Malarial diagnosis with RDT MAbs

Point of this post: why are patenting officers giving provisional patents when the idea has been described in earlier papers (lack of novelty) and is a logical consequence of earlier work?  

Why HRP2 for detection?

Plasmodium falciparum (Pf) HRP epitopes recognised by MAbs

abundance of HRP2 motifs

HRP3

Other useful characteristics of RDT MAbs (malaria)

Conformational epitope vs linear epitope

HRP2 missing!

HRP2 and HRP3 missing in some South American P. falciparum parasites

Selection of last 105 amino acids in HRP2 as epitope

band deletions by brightness-contrast adjustments 

Why are the lane bands not the same colour as the marker lane? 

Too much whitening ....

Lee N., A. Pelecanos, M. Bubb, I. Gonzalez, D. Bell, Q. Cheng & J. S. McCarthy (2012). Identification of Optimal Epitopes for Plasmodium falciparum Rapid Diagnostic Tests That Target Histidine-Rich Proteins 2 and 3, Journal of Clinical Microbiology, 50 (4) 1397-1405. DOI: http://dx.doi.org/10.1128/jcm.06533-11

Lee N., K. T. Andrews, M. L. Gatton, D. Bell, Q. Cheng & J. McCarthy (2006). Effect of Sequence Variation in Plasmodium falciparum Histidine- Rich Protein 2 on Binding of Specific Monoclonal Antibodies: Implications for Rapid Diagnostic Tests for Malaria, Journal of Clinical Microbiology, 44 (8) 2773-2778. DOI: http://dx.doi.org/10.1128/jcm.02557-05

Cheng Q., John Barnwell, Peter Chiodini, James McCarthy, David Bell & Jane Cunningham (2014). Plasmodium falciparum parasites lacking histidine-rich protein 2 and 3: a review and recommendations for accurate reporting, Malaria Journal, 13 (1) 283. DOI: http://dx.doi.org/10.1186/1475-2875-13-283

Verma R., M.A. Vijayalakshmi & Krishnan Venkataraman (2015). Novel monoclonal antibody against truncated C terminal region of Histidine Rich Protein2 (PfHRP2) and its utility for the specific diagnosis of malaria caused by Plasmodium falciparum, Experimental Parasitology, 150 56-66. DOI: http://dx.doi.org/10.1016/j.exppara.2015.01.001

FVIIILC from Pichia

Here is a 2013 paper on FVIII LC purification from Pichia pastoris....

In the two figures below, how do we know that this is really FVIII LC and what the antibody has detected really is the LC?  No positive control for Western or SDS PAGE?  

 IN ELISA procedure described below, note that the well surface is coated with PS or PC, then FVIII-LC is added and then the antibody.  

So these authors have no problem using PL/PS and the antibody against C-2 region in that order....

 The authors claim that ethylene glycol is able to disrupt the hydrophobic interactions between the lipid and the protein and hence decrease the ELISA signal.

 Now take a look at some papers reporting that antibodies binding to the C-2 region prevent FVIII-PS interaction.  here's a 1989 paper (just the abstract) and then the 2001 paper.  Hydrophobic residues in C-2 play a role in interrupting this interaction between FVIII C-2 (LC) and the phospholipid because this is where the antibody is supposed to bind.  Yet, the authors of the 2013 paper above manage to use an ELISA  using an antibody against C-2 AND get the LC to bind both the antibody AND the PL. But they claim that hydrophobic interactions play a role in the lipid-FVIII interaction.  There is no discussion on the possibility of a shared epitiope on C-2 that would bind to both lipid and antibody and no evidence that this particular polyclonal antibody does not fall under this category.

 

Why do reviewers not ask for positive controls and why do they not compare what is done in a manuscript to what is in the field?   

Sudheer Reddy AR, Padikara Kutty Satheeshkumar, & Mookambeswaran A. Vijayalakshmi (2013). Expression, purification, and partial in vitro characterization of biologically active human coagulation Factor VIII chain (A3-C1-C2) in Pichia pastoris Applied Biochemistry and Biotechnology DOI: 10.1007/s12010-013-0338-4

A.R. S.R., Satheeshkumar P.K. & Vijayalakshmi M.A. (2013). Expression, Purification, and Partial In Vitro Characterization of Biologically Active Human Coagulation Factor VIII Light Chain (A3-C1-C2) in Pichia pastoris, Applied Biochemistry and Biotechnology, 171 (1) 10-19. DOI: 10.1007/s12010-013-0338-4