title: Optimization and standardization of an enzyme-linked immunosorbent assay protocol for serodiagnosis of Actinobacillus pleuropneumoniae serotype 5.
abstract: An indirect enzyme-linked immunosorbent assay protocol has been optimized with special emphasis given to assay standardization and quality control. Technical aspects such as choice of a microplate, antigen immobilization, buffer composition, optimal screening dilution of sera, and kinetics of the enzymatic reaction were studied and evaluated in order to design a standard protocol offering maximal analytical sensitivity and specificity, as well as to obtain minimal within- and between-plate variability. Among the 27 plates tested, the Nunc 475-094 and 269-620 immunoplates were found to be the best in terms of high positive-to-negative ratio and low variability. No significant differences in antigen immobilization were found by using buffers of various compositions or pHs; however, the presence of magnesium ions (Mg2+; 0.02 M) resulted in a twofold increase in nonspecific background. An optimal screening dilution of sera was established at 1:200. A 1-h incubation period for test serum was found to be optimal. Maximum enzymatic activity for peroxidase was obtained by adjusting both substrate (H2O2) and hydrogen donor [2,2' -azinobis(3-ethylbenz-thiazoline sulfonic acid)] concentrations to 4 and 1 mM, respectively. To control between-plate variability, a timing protocol was adopted. Within-plate variability was also controlled by using a sample placement configuration pattern. Sliding scales were determined by repeated testing of a cross section of samples to set acceptance limits for both within- and between-plate variability. These limits were used in a quality control program to monitor assay performance. The results obtained suggest that this standardized protocol might be useful in the serodiagnosis of Actinobacillus pleuropneumoniae serotype 5.
authors.affiliation: Groupe de Recherches sur les Maladies Infectieuses du Porc, Faculté de Médecine Vétérinaire, Université de Montréal, Saint Hyacinthe, Quebec, Canada.
authors.forename: Y L
authors.initials: YL
authors.lastname: Trottier
authors.forename: P F
authors.initials: PF
authors.lastname: Wright
authors.forename: S
authors.initials: S
authors.lastname: Larivière
mesh_terms: D000189:Actinobacillus Infections / Q000097:blood / Q000175:diagnosis* / Q000201:enzymology; D016977:Actinobacillus pleuropneumoniae / Q000276:immunology / Q000302:isolation & purification*; D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D007700:Kinetics; D011002:Pleuropneumonia, Contagious / Q000097:blood / Q000175:diagnosis* / Q000201:enzymology; D011786:Quality Control; D012015:Reference Standards; D012703:Serotyping; D013552:Swine; D013553:Swine Diseases / Q000097:blood / Q000175:diagnosis* / Q000201:enzymology
chemical_list: D000942:Antigens, Bacterial