Ribose (30 mM) was mixed with 2 mg/mL gelatin in 200 mM NaPB in the presence of AMG (0.1, 1, 10 and 100 µM) or TBE (0.01, 0.1, 1, 10, 100 µg/mL), and incubated at 37 °C for 7 days. MG-gelatin was prepared by incubating 2 mg/mL gelatin with 100 µM MG in PBS at 37 °C for 3 days in the presence of AMG or TBE. MG-H1 formation was measured by ELISA as previously described [37 (link)]. In brief, for noncompetitive ELISA, each well of a 96-well immune plate (Thermo Fisher Scientific, Waltham, MA, USA) was coated with 0.1 mL of the 1 µg/mL sample in PBS and blocked with 0.5% gelatin hydrolysate in PBS. The wells were incubated for 1 h with 0.1 mL of 1 µg/mL MG-H1 antibody [19 (link)]. Antibodies bound to the wells were detected using horseradish peroxidase-conjugated anti-mouse IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA). Then, stained with 100 μL of 500 µg/mL O-phenylenediamine dihydrochloride (Fuji Film Wako Pure Chemical, Japan) in citrate-phosphate buffer (pH 5.0) containing 5.9 mM hydrogen peroxide for 3 min. The reaction was terminated with 100 μL of 1.0 M sulfuric acid, and the absorbance was measured at 492 nm using a Sunrise RAINBOW THERMO RC system (TECAN, Männedorf, Switzerland).
96 well immune plate
Manufactured by Thermo Fisher Scientific
Sourced in China
The 96-well immune plates are a versatile laboratory tool designed for a variety of immunoassay applications. These plates feature a standardized 96-well format, providing a consistent and reliable platform for conducting various immune-related experiments and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Np25 bsa, by Biosearch Technologies (3 mentions)
P nitrophenylphosphate, by Southern Biotech (3 mentions)
Mouse igm and igg3, by Southern Biotech (2 mentions)
Ap conjugated anti mouse igm and igg3 antibodies, by Southern Biotech (2 mentions)
Horseradish peroxidase conjugated anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
O phenylenediamine dihydrochloride, by Fujifilm (1 mentions)
Epoch 2, by Agilent Technologies (1 mentions)
Anti mouse ig h l, by Southern Biotech (1 mentions)
Np9 bsa, by Biosearch Technologies (1 mentions)
Nbt bcip, by Roche (1 mentions)
Tmb substrate reagent set, by BD (1 mentions)
6 protocols using 96 well immune plate
1
Quantification of MG-H1 Formation in Ribose-Gelatin
2
ELISA-based SARS-CoV-2 Spike Antibody Titer
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The antibody titer was estimated using enzyme-linked immunosorbent assay (ELISA) according to a previous paper [12 (link)]. Briefly, 100 µL of SARS-CoV-2 spike-receptor-binding domain (RBD) protein (adjusted as 0.1 µg/mL; purchased from AIVD Biotech Inc., Shenzhen, China) was added to a 96-well immune plate (Thermo Fisher Scientific, Roskilde, Denmark). The sera of HD patients, infected patients, and healthy, unvaccinated individuals (each diluted 1:100 with phosphate-buffered saline) were applied as test samples, a positive, and negative control, respectively. After final incubation, the goat anti-human whole IgG and immunoglobulin M (1:5,000 dilution) conjugated with alkaline phosphate in a substrate buffer containing 20 mg of p-nitrophenyl phosphate tablet (Sigma-Aldrich, St. Louis, MO, USA) was added. The absorbance was measured at 405 nm using an ELISA reader (EPOCH2; BioTek, Santa Clara, CA, USA).
3
ELISA and ELISPOT Assays for Antibody Responses
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For SRBC immunization experiments, 96-well immune plates (Thermo Fisher Scientific) were coated with anti–mouse Ig(H+L) (Southern Biotech). For NP immunization experiments, plates were coated with NP9-BSA or NP25-BSA (Biosearch Technologies). Mouse serum samples were incubated for 2 h at room temperature. Standard curves were generated using mouse IgM and mouse IgG1 (Southern Biotech). Bound antibodies were detected by AP-conjugated anti–mouse IgM and anti–mouse IgG1 antibodies (Table S5). Plates were developed with p-nitrophenylphosphate (Southern Biotech) dissolved in substrate buffer. For ELISPOT analysis, the spleens and BM of NP-KLH–immunized mice were removed at the indicated time points, and single cell suspensions were subjected to hypotonic lysis. The samples were plated overnight on 96-well filtration plates (Millipore) coated with NP25-BSA (Biosearch Technologies). The cells were plated in 1:2 serial dilutions, starting at 8 × 105 cells for BM-derived samples, and at 105 cells for spleen-derived fractions. NP-specific IgG1-secreting cells were detected by AP-conjugated anti–mouse IgG1 antibody (Table S5). Plates were developed with nitro blue tetrazolium chloride-5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP; Roche).
4
NP-BSA Immunization Antibody Assay
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For NP-LPS and NP-Ficoll immunization experiments, 96 well immune-plates (Thermo Fisher Scientific) were coated with NP25-BSA (Biosearch Technologies). Mouse serum samples were incubated for 2h at RT. Standard curves were generated using mouse IgM and IgG3 (Southern Biotech). Bound antibodies were detected by AP-conjugated anti-mouse IgM and IgG3-antibodies (Southern Biotech). Plates were developed with p-nitrophenylphosphate (Southern Biotech) dissolved in substrate buffer.
5
NP-BSA Immunization Antibody Assay
6
TLR4-SP-A Binding Assay Protocol
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The binding assay procedures have been described previously40 (link). 500 ng recombinant human TLR4 (hTLR4) (Sino Biological Inc., Beijing, China) was coated onto 96-well immune plates (Thermo Fisher scientific), and nonspecific binding was blocked with 10 mM HEPES buffer (pH 7.4) containing 0.15 M NaCl, 5 mM CaCl2, and 2% fatty acid-free BSA (buffer A). 5 μg SP-A in buffer A were added and incubated at 37 °C for 5 h. The wells were washed and then incubated with anti-human SP-A polyclonal antibody followed by HRP-labeled anti-rabbit IgG in PBS containing 2% fatty acid-free BSA. The binding of hSP-A to the plates was detected using a TMB Substrate Reagent Set (BD Biosciences). The absorbance intensity of each well was measured at 450 nm.
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