Sba clonotyping system hrp kit

Mice were immunized with 100 ug NP19-OVA in alum via intraperitoneal injection. Serum collected at different days after immunization was subjected to ELISA. Briefly, serum was incubated in 96-well plates coated with NP28-BSA or NP8-BSA to capture low and high affinity NP-specific antibodies, respectively, followed by quantification of IgG1 abundance using the SBA Clonotyping System-HRP kit (SouthernBiotech; 5300-05) following the manufacturer’s instruction. For ELISPOT assay, bone marrow cells were collected on day 85 after immunization. 3 million BM cells were seeded into a 96-well MultiScreen IP Filter Plate (MilliporeSigma; MSIPS4510) coated with NP8-BSA and incubated overnight at 37 °C. NP-specific LLPC spots were visualized using AP-conjugated goat anti-mouse IgG1 (SouthernBiotech; 1071-04) with enzyme substrates.

Anti-ADAM8 Abs were made by the hybridoma method [17 (link)] using recombinant human ADAM8 [rHuADAM8] (AD8-H5223, ACRO Biosystems, Newark, DE, USA) injection into Balb/c and SJL mice and fusion of B cells to Sp2/0-Ag14 (RRID: CVCL_2199) myeloma cells. Hybridoma supernatants were screened for clones producing Abs with highly specific ADAM8-binding and dual MP and DI inhibitory activity, as described below. Following subcloning by serial dilution, 18 clones making such mAbs (termed ADPs) were isolated. Abs were purified from hybridoma supernatants using protein A columns. Sterile filtered, purified Abs in phosphate-buffered saline (PBS) had low endotoxin (<2 EU/mg) and >95% purity (SDS-PAGE). Isotype and light chain type were determined using an SBA Clonotyping System-HRP kit (5300-05, Southern Biotech, Birmingham, AL, USA).

Levels of IgG, IgG1 and IgG2a antibody in serum were determined by ELISA using SBA Clonotyping System-HRP Kit according to the manufacture’s instruction (Southern Biotech Co., LTD, Birmingham, USA). Microtiter plates were coated with capture antibody (10 μg/ml; provided by the commercial Kit) in 100 μl of phosphate buffered saline (PBS; pH7.4) at 4°C overnight. Plates were washed twice with PBS plus 0.05% Tween20 (PBS-T) and blocked with PBS containing 1% BSA (PBS-1% BSA) for 1 h at room temperature. After being washed with PBS-T, the wells were incubated with 100 μl of horseradish-peroxidase (HRP) conjugated anti-mouse IgG (diluted in 1:250 in PBS-1% BSA), anti-mouse IgG1 or IgG2a (1:500) at 37°C for 60 min. After incubation with 100 μl substrate solution (pH4.0) (1.05% citrate substrate buffer; 1.5% ABTS; 0.03% H₂O₂) for 20 min, the absorbance was measured at 405 nm using an ELISA reader (Bio-TekEL × 800, USA). All samples were run in triplicate.

A dose of 100 μg of prokaryotically expressed pB602L mixed with an equal volume of complete Freund’s adjuvant or incomplete Freund’s adjuvant (Sigma, USA) was used to immunize mice. Four six-week-old female BALB/c mice were immunized four times at 14-day intervals. Three days after the fourth immunization, spleen cells were harvested and fused with SP2/0 cells. The fused cells were cultured in DMEM medium containing HAT Supplement (Sigma, USA) and 20% FBS. After 10 days, the supernatant of the fused cells was collected, and antibodies against pB602L were detected using an indirect ELISA (iELISA). Positive clones were selected and subcloned three times by limiting dilution. Monoclonal cells were cultured in DMEM medium containing 20% FBS and 1% penicillin-streptomycin. Ten-week-old female BALB/c mice were used for the preparation of ascites containing antibodies against the pB602L protein. The antibody titers in the ascites were determined using iELISA. An SBA Clonotyping System-HRP kit (Southern Biotech, USA) was used to identify the subtypes of the monoclonal antibodies. The specificity of the monoclonal antibodies against the recombinant protein and virions was evaluated using Western blot and IFA.

In immunized mice, sera were obtained at 21and 56 days after immunization. Plate-bound NP(7)-BSA and NP(25)-BSA (10 μg/ml) was used to measure antigen-specific antibodies. Class-switched serum immunoglobulin levels were detected using SBA Clonotyping System HRP kit (Southern Biotech). Antibody titers are given as -log2(dilution) x 40. Positive values were defined as those 3 s.d above mean values of the negative controls^{6 (link)}.

Serum from WT NOD, B1411-Igμ^homIgκ^hom and B1411-Rag1^−/− mice were analyzed using the SBA Clonotyping System-HRP Kit according to manufacturer's protocol (SouthernBiotech). Briefly, the capture antibody was diluted 1:500 and coated on 96-well plates overnight. Serum of indicated mice was then incubated for 1 h at room temperature. After incubation with HRP-conjugated detection antibodies, TMB substrate was added to evoke the colorimetric response and sulfuric acid was used to stop the reaction. The optical density was read at a wavelength of 450 nm.