Western Blot Assay for Quantitative and Qualitative Antigen Detection in Vaccine Development

Sanjai Kumar1, Hong Zheng1, Babita Mahajan1, Yukiko Kozakai1, Merribeth Morin2, Emily Locke2

1 Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, Maryland, 2 PATH Malaria Vaccine Initiative, Washington, D.C.
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 18.4
DOI:  10.1002/9780471729259.mc1804s33
Online Posting Date:  May, 2014
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Immunological methods for quantitative measurement, antigenic characterization, and monitoring the stability of active immunogenic component(s) are a critical need in the vaccine development process. This unit describes an enhanced chemiluminescence‐based western blot for quantitative detection of Plasmodium falciparum circumsporozoite protein (PfCSP), a major malaria candidate vaccine antigen. The most salient features of this assay are its high sensitivity and reproducibility; it can reliably detect ∼5 to 10 pg PfCSP expressed on native parasites or recombinantly expressed in Escherichia coli. Although described for a specific vaccine antigen, this assay should be applicable for any antigen‐antibody combination for which relevant detection reagents are available. Detailed stepwise experimental procedures and methods for data acquisition and analysis are described. Curr. Protoc. Microbiol. 33:18.4.1‐18.4.11. © 2014 by John Wiley & Sons, Inc.

Keywords: western blot; malaria; vaccine; antigen; stability

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: P. falciparum CSP Western Blot Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: P. falciparum CSP Western Blot Assay

  • Recombinant P. falciparum CSP (rPfCSP; Rathore et al., ; Kumar et al., ) or P. falciparum sporozoites (PfSPZ; Kumar et al., )
  • Loading dye solution (see recipe)
  • 4‐12% NuPAGE gel (Life Technologies, cat. no. NP0321BOX)
  • 1× NuPAGE MES SDS running buffer (dilute from 20× in deionized water; Life Technologies, cat. no. NP0002)
  • Benchmark protein molecular weight marker (Life Technologies, cat. no. 10748‐010)
  • 1× transfer buffer (see recipe)
  • Methanol (Mallinckrodt Chemicals, cat. no. 3016‐02)
  • Blocking buffer (see recipe)
  • Anti‐PfCSP mAb 2A10 (produced as ascites in mice and purified by protein G affinity chromatography; Harlan Laboratories)
  • 10× phosphate‐buffered saline (PBS, Lonza, cat. no. 17‐515Q)
  • ECL kit: e.g., Western‐Star Immunodetection System (Applied Biosystems, cat. no. T1046) including blocking buffer, alkaline phosphatase (AP)−conjugated goat anti‐mouse IgG+IgM, detection reagents, and plastic folders for autoradiography
  • Gel apparatus (e.g., XCell SureLock Mini‐Cell, Life Technologies, cat. no. EI0001)
  • Power supply (Life Technologies, cat. no. EI8600)
  • Gel‐cutting knife
  • Western blot chamber (XCell II Blot Module, Life Technologies, cat. no. EI9051) with four sponges (cat. no. EI9052)
  • Filter paper (Schleicher & Schuell, cat. no. 31550N)
  • Polyvinylidene fluoride (PVDF) membrane (Millipore, cat. no. IPVH00010)
  • Plastic staining tray
  • Rocker platform (Labnet International, cat. no. Rocker 35)
  • Forceps
  • Film cassette
  • AR X‐ray film (Kodak, cat. no. 165‐1579)
  • Film developer (Kodak, cat. no. O‐XAT‐1000A)
  • Scanner (Hewlett‐Packard Scanjet G4050)
  • Adobe Photoshop software
  • ImageJ image analysis software (http://imagej.nih.gov/ij/download.html)
  • GraphPad Prism software (http://www.graphpad.com)
PDF or HTML at Wiley Online Library



Literature Cited

  Blackman, M.J., Heidrich, H.G., Donachie, S., McBride, J.S., and Holder, A.A. 1990. A single fragment of a malaria merozoite surface protein remains on the parasite during red cell invasion and is the target of invasion‐inhibiting antibodies. J. Exp. Med. 172:379‐382.
  Dutta, S., Lee, S.Y., Batchelor, A.H., and Lanar, D.E. 2007. Structural basis of antigenic escape of a malaria vaccine candidate. Proc. Natl. Acad. Sci. U.S.A. 104:12488‐12493.
  Gallagher, S., Winston, S.E., Fuller, S.A., and Hurrell, J.G. 2008. Immunoblotting and immunodetection. Curr. Protoc. Immunol. 83:8.10.1‐8.10.28.
  Gangnard, S., Badaut, C., Ramboarina, S., Baron, B., Ramdani, T., Gamain, B., Deloron, P., Lewit‐Bentley, A., and Bentley, G.A. 2013. Structural and immunological correlations between the variable blocks of the VAR2CSA domain DBL6epsilon from two Plasmodium falciparum parasite lines. J. Mol. Biol. 425:1697‐1711.
  Kumar, S., Zheng, H., Sangweme, D.T., Mahajan, B., Kozakai, Y., Pham, P.T., Morin, M.J., Locke, E., and Kumar, N. 2013. A chemiluminescent‐western blot assay for quantitative detection of Plasmodium falciparum circumsporozoite protein. J. Immunol. Methods 390:99‐105.
  Mahajan, B., Noiva, R., Yadava, A., Zheng, H., Majam, V., Mohan, K.V., Moch, J.K., Haynes, J.D., Nakhasi, H., and Kumar, S. 2006. Protein disulfide isomerase assisted protein folding in malaria parasites. Int. J. Parasitol. 36:1037‐1048.
  Michaelis, L. and Menten, M.L. 1913. The kinetics of invertin action. Biochem. Z. 49:333‐369.
  Nardin, E.H., Nussenzweig, V., Nussenzweig, R.S., Collins, W.E., Harinasuta, K.T., Tapchaisri, P., and Chomcharn, Y. 1982. Circumsporozoite proteins of human malaria parasites Plasmodium falciparum and Plasmodium vivax. J. Exp. Med. 156:20‐30.
  Rathore, D., Hrstka, S.C., Sacci, J.B.Jr., De la Vega, P., Linhardt, R.J., Kumar, S., and McCutchan, T.F. 2003. Molecular mechanism of host specificity in Plasmodium falciparum infection: Role of circumsporozoite protein. J. Biol. Chem. 278:40905‐40910.
  Weber, J.L. and Hockmeyer, W.T. 1985. Structure of the circumsporozoite protein gene in 18 strains of Plasmodium falciparum. Mol. Biochem. Parasitol. 15:305‐316.
  Zeeshan, M., Alam, M.T., Vinayak, S., Bora, H., Tyagi, R.K., Alam, M.S., Choudhary, V., Mittra, P., Lumb, V., Bharti, P.K., Udhayakumar, V., Singh, N., Jain, V., Singh, P.P., and Sharma, Y.D. 2012. Genetic variation in the Plasmodium falciparum circumsporozoite protein in India and its relevance to RTS,S malaria vaccine. PloS One 7:e43430.
  Zhu, D., McClellan, H., Dai, W., Gebregeorgis, E., Kidwell, M.A., Aebig, J., Rausch, K.M., Martin, L.B., Ellis, R.D., Miller, L., and Wu, Y. 2011. Long term stability of a recombinant Plasmodium falciparum AMA1 malaria vaccine adjuvanted with Montanide((R)) ISA 720 and stabilized with glycine. Vaccine 29:3640‐3645.
Key Reference
  Kumar et al., 2013. See above.
Internet Resource
  ImageJ software used for analysis of digital image is available in the public domain.
PDF or HTML at Wiley Online Library