2.3. Antileishmanial Antibody Quantification via ELISA

Antibody levels against L. infantum were assessed using an in-house Enzyme-Linked ImmunoSorbent Assay (ELISA), as outlined in previous studies [3,40]. Dog sera were diluted to a ratio of 1:800 in a phosphate buffer solution (PBS) containing Tween 20 and 1% dry milk. This mixture was then incubated in plates previously coated overnight with sonicated promastigotes of L. infantum (MHOM/MON-1/LEM-75) obtained from an infected dog at a concentration of 20 µg/mL, and the incubation took place at 37 °C for 1 h [41]. Following this, the plates underwent a series of washes using PBS-Tween and PBS. Next, Protein A conjugated to horseradish peroxidase (Peroxidase Conjugate Protein A; Merck KGaA, Darmstadt, Germany) was added to the plates at a dilution of 1:30,000 and incubated for 1 h at 37 °C. After another round of washing, the plates were treated with o-phenylenediamine and substrate buffer (SIGMAFAST OPD; Merck KGaA, Darmstadt, Germany). The reaction was terminated using 5 M H₂SO₄. Absorbance readings were taken at 492 nm using a spectrophotometer (MB-580 HEALES; Shenzhen Huisong Technology Development Co., Ltd., Shenzhen, China). The quantification of results was performed in terms of ELISA units (EUs), normalized against positive canine serum utilized as a calibrator and set at 100 EU.

The threshold was established at 35 EU [39]. Serum samples were categorized as negative when their value fell below 35 EU. If the result ranged from 35 EU up to but not exceeding 150 EU, it was considered low positive. Similarly, if the result fell between 150 EU and 300 EU, it was categorized as medium positive. Finally, if the value equaled or surpassed 300 EU, it was classified as high positive.

For a more in-depth examination of samples identified as medium or high positive, an ELISA involving a two-fold serial dilution was conducted. This process was initiated at a dilution of 1:800 and continued with an additional 7 to 11 dilutions. Quantification was predicated on arbitrary units (EU) in relation to a calibrator set at 100 EU, which corresponded to an optical density (OD) value of 1 at the 1:800 dilution. The mean values of dilutions displaying an OD approximate to one were chosen for calculating the EU. This computation was executed using the following formula: (sample OD/calibrator OD) × 100 × dilution factor [40].