Ap conjugated streptavidin

Manufactured by Jackson ImmunoResearch

Sourced in United States

AP-conjugated streptavidin is a reagent used in various immunoassay and biotechnology applications. Streptavidin, a protein derived from the bacterium Streptomyces avidinii, has a strong affinity for the small molecule biotin. In this product, streptavidin is chemically conjugated to alkaline phosphatase (AP), an enzyme that can be used to detect the presence of the biotin-streptavidin complex through a colorimetric or chemiluminescent reaction.

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Lab products found in correlation

Goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions) Goat anti mouse igg1, by Southern Biotech (1 mentions) Goat anti mouse igg2a, by Southern Biotech (1 mentions) Diethanolamine, by Merck Group (1 mentions) Spectramax plus 384, by Molecular Devices (1 mentions) Ez link nhs biotin, by Thermo Fisher Scientific (1 mentions) Phosphatase substrate, by Merck Group (1 mentions)

2 protocols using ap conjugated streptavidin

ELISA for Virus-Specific Antibody Titers

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Purified β-propiolactone-inactivated rWT virus (2.5 μg/mL) was coated onto 96-well Immulon-2 plates (Dynex Technology Inc., Chantilly, VA, USA) and incubated overnight at 4°C. Serially diluted mouse sera was added and left to incubate at room temperature for 1.5 h. Goat anti-mouse IgG (H+L) (Invitrogen, B2763, Carlsbad, CA, USA), goat anti-mouse IgG1 (Southern Biotech, 1070-08, Birmingham, AL, USA), or goat anti-mouse IgG2a (Southern Biotech, 1080-08, Birmingham, AL, USA) was added for 1 h at room temperature to detect IgG, IgG1, or IgG2a, respectively. Color development was initiated by the addition of alkaline phosphatase (AP)-conjugated streptavidin (Jackson ImmunoResearch) and p-nitrophenyl phosphate (PNPP) substrate [10 mg/mL p-nitrophenyl phosphate di (tris) salt crystalline (Sigma-Aldrich, St. Louis, MO, USA), 1% diethanolamine (Sigma-Aldrich, St. Louis, MO, USA), 0.5 mg/mL MgCl₂, pH 9.8]. Detection of the optical density (OD) occurred at 405 nm (using a reference filter of 490 nm) on a microplate reader (Molecular Devices SpectraMax Plus 384, San Jose, CA, USA). The titres of each sample were determined as the highest dilution at which the OD of the sample was larger than the defined cut-off. The cut-off was defined as the mean OD of a known negative sample plus twice the standard deviation.

Landreth S., Lu Y., Pandey K, & Zhou Y. (2020). A Replication-Defective Influenza Virus Vaccine Confers Complete Protection against H7N9 Viral Infection in Mice. Vaccines, 8(2), 207.

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Characterization of Recombinant Zika Antibodies

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To further determine the domain specificity of our recombinant nmAbs, a competition ELISA was performed. Plates were coated overnight with 2 μg ml⁻¹ ZIKV Envelope protein (ZIKV-SU-ENV100, Native Antigen) diluted in PBS. Each plate was washed 3-times with PBS-Tween20 and the wells were blocked with 3% BSA in water for 1.5 h at room temperature. Subsequently, the plate was washed 3-times with PBS-Tween20 and antibodies, 4G2 and a known domain III binding mAb characterized in our laboratory, ADI24255, were added to their designated wells at concentrations of 25 μg ml⁻¹ with subsequent 10-fold dilutions (35 ). After a 1.5 h incubation at room temperature the plate was washed and our previously biotinylated nmAbs (EZ-LINK NHS-Biotin, ThermoFisher Scientific) were added to their designated wells. After a 1.5 h incubation at room temperature the plate was washed and alkaline phosphatase (AP)-conjugated streptavidin (016-059-084, Jackson ImmunoResearch Laboratories) was added to all wells at a dilution of 1:2,000. Following a 1 h incubation at room temperature, the plate was washed and developed with phosphatase substrate (MilliporeSigma) at room temperature. The plates were immediately read at 405 nm with 5 min incubations at room temperature between reads.

Magnani D.M., Rogers T., Beutler N., Ricciardi M.J., Bailey V.K., Gonzalez-Nieto L., Briney B., Sok D., Le K., Strubel A., Gutman M.J., Pedreño-Lopez N., Grubaugh N.D., Silveira C.G., Maxwell H.S., Domingues A., Martins M.A., Lee D.E., Okwuazi E.E., Jean S., Strobert E.A., Chahroudi A., Silvestri G., Vanderford T.H., Kallas E.G., Desrosiers R.C., Bonaldo M.C., Whitehead S.S., Burton D.R, & Watkins D.I. (2017). Neutralizing human monoclonal antibodies prevent Zika virus infection in macaques. Science translational medicine, 9(410), eaan8184.

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