The hybridoma supernatants were screened by ELISA for identifying the immunoglobulin-secreting wells. Each immunoglobulin secreting clone was evaluated for binding to the vaccine peptides. Briefly, maxiSorp® immuno™ flat-bottom 96 well plates (Thermo Fisher Scientific) were coated with goat anti-human IgG (H + L) antibody (Southern Biotech, Birmingham, AL) or 0.5 μg peptides in carbonate buffer (pH 9.6) and blocked with 1% casein solution in TBS (Thermo Fisher Scientific) for screening IgG-secreting wells and vaccine peptide binding antibodies by ELISA. The target-binding antibodies were detected with HRP-conjugated goat anti-human IgG (H + L) antibody (Thermo Fisher Scientific) and 3,3′,5,5′-tetramethylbenzidine soluble substrate (EMD Millipore, Darmstadt, Germany). The formation of blue-colored horseradish peroxidase product was read immediately at 650 nm for 15 min at 3-min intervals using a Synergy HT plate reader (BioTek Instruments, Inc., Winooski, VT).
Hrp conjugated goat anti human igg h l antibody
Manufactured by Thermo Fisher Scientific
The HRP-conjugated goat anti-human IgG (H + L) antibody is a laboratory reagent used to detect and quantify human immunoglobulin G (IgG) in various immunoassays. It is a polyclonal antibody raised in goats and conjugated with horseradish peroxidase (HRP), an enzyme that can be utilized for colorimetric or chemiluminescent detection.
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Lab products found in correlation
Tris buffered saline (tbs), by Thermo Fisher Scientific (1 mentions)
Synergy ht plate reader, by Agilent Technologies (1 mentions)
Sodium caseinate, by Merck Group (1 mentions)
Streptavidin coated plate, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
2 protocols using hrp conjugated goat anti human igg h l antibody
1
Screening Hybridoma Supernatants for Vaccine Peptide-Binding Antibodies
2
Peptide-Based ELISA for Epitope Determination
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Epitope determination was performed via peptide‐based ELISA as previously described.24 , 25 , 27 Briefly, streptavidin‐coated plates (Thermo Fisher Scientific) were blocked with 0.1% PBST (0.1% Tween‐20 in PBS) containing 1% sodium caseinate (Sigma‐Aldrich) and 1% bovine serum albumin (BSA; Sigma‐Aldrich) overnight at 4°C, before addition of biotinylated peptides (1:1000 dilution in 0.1% PBST), followed by heat‐inactivated pooled healthy control and patient plasma/serum samples (1:2000 dilution in 0.1% PBST). HRP‐conjugated goat anti‐human IgG (H+L) antibody (Thermo Fisher Scientific) prepared in 0.1% blocking buffer was used for detection of peptide‐bound antibodies. TMB substrate and Stop reagent (Sigma‐Aldrich) were used for development, prior to absorbance measurements at 450 nm (Tecan).24 , 25 , 27 All incubation steps were at room temperature for 1 h on a rotating shaker, and ELISA readings were conducted in duplicates.
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