Open Access
Research (Published online: 06-09-2018)
4. Assessment of in vitro potency of inactivated Newcastle disease oil adjuvanted vaccines using hemagglutination test and blocking ELISA
Saleh E. Aly, Hussein Ali Hussein, Abdel-Hakim M. Aly, Mansour H. Abdel-Baky and Ahmed A. El-Sanousi
Veterinary World, 11(9): 1222-1228

Saleh E. Aly: Central Laboratory for Evaluation of Veterinary Biologics, Abbasia 11381, Egypt.
Hussein Ali Hussein: Department of Virology, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt.
Abdel-Hakim M. Aly: Central Laboratory for Evaluation of Veterinary Biologics, Abbasia 11381, Egypt.
Mansour H. Abdel-Baky: Central Laboratory for Evaluation of Veterinary Biologics, Abbasia 11381, Egypt.
Ahmed A. El-Sanousi: Department of Virology, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt.

doi: 10.14202/vetworld.2018.1222-1228

Share this article on [Facebook] [LinkedIn]

Article history: Received: 08-11-2017, Accepted: 20-07-2018, Published online: 06-09-2018

Corresponding author: Saleh E. Aly


Citation: Aly SE, Hussein HA, Aly AM, Abdel-Baky MH, El-Sanousi AA (2018) Assessment of in vitro potency of inactivated Newcastle disease oil-adjuvanted vaccines using hemagglutination test and blocking ELISA, Veterinary World, 11(9): 1222-1228.

Aim: The present study was aimed to establish a protocol for the evaluation of the in vitro potency of commercial inactivated Newcastle disease virus (NDV) oil-adjuvanted vaccines using hemagglutination test (HA) and blocking ELISA (B-ELISA) based on polyclonal antibodies.

Materials and Methods: Aqueous phases from a total of 47 batches of inactivated NDV vaccines manufactured by 20 different companies were extracted with isopropyl myristate. The viral antigen in each sample was detected and quantified by a standard HA test and a B-ELISA assay. To verify the efficiency of the antigen extraction method used in the batches which showed HA and to test the validity of using in vitro antigen quantification by HA and B-ELISA tests, a subset of 13 batches (selected from the total 47 batches) was inoculated in groups of 3-4-week-old specific pathogen-free chickens using the recommended vaccine dose. The immunogenicity of the selected vaccine batches was assessed by the NDV-hemagglutination inhibition antibody titers in individual serum samples collected 4 weeks after vaccination. Further, the efficacy of the vaccines and their protection rates were determined by a challenge test carried out for the vaccinated chickens with the Egyptian 2012 isolate of the virulent NDV genotype VII.

Results: A strong correlation was observed between HA titers and B-ELISA mean titers in the tested 47 batches (R2=0.817). This indicated the possibility of using the latter in vitro assays for vaccine potency assessment. The recommended protective NDV antigen titer measured by B-ELISA was determined to be 28 ELISA units per dose. The comparison between the HA titers of the aqueous extracts of test vaccines and the corresponding results of in vivo potency assays (i.e., immunogenicity and efficacy), including antibody titers in the serum of vaccinated birds, indicated that the efficiency of the antigen extraction used may interfere with obtaining a strong correlation between the in vitro and in vivo results.

Conclusion: HA or B-ELISA tests can be used as rapid and cost-effective alternatives to traditional in vivo potency tests for vaccine potency assessment by quantifying the NDV antigen present in aqueous phase extracts of the tested vaccines. The latter in vitro protocol, however, requires efficient extraction of the antigen to be able to obtain good correlation with the traditional in vivo potency tests.

Keywords: blocking ELISA, inactivated vaccines, in vitro, Newcastle disease virus, vaccine potency.


1. World Organization for Animal Health (OIE). (2012) Newcastle disease (Infection with Newcastle disease virus). Man. Diagn. Tests Vaccines Terr. Anim. (Mammals, Birds Bees), 1: 555-574.

2. King, A., Lefkowitz, E., Adams, M.I. and Carstens, E.B. (2012) Virus Taxonomy: 9th Report of the International Committee on Taxonomy of Viruses. Academic Press, London, United Kingdom.

3. Archetti, R.I. and Horsfall, F.L. (1950) Persistent antigenic variation of influenza A viruses after incomplete neutralization in ovo with heterologous immune serum. J. Exp. Med., 92: 441-462. [Crossref] [PubMed] [PMC]

4. Alexander, D.J. (2003) Paramyxoviridae. Iowa State University Press, Iowa.

5. Miller, P.J., Afonso, C.L., Spackman, E., Scott, M.A., Pedersen, J.C. and Senne, D.A, Brown, J.D., Fuller, C.M., Uhart, M.M., Karesh, W.B., Brown, I.H., Alexander, D.J. and Swayne, D.E. (2010) Evidence for a new avian Paramyxovirus serotype 10 detected in rockhopper penguins from the Falkland Islands. J. Virol., 84: 11496-11504. [Crossref] [PubMed] [PMC]

6. Briand, F.X., Henry, A., Massin, P. and Jestin, V. (2012) Complete genome sequence of a novel avian Paramyxovirus. J. Virol., 86: 7710. [Crossref] [PubMed] [PMC]

7. Terregino, C., Aldous, E.W., Heidari, A., Fuller, C.M., De Nardi, R. and Manvell, R.J., Beato, M.S., Shell, W.M., Monne, I., Brown, I.H., Alexander, D.J. and Capua, I. (2013) Antigenic and genetic analyses of isolate APMV/wigeon/Italy/3920-1/2005 indicate that it represents a new avian Paramyxovirus (APMV-12). Arch. Virol., 158: 2233-2243. [Crossref]

8. Goraichuk, I., Sharma, P., Stegniy, B., Muzyka, D., Pantin-Jackwood, M.J. and Gerilovych, A., Solodiankin, O., Bolotin, V., Miller, P.J., Dimitrov, K.M. and Afonso, C.L. (2016) Complete genome sequence of an avian Paramyxovirus representative of putative new serotype 13. Genome Announc., 4: e00729-e0016. [Crossref]

9. Yamamoto, E., Ito, H., Tomioka, Y. and Ito, T. (2015) Characterization of novel avian Paramyxovirus strain APMV/Shimane67 isolated from migratory wild geese in Japan. J. Vet. Med. Sci., 77: 1079-1085. [Crossref] [PubMed] [PMC]

10. Karamendin, K., Kydyrmanov, A., Seidalina, A., Asanova, S., Sayatov, M. and Kasymbekov, E., Khan, E., Daulbayeva, K., Harrison, S.M., Carr, I.M., Goodman, S.J. and Zhumatov, K. (2016) Complete genome sequence of a novel avian Paramyxovirus (APMV-13) isolated from a Wild Bird in Kazakhstan. Genome Announc., 4: 13-14. [Crossref] [PubMed] [PMC]

11. Thampaisarn, R., Bui, V.N., Trinh, D.Q., Nagai, M., Mizutani, T. and Omatsu, T., Katayama, Y., Gronsang, D., Le, D.H.T., Ogawa, H. and Imai, K (2017) Characterization of avian Paramyxovirus serotype 14, a novel serotype, isolated from a duck fecal sample in Japan. Virus. Res., 228: 46-57. [Crossref] [PubMed]

12. Lee, H.J., Kim, J.Y., Lee, Y.J., Lee, E.K., Song, B.M. And Lee, H.S. and Choi, K.S. (2017) A novel avian Paramyxovirus (putative serotype 15) isolated from wild birds. Front Microbiol., 8: 1-9. [Crossref] [PubMed] [PMC]

13. European Pharmacopoeia (Ph. Eur.). (2017) Monograph No. 870 Inactivated Newcastle Disease Vaccines. 9th ed. Iowa State University Press, Ames, IA.

14. Maas, P.A., de Winter, M.P.M., Venema, S., Oei, H.L. and Claassen, I.J.T.M. (2000) Antigen quantification as in vitro alternative for potency testing of inactivated viral poultry vaccines. Vet. Q., 22: 223-227. [Crossref] [PubMed]

15. Kulpa-Eddy, J., Srinivas, G., Halder, M., Hill, R., Brown, K., Roth, J., Draayer, H., Galvin, J., Claassen, I., Gifford, G., Woodland, R. and Doelling, V. (2011) Non-animal replacement methods for veterinary vaccine potency testing: state-of-the-science and future directions. Proc. Vaccinol., 5: 60-83. [Crossref]

16. Abdel Rahman, S.S., Abel Wanis, N.A., Aly, M.A., Abdel-Rahman, A. and Sae, A. (2006) Detection of antigen content of inactivated oil Newcastle disease vaccines by a new laboratory method. Kafer El-Sheikh Vet. Med. J., 4: 267-276.

17. Motitschke, A.J.C. (2011) The quantitative ELISA for inactivated Newcastle antigen: Experience report from an OMCL. Dev. Biol., 134: 55-66.

18. Basagoudanavar, S.H., Hosamani, M., Tamil, S.R.P., Sreenivasa, B.P., Saravanan, P., Chandrasekhar, S.B.K., Venkataramanan, R. (2013) Development of a liquid-phase blocking ELISA based on foot-and-mouth disease virus empty capsid antigen for seromonitoring vaccinated animals. Arch. Virol., 158: 993-1001. [Crossref]

19. Animal and Plant Health Inspection Service, editor. (2007) Title 9-Animals and Animal Products. Part 113.205-Newcastle disease Virus. Vol. 1. U.S. Department of Agriculture, Washington, DC.

20. Liljebjelke, K.A., King, D.J., Kapczynski, D.R. (2008) Determination of minimum hemagglutinin units in an inactivated Newcastle disease virus vaccine for clinical protection of chickens from exotic Newcastle disease virus challenge. Avian Dis., 52: 260-268. [Crossref] [PubMed]

21. Brugh, M. (1978) A simple method for recording and analyzing serological data. Avian Dis., 22: 362-365. [Crossref] [PubMed]

22. Jagt, H.J.M., Bekkers, M.L.E., van Bommel, S.A.J.T., van der Marel, P., Schrier, C.C. (2010) The influence of the inactivating agent on the antigen content of inactivated Newcastle disease vaccines assessed by the in vitro potency test. Biologicals, 38: 128-134. [Crossref] [PubMed]

23. Minor, P. (2015) Assaying the potency of influenza vaccines. Vaccines, 3: 90-104. [Crossref] [PubMed] [PMC]

24. Bodle, J., Verity, E.E., Ong C., Vandenberg, K., Shaw, R., Barr, I.G. and Rockman, S. (2013) Development of an enzyme-linked immunoassay for the quantitation of influenza haemagglutinin: An alternative method to single radial immunodiffusion. Influenza Other Respi. Viruses, 7: 191-200. [Crossref] [PubMed] [PMC]

25. Wood, J.M., Mumford, J.A., Dunleavy, U., Seagroatt, V., Newman, R.W., Thornton, D., Schild, G.C. (1988) Single radial immunodiffusion potency tests for inactivated equine influenza vaccines. In: Powell, D.G.E., editor. Equine Infect. Dis. V. Proceeding Fifth International Conference. The University Press of Kentucky, KY USA. p74-79.

26. Williams, T.L., Pirkle, J.L., Barr, J.R. (2012) Simultaneous quantification of hemagglutinin and neuraminidase of influenza virus using isotope dilution mass spectrometry. Vaccine, 30: 2475-2482. [Crossref] [PubMed]

27. Yang, Y., Li, H., Li, Z., Zhang, Y., Zhang, S., Chen, Y, Yu, M., Ma, G. and Su, Z. (2015) Size-exclusion HPLC provides a simple, rapid, and versatile alternative method for quality control of vaccines by characterizing the assembly of antigens. Vaccine, 33: 1143-1150. [Crossref] [PubMed]