Streptavidin hrp

We coated 96-well plates (Nunc) with 5 μg/mL goat anti-human C1q antibody (Accurate Chemical & Scientific Corporation) for 16 h at 4°C and blocked with PBST/3% BSA for 2 h at room temperature; a 100 μL/well of 3 μg/mL C1q (Quidel Corporation) was added to each well and incubated for 2 h at room temperature. Varying concentrations of TNFα antagonists and 133.3 nM biotin-rituximab were added to the plates alone or in the presence of a 0.8-fold molar excess sTNFα. Plates were incubated at room temperature for 15 h. The streptavidin-HRP (Beyotime) was added to the plates for 1 h at room temperature. Plates were washed with PBS containing 0.05% Tween-20 after each incubation step. Peroxidase activity was detected with TMB reagent (Thermo Scientific), and the plates were incubated at room temperature for 0.5 h to allow color development. The reaction was terminated with 1 M H₂SO₄, and the absorbance was measured at 450 nm (the background measured at 650 nm was subtracted for each well) using a SpectraMax M2^e microplate reader. Cells incubated with biotin-IgG1 in the absence of drugs served as controls. For data analysis, percentage competition was calculated as follows: percentage competition (%) = (Value _control − Value _drug) / Value _control ×100.

For TUNEL assay in tissues, the assay was performed in 3-mm-thick sections of paraffin-embedded tissue with the TUNEL assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Briefly, the paraffin-embedded tissue sections were dewaxed in dimethylbenzene and ethanol successively. Then the sections were treated with Protein Kinase K (Beyotime, Shanghai, China) for 30 min. After endogenous peroxidase was deactivated by using 3% H₂O₂, the sections were incubated with the biotin-dUTP buffer for 60 min. The sections were observed after treated with Streptavidin-HRP solution for 30 min, and the DAB buffer (Beyotime, Shanghai, China) for 5 min. For TUNEL assay in cells, the cells were washed with PBS and then fixed in 4% paraformaldehyde for 30 min following the 5 min-treatment with 0.3% Trition X-100. Then the cells were incubated with the TUNEL detection buffer for 60 min. DAPI was used to stain the nucleus. The cells were observed the fluorescence intensity under the fluorescence microscope (Leica DM1000).

To dectect prM-AIDs titer in the immunized sera, serially diluted control and immunized sera were coated in 96-well plates at 4°C overnight. And to identify prM-AIDs in purify antibodies, antibodies purified from naive mice as negative control, and purify anti-idiotypic antibodies from immunized mice were coated in 96-well plates at 4°C overnight. The plates were blocked by 2% BSA in PBST for 1 h at room temperature. The prM mAb was biotinylated by using NH2-reactive biotin (Elabscience, Cat. No#EBLK0002). After washing with TBST, biotinylated prM mAb was added and incubated for 1 h at 37°C. Plates were washed four times with TBST. Streptavidin-HRP (1:10,000 diluted, Beyotime, Cat. No#A0303) were added and incubated for 30 h at 37°C. Wash the plates as above. TMB was added as the substrate, and the reaction was stopped by H₂SO₄ stop solution. The OD at 450 nm was measured with an iMarkTM Microplate Reader. Samples were considered positive if P/N ration>2.1.