Mouse elisa kit

The effects of the extracts on levels of TNF-α, IL1 β and IL10 were determined and quantified using the mouse ELISA kits with catalogue numbers E-EL-M0049, E-EL-M0037 and E-EL-M0046, (Elabscience, Biocom Africa, Johannesburg South Africa)), respectively, following the same protocol as above using the instructor manual on the cell culture medium.

Blood samples were collected and left to stand for 30 min, then were centrifuge for 15 min at 3000r at 4°C. BNP, NT-ProBNP, Ang-1, Ang-2 and Tie-2 in the supernatant was measured using the Mouse ELISA Kit (Elabscience, Wuhan, China). The absorbance was recorded at 450 nm with a plate reader. Based on the standard curve OB values of the samples, the corresponding BNP, NT-ProBNP, Ang-1, Ang-2 and Tie-2 were calculated.

24 h after operation, 6 mice in each group were randomly collected and blood samples were collected under sevoflurane anesthesia. These blood samples were left at room temperature for 30 min, centrifuged at 3000 RPM for 10 min, and then the supernatant was collected. According to the manufacturer’s instructions, the serum cTnI concentration was detected using mouse ELISA kit (Elabscience Biotechnology Co., LTD., Wuhan, China).

The antibody levels were detected by the indirect enzyme-linked immunosorbent assay (ELISA) as previously described with slight modifications [25 (link)]. Briefly, 96-well plates (Costar, USA) were coated with purified recombinant protein [200 ng/100 μl diluted in 0.02 M carbonate-bicarbonate buffer (PH 9.6)] and incubated overnight at 4°C. Wells were subsequently washed thrice with PBST, then incubated with 5% (w/v) bovine serum albumin (BSA) in PBST for 1.5 h at 37 h, followed by washing(3x). Plates were incubated with 100 μl sera diluted in 1:100 for 1.5 h at 37 h. PBST was used to wash the plates (4x); biotinylated goat anti-mouse HRP-IgG (1:5000, Bryotime, China) was then added to the wells (100 μl) and plates were incubated for 1 h at 37 h. After washing 5 times with PBST, TMB was added to the plate wells and kept for 10 min in a dark room. The reaction was stopped by adding 50 μl of 2 M H2SO4 to each well. Absorbance (OD) of the plate wells was read at a wavelength of 450 nm using a spectrophotometer (Bio-Rad).
The levels of IFN-γ (Interferon γ), IL-2 (Interleukin 2) and IL-4 (Interleukin 4) in the immune serum were determined using mouse ELISA kit (Elabscience, China), according to the instructions of manufacturer. The absorbance of the plate wells was read at a wavelength of 450 nm using a spectrophotometer (Bio-Rad).

The blood samples from the mice of each group were collected by retro-orbital sinus puncture. The serum levels of IFN-γ (Beyotime Biotechnology, cat. No. PI508) and TNF-α (Beyotime Biotechnology, cat. No. PT512) were measured with commercial ELISA kits according to the manufacturer's instructions. 2×10⁶ macrophages (RAW 264.7 cells) and microglia (BV2 cells) were seeded in a 96-well plate, and then different concentrations of NAcp@CD47 or controls were co-incubated with the cells for 24 h. IFN-β was measured by mouse ELISA kit (Elabscience, cat. No. E-EL-M0033c).

Total serum IgG (total IgG) and fecal IgA were measured by indirect ELISA as previously described (Gao et al., 2009 (link)). ELISA plates were coated with 100 μl of 10 μg/ml purified espA-Tir-M protein (our laboratory stock). Serum and fecal extracts were prepared as described above and diluted 1:50 and 1:10, respectively. In addition, 5 μl serum (collected on day 34) was assayed for murine interferon (IFN)-γ and interleukin (IL)-4 and IL-10 by quantitative ELISA using a mouse ELISA kit (Elabscience, Hubei, China) according to the manufacturer’s instructions. ELISA results were obtained by measuring the OD₄₅₀ using an EL9800 ELISA microplate reader (BioTek, Winooski, VT, USA).

Blood was withdrawn by cardiac puncture of anesthetized mice. Concentrations of immunoglobulin A (IgA) and immunoglobulin G (IgG) in serum of mice were measured using the mouse ELISA kit (Elabscience Biotechnology), according to the manufacturer’s instructions.