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 withinand 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 (H202) 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 withinand 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.

Seroprevalence of Actinobacillus pleuropneumoniae serovars 2, 3 and 9 in slaughter pigs from Belgian fattening farms

“…An indirect ELISA, based on that described by Trottier and others (1992), was used to detect antibodies against the heat‐stable antigens of A pleuropneumoniae serovars 2, 3 and 9. The heat‐stable antigens were prepared by boiling a suspension of 10 10 colony‐farming units/ml of the A pleuropneumoniae strains for one hour.…”

Comparaison de Deux Antigénes de Nature polysaccharidique pour le Diagnostic Sérologique par ELISA de la Pleuropneumonie Porcine (Sérotype 5)

“…Plusieurs ipreuves ont it6 diveloppies pour la detection sirologique des troupeaux infectis: le test de fixation du complement (CFT) (1 8 26,28). Cette derniere mithode est avantageuse car elle est rapide, sensible et d'exicution facile.…”

A Novel Enzyme-Linked Immunosorbent Assay Using the RecombinantActinobacillus pleuropneumoniaeApxII Antigen for Diagnosis of Pleuropneumonia in Pig Herds

“…To control the disease, a variety of serological tests is being employed, with the complement fixation test (CFT) still being used as a reference test (9). In addition, several enzyme-linked immunosorbent assays (ELISAs) have been developed (6,(13)(14)(15)18). All these tests, however, are serotype specific.…”