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Sensitivity and Specificity of an In-house Sandwich Elisa Kit for Newcastle Disease Virus Antigen Detection

Md. Mostofa Kamal
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Md. Mortuza Ali
Department of Community Medicine, Gonoshasthaya Samaj Vittik Medical College, Gono Bishwabidyalay, Mirzanagar, Savar, Dhaka-1344

Md. Ehsanul Haque
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Mohammad Muradul Islam Chowdhury
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Mohammad Aynul Haque
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Alina Aktar
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Most. Jesmin Ara
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Md. Enamul Haque
Research and Development division, FnF Pharmaceutical Ltd., Rautail, Nagarbathan, Jhenaidah, Bangladesh

Mohammed Alimul Islam*
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Of all serological tests enzyme-linked immunosorbent assay (ELISA) is still considered the gold standard for the detection of antigens and antibodies of either macro or micro-organisms worldwide. The ELISA kits for serum antibody detection against many viruses and other micro-organisms of both man and animals are available in the market. Whereas, antigen detection ELISA kits for Newcastle disease virus (NDV) is not yet available in Bangladesh. The Present study was designed for the development of an economically feasible In-house Sandwich ELISA and to test its sensitivity and specificity for the detection of NDV antigens from clinically suspected field samples. 96- well flat bottom polystyrene plates coated with hyperimmune polyclonal serum against NDV raised in rabbits was used to capture NDV antigens. The anti-rabbit IgG and DAB with 30% H₂O₂ were used as conjugate and substrate respectively for standardization of the test method. The plate coated with serum diluted 10-3 was found suitable for capturing maximum antigen of NDV by the In-house Sandwich ELISA. The cut-off value of the present ELISA test was calculated as 0.855 and was able to capture the viral antigen present in the 10-4 fold dilution of allantoic fluid (AF) which is equivalent to 1HA unit, indicating the highest degree of sensitivity of the newly developed ELISA. In case of field samples, the newly developed ELISA kit was able to detect 100% viral antigens of NDV present in the feces, 95.50% of the brain tissue and oro-nasal swab and 94.12% of colon swab samples of either naturally and experimentally infected birds in this study. The ND virus specific polyclonal antibody used in the kit bind only with ND virus without any cross reactive antigens of other viruses of chicken like Avian influenza virus (AIV) and Infectious bursal disease viruses (IBDV). Therefore, findings of the present study clearly indicates that the newly developed In-house Sandwich ELISA kit can be used for rapid confirmatory diagnosis of Newcastle disease (ND) with minimum cost, using any kind of field samples from either sick or dead birds.

Keywords: In-house, Sandwich ELISA, Newcastle disease virus, Antigen detection, Polyclonal serum

Location:

Start Date:

End Date:

Program Area: Pest Management

Commodity: Poultry

Objectives:

The present study was undertaken to develop a reliable, rapid and cost effective serological tool for the detection of ND viral antigen directly from field samples and to evaluate its sensitivity and specificity to avoid confusion with other deadly diseases (ND/AI/IBD) of poultry in the commercial and breeder flock of either layer or broiler farms in Bangladesh.

Methodology:

Reference NDV Previously characterized velogenic strain of NDV was obtained from the repository of the Dept. of Microbiology and Hygiene, BAU, Mymensingh and used for large scale production of purified antigens for preparation of hyper immune serum in rabbits. Embryonated eggs Sero-negative eggs from 65 week old parent stock of broiler (Cob 500) chickens were used for the propagation the velogenic NDV. Carrier adjuvant Freund’s complete adjuvant (Sigma, USA) was used to bind the inactivated purified antigen of NDV for the production of hyper immune serum (polyclonal antibody) in rabbits. 96-well Microplate Flat bottomed polystyrene 96-well plate was used to coat each well of the plate with primary antibody (anti-NDV) raised in the rabbit. Rabbits American white male, young (4 months old) and healthy rabbits were used for the production of hyperimmune serum against NDV. Anti-Rabbit IgG Horse radish conjugated commercial anti-rabbit IgG was used as a source of conjugate (Sigma, USA). Substrates 3,3'-Diaminobenzidine (DAB) with 30% H₂O₂ was used as a source of substrate ELISA Reader ELISA reader (Thermo ELECTRON CORPORATION, ORIGINAL MULTISKAN EX, Japan) at 405 nm filter. Specificity test of the newly developed In-house sandwich ELISA kit The specificity test of the newly developed In-house sandwich ELISA kit was performed using two known viruses NDV and IBDV. Samples were diluted 10-1-10-4 to show the specificity of the newly developed In-house sandwich ELISA kit designed for specific binding of viral antigen of NDV only. For the specificity test, 100 µl of 10-1,10-2, 10-3 and 10-4 concentration of prepared inoculum of known ND & IBD suspected field samples were dispensed on A & B, C & D, E & F and G & H columns up to 10 rows of antibody coated microtiter plate respectively. The negative and positive control samples were dispensed on rows 11 and 12. After adding the viral antigen the plate was incubated for 1 hour at 37^oC. Excess antigen was removed and the plate was washed 5 times with 200 µl of washing buffer (PBST20). After that 100 µl of 10-3 diluted hyperimmune serum raised in rabbit was dispensed on the A & B, C & D, E & F and G & H rows of the microtiter plate. The plate was incubated at 37^oC for 1 hour. Hyperimmune serum was removed and the plate was washed 5 times with washing buffer (PBST20). A 100 µl conjugate solution was added to each well of the ELISA plate. The plate was incubated again for 1 hour at 37^oC. The conjugate solution was removed and the plate was washed 5 times with washing buffer (PBST20). Finally, 100 µl of substrate solution was added in each well of the ELISA plate. The plate was then incubated for 30 minutes at room temperature until the color reaction appeared. After establishment of positive reaction between the substrate and conjugate an orange colour developed and the reaction was stopped by adding 100 µl of stop solution with 1N H₂SO₄ to each well of the ELISA plate. The intensity of color development was measured by determination of OD (optical density) value using an ELISA reader (Thermo ELECTRON CORPORATION, ORIGINAL MULTISKAN EX, Japan) at 405 nm filter.

Source of Information: Microbes and Health, December 2012, 1(2): 65-71

Article Link (Online Journal): http://www.banglajol.info/index.php/MH/article/view/14093/10532

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Commercially available sandwich ELISA kits which are just for the detection of serum antibody are highly expensive. To import these types of kits for the detection of anti NDV antibody in the serum of vaccinated birds in Bangladesh will require 45,000 BDT for 450 samples only. The newly developed sandwich ELISA kit is economically feasible and requires less than 500 BDT for 450 samples. The In-house Sandwich ELISA kit is designed for the detection of viral antigens from field samples and is cheaper than other commercially available sandwich ELISA kits. It is hoped the farmers will used the kit for rapid diagnoses of a large number of ND suspected samples at the farm level. The Government of the People’s Republic of Bangladesh and a number of semi-government and non-government organizations can easily earn foreign currency by marketing and exporting this newly developed In-house sandwich ELISA kit designed for NDV after fulfilling the demand for this country.

Knowledge Management: Journal