TY - JOUR
T1 - A chemiluminescent-western blot assay for quantitative detection of Plasmodium falciparum circumsporozoite protein
AU - Kumar, Sanjai
AU - Zheng, Hong
AU - Sangweme, Davison T.
AU - Mahajan, Babita
AU - Kozakai, Yukiko
AU - Pham, Phuong T.
AU - Morin, Merribeth J.
AU - Locke, Emily
AU - Kumar, Nirbhay
N1 - Funding Information:
We thank the PATH Malaria Vaccine Initiative for providing the financial support for this study.
PY - 2013/4/30
Y1 - 2013/4/30
N2 - Highly sensitive and reliable assays based on the quantitation of immunologically relevant component(s) in recombinant or whole parasite-based vaccines would facilitate pre-clinical and clinical phases and the monitoring of malaria vaccine deployment. Here we report a laboratory-grade Western Blot assay for quantitative detection of Plasmodium falciparum circumsporozoite protein (PfCSP) in P. falciparum sporozoite (PfSPZ) and in recombinant (rPfCSP) product. This assay is based on the immuno-reactivity of an anti-P. falciparum CSP monoclonal antibody (mAb 2A10) with the NANP-repeat units on PfCSP. The antigen-antibody complex is detected by reaction with a commercially obtained chemiluminescence-linked Immunodetection system. The linear range for detecting the recombinant P. falciparum CSP (rPfCSP) in this assay is 3-12pg (R2=0.9399). The range for detecting the day 15 salivary-gland PfSPZ is between 0.0625 and 1 parasite (R2=0.9448) and approximately 10.0pg of PfCSP was detected on each sporozoite. The assay was highly reproducible in measuring the PfCSP on PfSPZ. The inter-assay Coefficient of Variation (CV%) was 10.31% while the intra-assay CV% on three different days was 6.05%, 2.03% and 1.42% respectively. These results suggest that this ECL-WB assay is highly sensitive and robust with a low degree of inter-assay and intra-assay variations. To our knowledge, this is the most sensitive immunoassay for the detection of a recombinant or native malarial protein and may have a wider range of applications including the quantification of immunological component(s) in a vaccine formulation, determination of the antigenic integrity in adjuvanted-vaccine and in stability studies. In addition, this assay can be applied to measure the mosquito infectivity in malaria transmission areas and to determine the effects of intervention measures on malaria transmission.
AB - Highly sensitive and reliable assays based on the quantitation of immunologically relevant component(s) in recombinant or whole parasite-based vaccines would facilitate pre-clinical and clinical phases and the monitoring of malaria vaccine deployment. Here we report a laboratory-grade Western Blot assay for quantitative detection of Plasmodium falciparum circumsporozoite protein (PfCSP) in P. falciparum sporozoite (PfSPZ) and in recombinant (rPfCSP) product. This assay is based on the immuno-reactivity of an anti-P. falciparum CSP monoclonal antibody (mAb 2A10) with the NANP-repeat units on PfCSP. The antigen-antibody complex is detected by reaction with a commercially obtained chemiluminescence-linked Immunodetection system. The linear range for detecting the recombinant P. falciparum CSP (rPfCSP) in this assay is 3-12pg (R2=0.9399). The range for detecting the day 15 salivary-gland PfSPZ is between 0.0625 and 1 parasite (R2=0.9448) and approximately 10.0pg of PfCSP was detected on each sporozoite. The assay was highly reproducible in measuring the PfCSP on PfSPZ. The inter-assay Coefficient of Variation (CV%) was 10.31% while the intra-assay CV% on three different days was 6.05%, 2.03% and 1.42% respectively. These results suggest that this ECL-WB assay is highly sensitive and robust with a low degree of inter-assay and intra-assay variations. To our knowledge, this is the most sensitive immunoassay for the detection of a recombinant or native malarial protein and may have a wider range of applications including the quantification of immunological component(s) in a vaccine formulation, determination of the antigenic integrity in adjuvanted-vaccine and in stability studies. In addition, this assay can be applied to measure the mosquito infectivity in malaria transmission areas and to determine the effects of intervention measures on malaria transmission.
KW - Chemiluminescent-western blot
KW - Circumsporozoite protein
KW - IOD
KW - Plasmodium falciparum
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U2 - 10.1016/j.jim.2013.02.001
DO - 10.1016/j.jim.2013.02.001
M3 - Article
C2 - 23399449
AN - SCOPUS:84875233699
SN - 0022-1759
VL - 390
SP - 99
EP - 105
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1-2
ER -