Development of antigen-capture ELISA using monoclonal antibodies for the detection of brucellae in milk

Abstract

In this study, a Brucella antigen-capture ELISA (Ag-cELISA) prototype was developed. To study the validity of the developed Ag-cELISA, milk samples collected from Brucella-positive goats (n=120) and cattle (n= 64), as well as from unknown Brucella-status cattle (n=105) and sheep (n=65) herds were tested by Ag-cELISA, I-ELISA, and culture method. All Brucella-positive samples were confirmed using PCR. It was found that the developed Ag-cELISA could detect 50-100 bacteria per well (equivalent to 103 to 2×103 cells per mL) as the lowest limit of detection (LOD) and was therefore considered moderately sensitive to detect brucellae in milk. In an infected goat herd, out of 120 milk samples, 41, 32, and 17 were positive by Ag-cELISA, I-ELISA, and culture, respectively. Ag-cELISA detected 15 positive cases out of 17 culture-positive milk samples. Two culture-positive milk samples were not detected in Ag-cELISA. The relative sensitivity and specificity between Ag-cELISA and I-ELISA were 78% and 100%, respectively. In an infected cow herd, out of 64 milk samples, 32, 23, and 11 were found positive by Ag-cELISA, I-ELISA, and culture, respectively. Ten out of 11 culturally positive milk samples were found positive by Ag-cELISA. The relative sensitivity and specificity between the Ag-cELISA and I-ELISA were 71.9% and 100%, respectively. From randomly collected 105 cow and 110 sheep milk samples from herds of unknown Brucella-infection status, three (2.85%) and five (4.5%) samples were found positive using Ag-cELISA, respectively. These results showed that Ag-cELISA could be used to detect brucellae in milk more practically and safely than bacterial culture. On the other hand, this information re-affirms that milk can be an important source of brucellosis and creates a public health risk in humans; therefore, increased public awareness is of utmost importance.