Monoclonal Antibodies Directed Against the Outer Membrane Protein of Bordetella avium

Abstract

Bordetella avium is the etiologic agent of coryza and rhinotracheitis in poultry. This respiratory disease is responsible for substantial economic losses in the poultry industry. Monoclonal antibodies (MAbs) were produced against the outer membrane proteins (OMPs) of B. avium isolated from diseased chickens. BALB/c mice were immunized with the extracted B. avium OMPs. Then the splenocytes from immunized mice and SP2/0 myeloma cells were fused using PEG 4000. Three stable hybridoma clones (designated as 3G₁₀, 4A₃, and 4E₈) were produced via indirect ELISA and three rounds of subcloning. The MAbs were classified as IgG1, and can recognize the 58 kDa OMP band by Western blot assays. No MAb cross-reactivity with chicken Proteus mirabilis, Escherichia coli, and Salmonella was observed. A double antibody sandwich ELISA (DAS-ELISA) was developed using the rabbit polyclonal antibodies as the capture antibody and MAb 4A₃ as the detection antibody. Under the DAS-ELISA, the minimum detectable concentration of B. avium was 1 × 10(4) CFU/mL, and no cross-reactivity occurred with chicken Proteus mirabilis, Escherichia coli, and Salmonella. Results showed that the DAS-ELISA has good sensitivity and specificity. Clinical application showed the DAS-ELISA was more sensitive than the plate agglutination test. This study may be used to develop a quick and specific diagnostic kit, analyze epitopes, and establish systems for typing B. avium.

The Preparation and Identification of a Monoclonal Antibody against Domoic Acid and Establishment of Detection by Indirect Competitive ELISA

“…by hybridoma cell line 1C3. The developed ascites were harvested and centrifuged at 10,000× g for 10 min, and caprylic/ammonium sulfate method [ 15 , 45 , 46 ] was used for mAb purification with minor modifications. After purification, SDS-PAGE was used for the analysis of purified mAb [ 39 ], and the experiment was repeated three times for obtaining distinct bands of light and heavy chain of purified mAb.…”

Development of ELISA and Lateral Flow Immunoassays for Ochratoxins (OTA and OTB) Detection Based on Monoclonal Antibody

“…About 1 week later, the developed ascites fluid was carefully harvested and centrifuged at 10,000 r/min for 30 min. The ascites were purified with two-step caprylic/ammonium sulfate precipitation methods (Liu et al, 2013). The purity of the mAb was assayed by SDS-PAGE (Chang and Gottlieb, 1988;Di Girolamo et al, 2010), and stained using Coomassie Brilliant Blue staining solution.…”