Evaluation of Indirect ELISA and IgG Avidity Assays Using 1 Protein A-Peroxidase Conjugate for Serological Distinction

1 This study aimed to evaluate the use of Protein A-peroxidase (HRPO) in indirect ELISA 2 (iELISA) and IgG avidity assays for serological distinction between Brucella abortus S193 vaccinated and infected cows. Four groups were analyzed: (GI) 41 non-vaccinated seropositive 4 cows; (GII) 79 S19-vaccinated heifers analyzed at 3 months post-vaccination; (GIII) 105 S195 vaccinated cows analyzed after 24 months of age; (GIV): 278 non-vaccinated seronegative cows. 6 IgG levels and avidity to B.abortus smooth lipopolysaccharide (S-LPS) were determined using 7 anti-bovine IgG/HRPO or Protein A/HRPO conjugates. Similar levels of IgG anti-S-LPS were 8 found in GI using both conjugates. Lower IgG levels were detected in GII, GIII and GIV using 9 Protein A/HRPO. Both conjugates showed high performance to discriminate GI from GIII, with 10 high sensitivity (Se, 97.6%) and specificity (Sp, 97.1%). Protein A/HRPO was better to 11 distinguish GI from GIV (Se, 97.6%; Sp, 94.6%) and GI from GII (Se, 80.5%; Sp, 94.9%). 12 Protein A/HRPO excluded higher number of positive samples in GII and GIV. IgG avidity 13 showed that Protein A/HRPO, but not anti-IgG/HRPO, was able to distinguish non-vaccinated 14 from vaccinated cattle, showing higher avidity index (AI) in GI than GII, with 78% of sera 15 showing AI<50% in GII. Therefore, iELISA using B.abortus S-LPS antigen and Protein A/HRPO 16 conjugate for preferential detection of IgG2 subclass showed to be suitable for serological 17 distinction between S19-vaccinated and infected cows. Also, antibodies generated after 18 vaccination showed lower avidity, suggesting a role for IgG2 subclass as antibody of higher 19 affinity maturation after infection, constituting an additionaltool to differentiate vaccinated from 20 infected cattle. 21 22