Refolding and characterization of a diabody against Pfs25, a vaccine candidate of Plasmodium falciparum

Jagannath, Deepak K; Valiyaparambil, Ashwathi; Viswanath, Vysakh K; Hurakadli, Manjunath A.; Kamariah, Neelagandan; Jafer, Alifia C.; Patole, Chhaya; Pradhan, Sabyasachi; Kumar, Naveen; Lakshminarasimhan, Anirudha

doi:10.1016/j.ab.2022.114830

Cited by 2 publications

(13 citation statements)

References 64 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…pET21a vector with codon optimized DNA sequence for the cassette V H -G 4 S-V L -His 6 from 4B7 and 1245 was expressed in E. coli BL21(DE3). The method optimised earlier for refolding 1269-Db (19), resulted in the recovery of the antibody fragments in solution after solubilization, refolding, dialysis and purification (Fig. 2A & 2B).…”

Section: Resultsmentioning

confidence: 98%

“…Diabody 4B7-Db shown in the second panel was evaluated alongside 1269-Db and mAb 4B7 as shown in figure 9 of Jagannath et. al (19). A small fraction of 1245-Db diabody observed in glutaraldehyde cross-linking assay could give rise to the weak signal observed in IFA.…”

Section: Resultsmentioning

confidence: 99%

“…4B7 monoclonal antibody, recognizes a linear epitope on Pfs25 (15), due to which it can recognize denatured Pfs25, in situ on a western blot (19). In line with its parent antibody, 4B7-Db recognized denatured Pfs25, with a western blot based binding assay (Fig 6A).…”

Section: Resultsmentioning

confidence: 99%

“…In line with its parent antibody, 4B7-Db recognized denatured Pfs25, with a western blot based binding assay (Fig 6A). We had earlier shown that 1269-Db, recognizing a conformational epitope on Pfs25, failed to exhibit binding with denatured Pfs25(19). In addition to recognizing denatured epitope, 4B7-Db also showed binding to native monomeric MBP-Pfs25 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In addition to recognizing denatured epitope, 4B7-Db also showed binding to native monomeric MBP-Pfs25 (Fig. 6B)(19).…”

Section: Resultsmentioning

confidence: 99%

See 4 more Smart Citations

Differential scanning fluorimetry as a measure of functionality of refolded anti-malarial antibody fragments

Valiyaparambil

Jagannath

Viswanath

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

Transmission blocking monoclonal antibodies, 4B7 and 1245, targeting Pfs25, have been demonstrated to block the developmental stages of the malarial parasite inside the mosquitoes. In this work, anti-plasmodium antibody fragments of 4B7 and 1245, designated as diabodies, were expressed in bacteria and refolded using a standardized method, with a yield of 0.1 and 0.4 mg per litre of culture, respectively. Dimeric species was detected for 4B7-Db, but not for 1245-Db, using glutaraldehyde cross-linking experiment. The quality of the purified refolded fraction was assessed with differential scanning fluorimetry (DSF). Refolded 4B7-Db, with a melting temperature of 59 degrees C, recognized Pfs25 expressed on the surface of ookinete and zygote stages of Plasmodium in an immunofluorescence-based assay, whereas, 1245-Db, exhibiting a skewed melt profile, showed weak recognition. Further, refolded 4B7-Db recognized the linear epitope, on purified Pfs25 protein, both in the denatured and native state. Differential scanning fluorimetry can be potentially employed as a qualitative measure of functionality, to evaluate refolded proteins, with applicability for antibody engineering in passive immunization.

show abstract

Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%