Vaccines that interrupt malaria transmission are of increasing interest and a 
robust functional assay to measure this activity would promote their development 
by providing a biologically relevant means of evaluating potential vaccine 
candidates. Therefore, we aimed to qualify the standard membrane-feeding assay 
(SMFA). The assay measures the transmission-blocking activity of antibodies by 
feeding cultured P. falciparum gametocytes to Anopheles mosquitoes in the 
presence of the test antibodies and measuring subsequent mosquito infection. The 
International Conference on Harmonisation (ICH) Harmonised Tripartite Guideline 
Q2(R1) details characteristics considered in assay validation. Of these 
characteristics, we decided to qualify the SMFA for Precision, Linearity, Range 
and Specificity. The transmission-blocking 4B7 monoclonal antibody was tested 
over 6 feeding experiments at several concentrations to determine four suitable 
concentrations that were tested in triplicate in the qualification experiments 
(3 additional feeds) to evaluate Precision, Linearity and Range. For 
Specificity, 4B7 was tested in the presence of normal mouse IgG. We determined 
intra- and inter-assay variability of % inhibition of mean oocyst intensity at 
each concentration of 4B7 (lower concentrations showed higher variability). We 
also showed that % inhibition was dependent on 4B7 concentration and the 
activity is specific to 4B7. Since obtaining empirical data is time-consuming, 
we generated a model using data from all 9 feeds and simulated the effects of 
different parameters on final readouts to improve the assay procedure and 
analytical methods for future studies. For example, we estimated the effect of 
number of mosquitoes dissected on variability of % inhibition, and simulated the 
relationship between % inhibition in oocyst intensity and % inhibition of 
prevalence of infected mosquitos at different mean oocysts in the control. SMFA 
is one of the few biological assays used in preclinical and early clinical 
development of transmission-blocking vaccines, and this study strongly supports 
its further development and application.