Hrp conjugated anti mouse ig

ELISA assays were performed using MaxiSorp 96-well plates (Nunc) coated with dilutions of purified mucin TR reporters starting from 100 ng/mL or fractions derived from C4 HPLC incubated o/n at 4 °C in 50 mL carbonate-bicarbonate buffer (pH 9.6). Plates were blocked with PLI-P buffer (PO₄, Na/K, 1% Triton-X 100, 1% BSA, pH 7.4) and incubated with mAbs 3C9, 5F4 and TKH2 (undiluted culture supernatants), biotinylated-lectins VVA (0.5 μg/mL), PNA (0.5 μg/mL), MAL II (2.0 μg/mL) (Vector Laboratories) or Pan Lectenz (2.0 μg/mL) (Lectenz Bio) for 1 h at RT, followed by extensive washing with PBS containing 0.05% Tween-20, and incubation with 50 mL of 1 μg/mL HRP conjugated anti-mouse Ig (Dako) or 1 μg/mL streptavidin-conjugated HRP (Dako) for 1 h. Plates were developed with TMB substrate (Dako) and reactions were stopped by the addition of 0.5 M H₂SO₄ followed by measurement of absorbance at 450 nm.

MaxiSorp 96-well plates (Nunc) were coated overnight at 4 °C in carbonate–bicarbonate buffer (pH 9.6) and blocked in PLI-P (PO₄³⁻, Na⁺/K⁺, 1% Triton, and 1% BSA) buffer at pH 7.4 for 1 h at RT. Neuraminidase treatment was performed with 50 mU neuraminidase in 50 mM sodium acetate buffer (pH 5.5) for 1 h at 37 °C. Plates were incubated with mAbs (undiluted culture supernatants), biotinylated PNA lectin (200 ng/ml) (Vector Laboratories), or biotinylated GAL-4 (0.5 μM) for 1 h at RT and followed by washing and incubation with HRP-conjugated antimouse Ig (Dako) or streptavidin–HRP (Dako) for 1 h at RT. Development was started by addition with 3,3′,5,5′-Tetramethylbenzidine substrate (Dako) and stopped with 0.5 M H₂SO₄, and absorbance read at 450 nm after 5 min.