A standard ELISA was used to determine binding of serum antibodies or monoclonal antibodies to RABV G protein (CVS‐11). Briefly, ELISA plates were coated with RABV G protein at 5 μg/ml, blocked with 10% FCS in PBS, incubated with sera or human antibodies, and washed. Bound antibodies were detected by incubation with AP‐conjugated goat anti‐human IgG (Southern Biotech). Plates were then washed, substrate (p‐NPP, Sigma) was added and plates were read at 405 nm. The relative affinities of sera binding or monoclonal antibody binding were determined by measuring the dilution of sera (ED50) or the concentration of antibody (EC50) required to achieve 50% maximal binding at saturation.
Ap conjugated goat anti human igg
Manufactured by Southern Biotech
AP-conjugated goat anti-human IgG is a secondary antibody that binds to human immunoglobulin G (IgG) antibodies. The antibody is conjugated with alkaline phosphatase (AP), which can be used for detection and quantification purposes in various immunoassays, such as ELISA.
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Lab products found in correlation
Erms da470, by Merck Group (2 mentions)
High binding 96 well plate, by Corning (2 mentions)
4 nitrophenyl phosphate disodium salt hexahydrate substrate, by Merck Group (1 mentions)
Ap substrate solution, by Merck Group (1 mentions)
α1 acid glycoprotein, by Merck Group (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
5 protocols using ap conjugated goat anti human igg
1
ELISA for RABV G Protein Antibodies
2
Quantitative ELISA for Vi Polysaccharide Antibodies
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The procedure carried out by Laboratory 7 is a modification of the ELISA by Szu et al. [18 (link)]. The plate surface is coated with directly with Vi PS from S. Typhi (lot 201308, Wuhan Institute of Biological Products Co. Ltd). Wells (Costar, 2592) were coated with 100 μL of 2 μg Vi mL−1 in PBS diluted from 1 mg mL−1 in Milli-Q. Plates were covered, incubated overnight at 4 °C, washed 5x with 1% Brij L23 in 0.8% NaCl (NaCl-Brij 23). Wells were blocked with BB2 for 2 h at 37 °C, washed 5x with NaCl-Brij 23 and shaken dry. Starting dilutions of test samples were prepared in BB2 with 0.1% Brij L23 (AD4). Wells of the first row received 200 μL of the starting dilution and all other wells received 100 μL AD4. Top down dilutions were prepared by transfer of 100 μL of the diluted sample to subsequent wells. Plates were covered, incubated overnight at 25 °C, washed 5x with NaCl-Brij 23 and shaken dry. Hundred μL of AP conjugated goat anti-human IgG (Southern Biotech, 2040-04) diluted 1:2000 in AD4 was added to each well and incubated at 37 °C for 2 h. Plates were washed 5x with NaCl-Brij 23 and shaken dry and 100 μL of 4-Nitrophenyl Phosphate disodium salt hexahydrate substrate (Sigma, 71768) in Tris/3 mM Mg-buffer (pH 9.8) was added to each well and incubated at RT for 90–120 mins, then stopped by 50 μL 3 M NaOH. Plates were read at OD405nm.
3
Quantifying IgG Antibody Binding to PfCSP
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Total IgGs were quantified using half-area, high-binding 96-well plates (Corning) with 10 μg ml−1 goat anti-human IgG (SouthernBiotech, 2040-01) using Certified Reference Material 470 (ERMs-DA470, Sigma-Aldrich) as a standard. To test specific antibody binding, ELISA plates were either directly coated with 1 μg ml−1 of recombinant PfCSP (Sanaria®, sequence previously shown21 (link)), 2 μg ml−1 of peptide 22–110 or 1 μg ml−1 of peptide 282–383, or first with 10 μg ml−1 of avidin (Sigma-Aldrich), followed by 10 μg ml−1 of NANP18 (NANPNANPNANPNANPNA), NPDP19 (KQPADGNPDPNANPNVDPN), NPDP15 (KQPADGNPDPNANPN) or various NPDP19 peptide mutants. Non-specific binding to plates coated with an irrelevant control peptide was tested to exclude polyreactivity of the antibodies. All peptides and mutants were synthesized with biotin attached to the C-terminus (A&A Labs). Plates were blocked with 1% bovine serum albumin (BSA) and incubated with titrated antibodies, followed by 1/500 AP-conjugated goat anti-human IgG (Southern Biotech, 2040-04). Plates were then washed, substrate (p-NPP, Sigma) was added and plates were read at 405 nm.
4
Quantification of CD22-Fc Binding to Ligands
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Recombinant proteins composed of the amino-terminal domains (domains 1–3) of mouse or human CD22 and the Fc region of human IgG1 (CD22-Fc) were described previously (41 (link)). Microtiter plates (96 well) were coated with 20 µg/ml α1-acid glycoprotein (Sigma). Alternatively, plates were coated with 50 µg/ml streptavidin followed by incubation with 4 µg/ml biotinylated synthetic CD22 ligand (42 (link)). Plates coated with α1-acid glycoprotein and synthetic CD22 ligand were then blocked with PBS containing 1% bovine serum albumin, followed by incubation with human or mouse CD22-Fc and compounds for 2 h, respectively. CD22-Fc bound to the plates was detected using alkaline phosphatase (AP)-conjugated goat anti-human IgG (Southern Biotechnology) and AP substrate solution (Sigma). The optical density at 405 nm was measured by a microplate reader (Molecular Devices). The concentrations of the compounds that reduce the binding of CD22-Fc to the biotinylated CD22 ligand and α1-acid glycoprotein by 50% (IC50) were determined.
5
Quantifying IgG Antibody Binding to PfCSP
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