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22 protocols using sba clonotyping system hrp kit

1

Subtyping and Characterization of Monoclonal Antibodies Against Mycoplasma hyorhinis

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The subtypes of mAbs produced were determined using the SBA Clonotyping System-HRP kit (Southern Biotech, Birmingham, AL, USA) according to the manufacturer’s instructions. The hybridoma supernatant was added as a primary antibody and horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody was used as the secondary antibody. Color development and screening were performed as described above. The reactivity of mAbs to Mhr was determined by Western blot analysis as described above and Mhr-specific antigen in the lung tissue of Mhr-infected pigs was detected using immunohistochemical assays. The proteolytic antigen retrieval step of the immunohistochemical assay was modified to include proteinase K (Merck Life Science Co. Ltd., Shanghai, China). Uninfected lung tissue (by PCR detection) was used as the negative control and the cross reactivity of mAbs to Mhp was tested by immunohistochemistry using Mhp positive tissues.
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2

Generating Monoclonal Antibodies to Norovirus VLPs

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For mouse immunization, VLPs of strain GII.17-KT were expressed in Pichia pastoris yeast as described above. The mouse immunization study was approved by the Institutional Animal Care and Use Committee at the Institut Pasteur of Shanghai and the animals were cared for in accordance with the institutional guidelines. Female Balb/c mice were immunized with 5 µg of the GII.17 VLP plus 500 µg of Alum adjuvant (Invivogen, USA) at weeks 0, 2, and 4. The mice were given a booster immunization with 15 µg of VLP at week 7. Three days later, splenocytes were isolated from the immunized mice and then fused with SP2/0 myeloma. The resulting hybridomas were screened for production of VLP-binding antibodies by ELISA as described below. MAbs were purified from positive hybridoma clones using HiTrapTM Protein G affinity column (GE Healthcare, USA). The isotypes of the MAbs were determined by ELISA using an SBA Clonotyping System-HRP Kit (SouthernBiotech, USA). A norovirus cross-reactive mAb, 7D8, was prepared from a mouse immunized with the insect cells-derived GII.4 VLP [33 (link)] according to the protocol described above.
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3

Determination of Immunoglobulin Isotypes

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The immunoglobulin isotypes of the A3 and G5 mAbs were determined using a mouse mAb isotyping kit (SouthernBiotech, SBA clonotyping System-HRP Kit), according to the manufacturer’s instructions.
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4

Monoclonal Antibody Isotype Determination

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The MAb isotype was determined by the SBA Clonotyping System-HRP kit according to the manufacturer's instructions (Southern Biotech, USA).
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5

Quantifying NP-specific IgG1 Titers and LLPCs

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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.
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6

Generation of Anti-ADAM8 Monoclonal Antibodies

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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).
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7

Quantification of Mouse Antibody Isotypes

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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.
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8

Production and Characterization of Anti-pB602L Monoclonal Antibodies

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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.
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9

Quantification of Antigen-Specific Antibodies in Mice

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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 controls6 (link).
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10

Serum Immunoglobulin Clonotyping Assay

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Serum from WT NOD, B1411-IgμhomIgκhom 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.
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