Table 2.

IgG ELISA protocol for the detection of Rubella virus antibodies in human serum samples used in this study (3D well and 96-well, ELISA).

Step 1: Coating 100 µL of Rubella virus HA antigen (1:2) in sodium carbonate buffer Incubation at 4 °C overnight

Washing: three rounds of washing with 300 µL of PBS-T per round

Step 2: Primary Antibody 100 µL of test samples, positive and negative reference antiserum diluted (1:100, 1:400, 1:1600 and 1:6400) in PBST-M; Incubation at room temperature (RT), 1 h.

Washing: three rounds of washing with 300 µL of PBS-T per round

Step 3: Secondary Antibody 100 µL of goat anti-human IgG – horseradish peroxidase (HRP) conjugate diluted in PBST-M (1:11,000); Incubation at RT, 1 h

Washing: three rounds of washing with 300 µL of PBS-T per round

Step 4: Substrate 100 µL of 2,2′-azino-bis-(3-ethylbenzthiazoline sulfonic acid) (ABTS) solution Incubation at RT, 30 min Absorbance Measurement Optical density at 405 nm (OD405) was measured against a reference of 490 nm