Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Feb 5;10(2):243.
doi: 10.3390/vaccines10020243.

Establishment of Sandwich ELISA for Quality Control in Rotavirus Vaccine Production

Cao Li  1 , Guoxing Luo  1 , Yuanjun Zeng  1 , Feibo Song  2 , Han Yang  1 , Shiyin Zhang  1 , Yingbin Wang  1 , Tingdong Li  1 , Shengxiang Ge  1 , Ningshao Xia  1   2
Affiliations

Establishment of Sandwich ELISA for Quality Control in Rotavirus Vaccine Production

Cao Li et al. Vaccines (Basel). .

Abstract

Non-replicating rotavirus vaccines are alternative strategies that may improve the protective efficacy of rotavirus vaccines in low- and middle-income countries. The truncated spike protein VP4 (aa26-476, VP4*)was a candidate antigen for the development of recombinant rotavirus vaccines, with higher immunogenicity and protective efficacy compared to VP8* and VP5* alone. This article describes the development of three genotype-specific sandwich ELISAs for P[4], P[6], and P[8]-VP4*, which are important for quality control in rotavirus vaccine production. Our results showed that the detection systems had good specificity for the different genotype VP4* and were not influenced by the E. coli host proteins. Moreover, the detection systems play an important role in determining whether the target protein was contaminated by VP4* proteins of other genotypes. They can also detect the adsorption rate of the adjuvant to the P[4], P[6], P[8]-VP4* protein during the process development. The three detection systems will play an important role in the quality control and process development of VP4* based rotavirus vaccines and facilitate the development of recombinant rotavirus vaccines.

Keywords: P[4], P[6], P[8]-VP4*; detection; enzyme-linked immunosorbent assay; vaccine.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The reactivity of the antibody pairs for P[4], P[6], P[8]-VP4*, and Bl21(DE3) E. coli lysate as examined by checkerboard. (A–D) The reactivity of P[4]-VP4*-specific mAb pairs for detecting 100 ng/mL P[4]-VP4*, 100 ng/mL P[6]-VP4*, 100 ng/mL P[8]-VP4*, and the cell lysate of Bl21(DE3); (E–H) the reactivity of P[6]-VP4*-specific mAb pairs for detecting 50 ng/mL P[4]-VP4*, 50 ng/mL P[6]-VP4*, 50 ng/mL P[8]-VP4*, and the cell lysate of Bl21(DE3); (I–L) the reactivity of P[8]-VP4*-specific mAb pairs for detecting 20 ng/mL P[4]-VP4*, 20 ng/mL P[6]-VP4*, 20 ng/mL P[8]-VP4*, and the cell lysate of Bl21(DE3).
Figure 2
Figure 2
The detection range and specificity of P[4], P[6], and P[8]-VP4*-specific detection systems. The purified VP4* proteins were 2-fold serially diluted from 2 μg/mL to 0.97 ng/mL by 20% NBS, and detected by P[4], P[6], and P[8]-specific VP4* detection system. (A) The reactivity of P[4]-VP4* antibody pair 7E3:5D8-HRP to different genotype VP4* proteins; (B) the reactivity of P[6]-VP4 antibody pair 1E9:3D4-HRP to different genotype VP4 proteins; (C) the reactivity of P[8]-VP4* antibody pair 15D9:5F10-1-HRP to different genotype VP4* proteins. All the experiments were performed in three replicates, and the error bars represent the standard deviation of each group.
Figure 3
Figure 3
The recovery rate of P[4], P[6], and P[8]-VP4* proteins in E. coli lysate. The purified P[4], P[6], and P[8]-VP4* proteins were added to the supernatant of Bl21(DE3) E. coli lysate to a final concentration of 100, 20, and 4 μg/mL, respectively, and detected by the corresponding genotype-specific antibody pairs. (A) The results detected by P[4]-VP4*-specific mAb pair (7E3: 5D8-HRP); (B) the results detected by the P[6]-VP4*-specific mAb pair (1E9: 3D4-HRP); (C) the results detected by the P[8]-VP4*-specific mAb pair (15D9: 5F10-1-HRP). All the experiments were performed in three replicates, and the error bars represent the standard deviation of each group. The concentration of each protein was calculated from the corresponding standard curve (Supplementary Figure S2). The recovery rate was calculated according to the formula: VP4* calculated/VP4*theoretical*100%.
Figure 3
Figure 3
The recovery rate of P[4], P[6], and P[8]-VP4* proteins in E. coli lysate. The purified P[4], P[6], and P[8]-VP4* proteins were added to the supernatant of Bl21(DE3) E. coli lysate to a final concentration of 100, 20, and 4 μg/mL, respectively, and detected by the corresponding genotype-specific antibody pairs. (A) The results detected by P[4]-VP4*-specific mAb pair (7E3: 5D8-HRP); (B) the results detected by the P[6]-VP4*-specific mAb pair (1E9: 3D4-HRP); (C) the results detected by the P[8]-VP4*-specific mAb pair (15D9: 5F10-1-HRP). All the experiments were performed in three replicates, and the error bars represent the standard deviation of each group. The concentration of each protein was calculated from the corresponding standard curve (Supplementary Figure S2). The recovery rate was calculated according to the formula: VP4* calculated/VP4*theoretical*100%.
Figure 4
Figure 4
The evaluation of the identity of purified VP4* proteins by the genotype-specific mAb pairs. In order to confirm whether the ELISA systems can be used to detect protein contamination, we mixed 10 μg/mL of the other two VP4* proteins into P[4], P[6], and P[8]-VP4* protein, respectively, and then the mixtures were detected by the three genotype-specific mAb pairs. As shown in the (A–C), the three ELISA detection systems can accurately identify the P[4], P[6], and P[8]-VP4* in the mixed samples. All the experiments were performed in two replicates, and the error bars represent the standard deviation of each group.
See this image and copyright information in PMC

References

    1. Troeger C., Forouzanfar M., Rao P.C., Khalil I., Brown A., Reiner R.C., Fullman N., Thompson R.L., Abajobir A., Ahmed M., et al. Estimates of global, regional, and national morbidity, mortality, and aetiologies of diarrhoeal diseases: A systematic analysis for the Global Burden of Disease Study 2015. Lancet Infect. Dis. 2017;17:909–948. doi: 10.1016/S1473-3099(17)30276-1. - DOI - PMC - PubMed
    1. Tate J.E., Burton A.H., Boschi-Pinto C., Parashar U.D., Global W.H.O.-C. Global, Regional, and National Estimates of Rotavirus Mortality in Children <5 Years of Age, 2000–2013. Clin. Infect. Dis. 2016;62:S96–S105. - PMC - PubMed
    1. Parashar U.D., Glass R.I. Rotavirus vaccines—Early success, remaining questions. N. Engl. J. Med. 2009;360:1063–1065. doi: 10.1056/NEJMp0810154. - DOI - PubMed
    1. Cunliffe N.A., Witte D., Ngwira B.M., Todd S., Bostock N.J., Turner A.M., Chimpeni P., Victor J.C., Steele A.D., Bouckenooghe A., et al. Efficacy of human rotavirus vaccine against severe gastroenteritis in Malawian children in the first two years of life: A randomized, double-blind, placebo controlled trial. Vaccine. 2012;30((Suppl. 1)):A36–A43. doi: 10.1016/j.vaccine.2011.09.120. - DOI - PMC - PubMed
    1. Zaman K., Anh D.D., Victor J.C., Shin S., Yunus M., Dallas M.J., Podder G., Thiem V.D., Mai L.T.P., Luby S.P., et al. Efficacy of pentavalent rotavirus vaccine against severe rotavirus gastroenteritis in infants in developing countries in Asia: A randomised, double-blind, placebo-controlled trial. Lancet. 2010;376:615–623. doi: 10.1016/S0140-6736(10)60755-6. - DOI - PubMed

LinkOut - more resources