Detection of False-Positive Sera in Contagious Agalactia with a Multiantigen ELISA and their Elimination with a Protein G Conjugate

Abstract

In serology, lack of specificity can generally be attributed to cross-reactions between different pathogens with antigens bearing similar epitopes. During seroepidemiologic surveys of contagious agalactia of sheep caused by Mycoplasma agalactiae infection, numerous sera were analyzed by enzyme-linked immunosorbent assay (ELISA). A few sera reacted with various antigens coated on plates, including the well with no antigen. This reactivity was not due to cross-reactions as initially suspected, and these multipositive sera were designated false-positive sera. Elimination of this false positivity was not possible by using covalent ELISA plates or different rabbit anti-sheep IgG conjugates. Only conjugates using monoclonal antibodies or protein G were efficient in elimination of false positivities without reducing the true specific positive titers. No false-positive sera have been observed since the implementation of protein G conjugates in the serologic diagnosis of contagious agalactia by ELISA for the past 2 years.

Inoculation of lactating ewes by the intramammary route with Mycoplasma agalactiae: comparative pathogenicity of six field strains

“…The antigen used in this ELISA was prepared from the reference strain P89. The results were expressed in units according to the curve of a titrated standard serum included in each microtitration plate [12].…”

“…In some French regions where contagious agalactia of ewes due to M. agalactiae is enzootic, the prevention of the disease is based on a strict control of infected flocks. The protocol includes culling of sheep with clinical signs, a bacteriological survey of bulk milk and a restriction of movements of flocks based on serology, using a quantitative ELISA leading to the classification of flocks with a serological index [9,12]. Most flocks in the infected areas of France are at present free of contagious agalactia using this sanitary control but a few remain infected at low levels [11,14].…”

Comparison of Three Enzyme-Linked Immunosorbent Assays for Serologic Diagnosis of Contagious Agalactia in Sheep

“…When contagious agalactia was quite prevalent, the AFSSA ELISA kit gave excellent results, leading to a significant decrease in disease prevalence in recent years, 6 whereas during these campaigns, several epidemiological observations have demonstrated this kit's lack of sensitivity. This limitation was demonstrated when the test failed to detect antibodies in young sheep experimentally infected with a fully virulent strain of M. agalactiae.…”

“…For the AFSSA kit, the cutoff value had been previously defined (Table 2). 4,6 A flock was considered positive when at least 1 out of 20 sera was positive or, in the absence of positive serum, when 2 sera were doubtful.…”

“…For this purpose, 20 randomly taken sera per flock are tested with an ELISA technique developed by the Agence Française de Sécurité Sanitaire des Aliments (AFSSA) Sophia Antipolis laboratory. 4,6 After transformation of the optical densities (ODs) into units using dilutions of a reference positive serum, individual titers are used to determine a flock index. This index, together with bulk-milk bacteriology in and around outbreak areas, is used to classify flocks into 4 categories: ''free of agalactia,'' ''presumed free,'' ''infected,'' and ''heavily infected.''…”