Hrp conjugated goat anti mouse igg

Antibodies of SaClpP (1:5,000, Cat#C11185), SaFtsZ (1:5,000, Cat#C11186), and SaGAPDH (1:5,000, Cat#C1399) were generated by Shanghai Immune Biotech Ltd using the purified protein as the antigen and validated by ELISA experiments. Antibodies of HsClpP (1:2,000, Clo#EPR7133, Cat#ab124822, Lot#GR3210822-8, Abcam), β-actin (1:5,000, Clo#2D4H5, Cat#66009-1-Ig, Lot#10004156, Proteintech), HRP-conjugated goat anti-rabbit IgG (1:10,000, Cat#CW0103, Cwbio), and HRP-conjugated goat anti-mouse IgG (1:10,000, Cat#CW0102, Cwbio) were commercially purchased.

The 3D7 strain of P. falciparum was preserved at the Laboratory of Pathogen Infection and Immunity (Jiangnan University, Wuxi, China). The parasites were grown at 37 °C in a mixed environment of 90% N₂, 5% O₂, and 5% CO₂, and were maintained in RPMI Medium 1640 (Gibco, New York, USA) containing O⁺ human erythrocytes (4% hematocrit), HEPES (Meilunbio, Dalian, China), NaHCO₃ (Meilunbio, Dalian, China), AlbuMax Ⅱ (Sigma, San Francisco, CA, USA), hypoxanthine (Sigma, San Francisco, CA, USA), and gentamicin (Solarbio, Beijing, China).
The schizonts were purified by 60% percoll (Solarbio, Beijing, China) gradient centrifugation, and lysed in 0.1% saponin (diluted in PBS) for 5 min on ice with intermittent mixing. The lysed material was centrifuged at 15,000× g for 5 min and washed thrice with PBS. Then, the parasite lysate was collected and boiled in a SDS-PAGE sample loading buffer (Meilunbio, Dalian, China) for 7 min [13 (link)]. The total protein was separated on SDS-PAGE gel, and native PfSRA in the P. falciparum crude protein was captured by the anti-rPfSRA mice sera at 1:1000 dilution overnight and detected by HRP-conjugated goat anti-mouse IgG (CWBio, Beijing, China).

For each SARS-CoV-2 variant, 7 mL of pseudotyped virus were added to 2 mL of 25% sucrose buffer and centrifuged at 10,0000 g for 3 h. Each pellet of purified pseudotyped virus was then re-suspended in 100 μL PBS. Samples were mixed with loading buffer and heated at 100°C for 5 min; a 30-μL aliquot of each sample was then used for SDS-PAGE and western blotting analysis. The primary antibodies were a homemade mouse anti-S2 antibody against SARS-CoV-2 spike protein and a custom anti-VSV M (KeraFast, EB0011) protein antibody; the secondary antibody was a 1:10000 dilution of HRP-conjugated goat anti-mouse IgG (CWbiotech). Immobilon western chemiluminescent HRP substrate (Millipore) was used to develop the immunoreactive bands. Band intensities were calculated using Alphaview software.

The purified recombinant RABV was further identified to confirm the expression of the F or G proteins of NiV by SDS-PAGE and Western blot as described previously [20 (link)]. The recombinant RABV was added to SDS-PAGE loading buffer (CWBIO, Taizhou, China), and viruses denatured in boiling water for 5 min were loaded onto a 10% polyacrylamide gel. For total protein analysis, the polyacrylamide gel was stained with Coomassie Brilliant Blue for 20 min, and the gel was then immersed in Coomassie Brilliant Blue destaining solution to destain until the gel background turned white. For Western blotting, the target protein was transferred to a 0.45 µm pore-size nitrocellulose (NC) membrane by the semi-dry transmembrane method. After blocking in 5% skimmed milk at RT for 2 h, the membrane was incubated overnight with the mAb of NiV glycoproteins (Mybiosource, Southern California, San Diego, CA, USA) at 1:1000 dilution and then incubated at RT for 1 h with the horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (CWBIO, Taizhou, China) at 1:20,000 dilution. Enhanced chemiluminescence (ECL) color developer was dropped onto the NC membrane for exposure and color development.

To determine the titer and subtype of specific antibodies against NiV in serum, we developed an indirect enzyme-linked immunosorbent assay (iELISA) [25 (link)]. In brief, 0.5 μg/mL of F or G proteins of NiV were coated onto ELISA plates overnight at 4 °C. After three washes with PBST, the plate was blocked with 3% bovine serum albumin (BSA) for 2 h. After three washes with PBST, 50 μL/well of the diluted inactivated serum samples were added and incubated for 1 h at 37 °C. After washing, a 1:10,000 dilution of HRP-conjugated goat anti-mouse IgG (CWBIO, Taizhou China), a 1:5000 dilution of HRP-conjugated goat anti-mouse IgG1 (Southern Biotech, Birmingham, AL, USA), or a 1:5000 dilution of HRP-conjugated goat anti-mouse IgG2a (Southern Biotech, Birmingham, AL, USA) was added and incubated at 37 °C for 1 h. After TMB color development, the OD_450nm value of each well was read. A test sample/negative sample with an OD_450nm greater than 2 was considered positive.

The purity of recombinant LppZ protein was determined by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Tanon, Shanghai, China) followed by Coomassie brilliant blue staining and further confirmed by Western blotting using anti-6 × His tag antibody (Abcam, Hong Kong, China) or human plasma (including TB patient, LTBI individual, HC and disease control). LppZ protein was electro-transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA). Human plasma was diluted at 1:200 ratio in the immunostaining assay. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Cwbio, Beijing, China) and HRP-conjugated goat anti-human IgA (Cwbio) were used as secondary antibodies. The ECL solution (Millipore) was used to visualize LppZ. The membranes were scanned on Tanon 5200S Chemiluminescence Imaging System (Tanon).

Twenty 6-week-old female BALB/c mice (Cavens, Changzhou, China) were randomly divided into four groups (five mice per group). Mice in experimental groups were immunized with rPfSRA-F1a, rPfSRA-F2a, and rPfSRA-F3a, respectively. Then, 50 µg of rPfSRA in PBS was emulsified with complete Freund’s adjuvant (CFA; Sigma, San Francisco, CA, USA) at a volume ratio of 1:1 with a total volume of 200 µL in the prime boost. The mixture was intraperitoneally injected into the mice. Additionally, incomplete Freund’s adjuvant (IFA; Sigma, San Francisco, CA, USA) was administered on day 21 and 42 postimmunization to boost the immunization. Control mice were immunized with CFA/IFA in emulsification with PBS. Blood was collected from each mouse on day 0, 7, 14, 28, 35, and 49 postimmunization by bleeding the tail vein, and sera were isolated for antibody detection by Western blot. The sera were used as primary antibodies at a dilution of 1:1500 in PBS to identify rPfSRA, and then HRP-conjugated goat anti-mouse IgG (CWBio, Beijing, China) was used as secondary antibody.