Goat anti rabbit igg

The native recombinant nucleocapsid proteins (NPs) of SARS-CoV, SARS-CoV-2 or variants, MERS-CoV, HKU1, OC43, NL63, and 229E (Sino Biological) (2 μg/mL) or denatured by treatment with the denaturing buffer containing 0.5% SDS and 40 mM DTT [18 (link)] (New England Biolabs) at 95 °C for 10 min were coated into 96-well half-area plates overnight at 4 °C. The plates were then blocked with blocking buffer (PBS containing 4% skim milk) at 37 °C for 1 h. Five-fold or three-fold serial-diluted NP-specific mAbs or Rabbit pAb against NP of SARS-CoV-2 (Sino Biological) were added to the plates in duplicate and incubated for 1 h at 37 °C. And then HRP-conjugated Goat anti-Human IgG (ZSGB-BIO) or Goat anti-Rabbit IgG (TransGen Biotech) secondary antibody was added to the plates and incubated at 37 °C for 1 h. For competitive ELISA, after blocking, serially diluted P301-F7, P301-H5, and human IgG1 were mixed with HRP-conjugated P301-F7, and then added to the plates and incubated at 37 °C for 1 h. The enzymatic reaction was developed with TMB substrate (BD Biosciences) and stopped by 2 M H₂SO₄. The optical density was measured at 450 nm (OD. 450 nm) with a Varioskan™ LUX Multimode Microplate Reader (Thermo Scientific).

The cell pellets obtained from 2 mL culture of expressed 16/8/10mer peptide were boiled at 95°C in 200 μL of 1×SDS-PAGE loading buffer for 10 min, and the proteins were resolved by 15% SDS-PAGE under reducing conditions. Gels were either stained with Coomassie brilliant blue G-250 for analyzing the bands corresponding to the target 16/8/10mer peptide, or used for Western blot by electro transferring the proteins onto a 0.2 μm nitrocellulose (NC) membrane [30 (link)]. Regarding the specific antigen-antibody reaction, the NC membrane was blocked with 5% (w/v) skimmed milk powder in Tris-buffered saline-Tween 20 (TBS-T), treated with rabbit pAb α-SGn (1:1000 dilution) or sheep serum (1: 100 dilution) as the primary antibody, and then reacted with goat anti-rabbit IgG or mouse anti-goat IgG conjugated to HRP (1:5000 dilution, Trans-Gen Biotech) as the secondary antibody. Finally, the blot was performed using the ECL plus Western blot detection reagent according to the manufacturer’s instructions, and it was then imaged by GE-Image Quant LAS 4000 (GE Healthcare, Buckinghamshire, UK).

We further verified the expression levels of SLC35A3 and CD4 proteins in tumor tissues and adjacent normal tissues of colorectal cancer patients through IHC. After deparaffinization, rehydration, and antigen retrieval by heating from the slides in sodium citrate buffer (pH 6.0), the antigens were retrieved. To inhibit endogenous peroxidase activity, they were blocked in 3% hydrogen peroxide for half an hour and then washed three times with PBS. Incubation with rabbit anti-SLC35A3 (SinoBiological, China) and rabbit anti-CD4 (Bioss, China) was performed overnight at 4 °C, followed by incubation with goat anti-rabbit IgG (TransGen Biotech, China) for 2 h at room temperature. After staining with 3,3′-diaminobenzidine (DAB), the sections were counterstained with hematoxylin, dehydrated, fixed, and sealed with a cover slip. The immunohistochemical results were analyzed in Image-Pro Plus 6.0, using the same brownish-yellow as the uniform standard for judging whether the image was positive. The cumulative optical density (IOD) and pixel area (area) of the tissue in each positive standard photo were analyzed, and the mean density was calculated as Mean density = IOD/area. The higher the Mean density value, the higher the expression level of the positive protein.