title: A slot blot immunoassay for quantitative detection of Plasmodium falciparum circumsporozoite protein in mosquito midgut oocyst.
abstract: There is still a need for sensitive and reproducible immunoassays for quantitative detection of malarial antigens in preclinical and clinical phases of vaccine development and in epidemiology and surveillance studies, particularly in the vector host. Here we report the results of sensitivity and reproducibility studies for a research-grade, quantitative enhanced chemiluminescent-based slot blot assay (ECL-SB) for detection of both recombinant Plasmodium falciparum circumsporozoite protein (rPfCSP) and native PfCSP from Oocysts (Pf Oocyst) developing in the midguts of Anopheles stephensi mosquitoes. The ECL-SB detects as little as 1.25 pg of rPfCSP (linear range of quantitation 2.5-20 pg; R2 = 0.9505). We also find the earliest detectable expression of native PfCSP in Pf Oocyst by ECL-SB occurs on day 7 post feeding with infected blood meal. The ECL-SB was able to detect approximately as few as 0.5 day 8 Pf Oocysts (linear quantitation range 1-4, R2 = 0.9795) and determined that one Pf Oocyst expressed approximately 2.0 pg (0.5-3 pg) of native PfCSP, suggesting a similar range of detection for recombinant and native forms of Pf CSP. The ECL-SB is highly reproducible; the Coefficient of Variation (CV) for inter-assay variability for rPf CSP and native PfCSP were 1.74% and 1.32%, respectively. The CVs for intra-assay variability performed on three days for rPf CSP were 2.41%, 0.82% and 2% and for native Pf CSP 1.52%, 0.57%, and 1.86%, respectively. In addition, the ECL-SB was comparable to microscopy in determining the P. falciparum prevalence in mosquito populations that distinctly contained either high and low midgut Pf Oocyst burden. In whole mosquito samples, estimations of positivity for P. falciparum in the high and low burden groups were 83.3% and 23.3% by ECL-SB and 85.7% and 27.6% by microscopy. Based on its performance characteristics, ECL-SB could be valuable in vaccine development and to measure the parasite prevalence in mosquitoes and transmission-blocking interventions in endemic areas.
authors.affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993, United States of America.
authors.forename: Sanjai
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993, United States of America.
authors.forename: Hong
authors.initials: H
authors.lastname: Zheng
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland 20852, United States of America.
authors.forename: Bingbing
authors.initials: B
authors.lastname: Deng
authors.affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993, United States of America.
authors.forename: Babita
authors.initials: B
authors.lastname: Mahajan
authors.affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993, United States of America.
authors.forename: Bryan
authors.initials: B
authors.lastname: Grabias
authors.affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993, United States of America.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Kozakai
authors.affiliation: Program for Appropriate Technology in Health (PATH) Malaria Vaccine Initiative, Washington, DC 20001, United States of America.
authors.forename: Merribeth J
authors.initials: MJ
authors.lastname: Morin
authors.affiliation: Program for Appropriate Technology in Health (PATH) Malaria Vaccine Initiative, Washington, DC 20001, United States of America.
authors.forename: Emily
authors.initials: E
authors.lastname: Locke
authors.affiliation: Program for Appropriate Technology in Health (PATH) Malaria Vaccine Initiative, Washington, DC 20001, United States of America.
authors.forename: Ashley
authors.initials: A
authors.lastname: Birkett
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland 20852, United States of America.
authors.forename: Kazutoyo
authors.initials: K
authors.lastname: Miura
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland 20852, United States of America.
authors.forename: Carole
authors.initials: C
authors.lastname: Long
mesh_terms: D000818:Animals; D000852:Anopheles / Q000502:physiology; D015153:Blotting, Western; D004064:Digestive System / Q000276:immunology / Q000378:metabolism* / Q000469:parasitology; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008163:Luminescent Measurements; D016778:Malaria, Falciparum / Q000175:diagnosis* / Q000276:immunology / Q000469:parasitology; D033661:Oocysts / Q000276:immunology / Q000378:metabolism* / Q000469:parasitology; D010963:Plasmodium falciparum / Q000235:genetics / Q000276:immunology; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D034101:Sporozoites / Q000276:immunology / Q000378:metabolism / Q000469:parasitology
chemical_list: D015800:Protozoan Proteins; D011994:Recombinant Proteins; C041595:circumsporozoite protein, Protozoan