRP2 protein from 1 μg/mL to 0.01 fg/mL detected by mAbs D2 and F9 are represented in Figure 6. From the results, both antibodies D2 and F9 showed binding to rPfHRP2 protein in heterologous configurations with mAb C1-13. However, the sensitivity of heterologous antibodies varied despite the use of the same mAbs D2 or F9 as either capture or detection antibodies. When using an OD cut-off value of 0.25, the detection limit for D2-Cap/C1-13-Detection was at 50 pg/mL, whereas C1-13-Cap/D2-Detection was at 1 pg/mL (Figure 6A). The configuration of mAbs F9 and C1-13 influenced different sensitivity. The lowest detection limit for F9-Cap/C1-13-Detection was 5 ng/mL, and C1-13-Cap/F9-Detection was ~0.5 ng/mL at the OD cut-off value of 0.25 (Figure 6B). Overall, the configuration of C1-13-Cap/D2-Detection was the most sensitive for detection of rPfHRP2 protein by ELISA. Neither heterologous mAbs D2 nor F9 reacted to 1% BSA protein.

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