Antibody-secreting cells were measured in peripheral blood according to our previously described enzyme-linked immunosorbent spot (ELISPOT) procedure (26 (link), 37 (link)). Briefly, nitrocellulose (bottom) plates (MSHAN-4550; Millipore, Bedford, MA) were coated with 100 μl of sialidase (25 μg/ml) in sodium bicarbonate buffer (pH 9.6), affinity-purified goat anti-human immunoglobulin (5 μg/ml; Jackson Immuno Research, West Grove, PA) to detect the total number of circulating ASCs, or keyhole limpet hemocyanin (KLH; 2.5 μg/ml [Pierce Biotechnology, Rockford, IL]) in PBS as a negative control. After blocking with RPMI solution, isolated PBMCs were incubated on the plates for 3 h, and secreted antibodies were detected with peroxidase-conjugated mouse anti-human IgA (1:500 dilution; Southern Biotech, Birmingham, AL) and alkaline phosphatase-conjugated IgG (1:500 dilution; Southern Biotech, Birmingham, AL). ASCs were detected with 3-amino-9-ethylcarbazole (AEC) (AEC premix solution; Sigma-Aldrich) and 5-bromo-4-chloro-3-indolylphosphate–nitroblue tetrazolium (BCIP/NBT; Sigma-Aldrich). The antigen-specific IgA and IgG isotypes of ASCs were expressed as the frequencies of the total circulating ASCs of the same isotype at the same time point.
Affinity purified goat anti human immunoglobulin
Manufactured by Jackson ImmunoResearch
Sourced in Cameroon, United States
Affinity-purified goat anti-human immunoglobulin is a laboratory reagent used to detect and quantify human immunoglobulins in various assays and experiments.
Automatically generated - may contain errors
Lab products found in correlation
5 bromo 4 chloro 3 indolyl phosphate nitro blue tetrazolium bcip nbt, by Merck Group (1 mentions)
Mshan4550, by Merck Group (1 mentions)
5 bromo 4 chloro 3 indolyl phosphate nitro blue tetrazolium, by Merck Group (1 mentions)
Keyhole limpet hemocyanin klh, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated goat anti human iga, by Southern Biotech (1 mentions)
2 protocols using affinity purified goat anti human immunoglobulin
1
ELISPOT Assay for Antibody-Secreting Cells
2
ELISPOT Assay for Antigen-Specific ASCs
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ASC responses were assessed through ELISPOT technique as described previously (35 (link)). Affinity-purified goat anti-human immunoglobulin (Jackson ImmunoResearch) as a positive control, V. cholerae O1 Ogawa OSP: BSA (10 µg/mL), GM1 ganglioside (3 nmol/mL) followed by recombinant CtxB (2.5 µg/mL), and as a negative control, keyhole limpet hemocyanin (KLH, 2.5 µg/mL; Pierce Biotechnology, Rockford, IL, USA) were used as coating antigen to coat nitrocellulose-bottom plates (Millipore, Bedford, MA, USA). Goat anti-human IgG (Southern Biotech, Birmingham, AL, USA) conjugated with alkaline phosphatase was used to detect IgG ASCs, and to detect IgA ASCs, horseradish peroxidase-conjugated goat anti-human IgA (Southern Biotech) was added to the plates, respectively. Plates were incubated overnight at 4°C, and on the following day, plates were developed using 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium (Sigma-Aldrich) and 3-amino-9-ethylcarbazole (pre-mix solution, Sigma-Aldrich) as substrate, respectively.
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