Antigen-specific antibody-secreting plasma cells in the spleen from immunized mice were determined by enzyme-linked immunosorbent spot (ELISpot) assay as described previously (52 (link)). Briefly, the ELISpot plates (MilliporeSigma, MAIPS4510) were coated with 10 µg/mL of NP(27)-BSA (Biosearch Technologies, N-5050H) at 4 °C overnight. The plates were washed with PBS and blocked with DMEM containing 10% FBS at room temperature for 1 h. Splenocyte suspensions were serially twofold diluted and plated, starting with 1 million cells per well, and then incubated at 37 °C for 4 h. After washes with PBST, the plates were incubated with biotin-conjugated goat anti-mouse isotype-specific antibodies (SouthernBiotech; IgM: 1021-08; IgG: 1015-08; IgG1: 1071-08; IgG2b: 1091-08; IgG2c: 1078-08; and IgG3: 1100-08) at 4 °C overnight. The plates were washed and then incubated with ExtrAvidin-alkaline phosphatase (MilliporeSigma, E2636) for 1 h. After final washes with PBST, the plates were incubated with BCIP/NBT Liquid Substrate System (MilliporeSigma, B1911) in the dark for 5 min. The plates were washed with water and dried overnight, and then the spots were enumerated.
Np27 bsa
Manufactured by Biosearch Technologies
NP27-BSA is a laboratory equipment product. It is a protein conjugate composed of bovine serum albumin (BSA) and a chemical compound. The core function of this product is to serve as a reference material or standard in various analytical applications, such as immunoassays or protein quantification.
Automatically generated - may contain errors
Lab products found in correlation
Np2 bsa, by Biosearch Technologies (3 mentions)
Np klh, by Biosearch Technologies (2 mentions)
Imject alum, by Thermo Fisher Scientific (2 mentions)
Multiskan fc, by Thermo Fisher Scientific (2 mentions)
Igg3 ap, by Southern Biotech (1 mentions)
Igm ap, by Southern Biotech (1 mentions)
Igg1 ap, by Southern Biotech (1 mentions)
Igg2b ap, by Southern Biotech (1 mentions)
Versamax spectrophotometer, by Molecular Devices (1 mentions)
Elisa plate, by Corning (1 mentions)
Tmb substrate, by BioLegend (1 mentions)
Microreader, by Bio-Rad (1 mentions)
Tmb microwell peroxidase substrate, by LGC (1 mentions)
Tween 20, by Nacalai Tesque (1 mentions)
6 protocols using np27 bsa
1
Quantifying Antigen-Specific Antibody-Secreting Plasma Cells
2
ELISA for Antibody Quantification
3
NP-specific IgG1 Antibody Quantification
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NP-specific IgG1 antibody were measured similar to what was described previously (Cho et al., 2018 (link)). In brief, 96-well plates were first coated overnight at 4°C with NP27-BSA (Biosearch Technologies), followed by blockade of non-specific binding by incubation with blocking buffer (1% of BSA in PBS) for 1 h at room temperature. Mouse serum was diluted to 10−4 of the original concentration in blocking buffer and then added to the plates, followed by incubation for 1 h at room temperature. Plates were washed with washing buffer (0.05% Tween-20 in PBS) three times. Bound antibodies were detected by HRP-conjugated anti-mouse IgG1 antibodies (5300-05; 1:1,000; Southern Biotech). The reactions were developed by incubation for 15 min at room temperature with TMB substrate (BioLegend) and were stopped by the addition of 2N H2SO4. Absorbance was measured by a microreader (Bio-Rad) at 450 nm.
4
NP-KLH Immunization and Antibody Affinity Analysis
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Mice were immunized subcutaneously in the flanks on day zero with 50 μg NP-KLH (Biosearch Technologies) emulsified in 100 μl Imject Alum (ThermoScientific). Serum and draining inguinal lymph nodes for flow cytometric analysis were collected on day 12 post-immunization. Affinity assays were performed as described previously29 (link). Briefly, plates (Corning, New York, NY) were coated at with 10 μg/ml of either NP-27-BSA (total) or NP-2-BSA (high-affinity) per well (Biosearch Technologies). Antibody titers are given as –log2 dilution × 40. Positive titers were defined as three standard deviations (SD) above the mean values of the negative controls41 (link).
5
NP-KLH Immunization and Antibody Affinity Analysis
6
Measurement of Anti-Immunoglobulin Titers by ELISA
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To measure anti-NP or anti-SRBC immunoglobulin titers, a 96-well microtiter plate for ELISA (Corning) was coated with 10 µg ml−1 NP27-BSA (Biosearch Technologies) or extracts of SRBC in a carbonate-bicarbonate BupH buffer (Thermo Fisher Scientific). An immunogen-coated microtiter plate was treated with 1% BSA in PBS to prevent non-specific background staining and then washed with PBS washing buffer containing 0.02% Tween-20 (Nacalai Tesque, Kyoto, Japan). Properly diluted serum was incubated at room temperature for 2 h. HRP-conjugated IgM, IgG1, IgG2a, IgG2b, and IgG3 antibodies (SouthernBiotech, Birmingham, AL) were incubated separately on plates at room temperature for 2 h. TMB microwell peroxidase substrate (Sera Care, Milford, MA) and 0.2 N H2SO4 as a stop buffer were used for colorimetric detection. The OD value was measured as the absorbance at 450 nm using a microplate reader (Multiskan FC, Thermo Fisher Scientific). HDM-specific IgG1 was detected using a high-binding plate coated with 5 μg ml−1 HDM (Greer Laboratories). HDM-specific IgE in sera was analyzed by an ELISA kit for anti-HDM IgE antibodies in mouse serum according to the manufacturer’s protocols (Chondrex, Woodinville, WA).
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