Ap conjugated secondary antibody

Manufactured by Southern Biotech

The AP-conjugated secondary antibody is a laboratory reagent used in various immunoassay techniques. It is designed to detect and bind to the primary antibody, which is specific to the target antigen. The AP (Alkaline Phosphatase) enzyme conjugated to the secondary antibody serves as a detection system, enabling signal amplification and visualization of the target molecule.

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Lab products found in correlation

Ap yellow elisa substrate, by Merck Group (2 mentions) Serotype specific antibodies, by Statens Serum Institut (1 mentions) Tnp conjugated bsa, by Biosearch Technologies (1 mentions) Tnp lps, by Biosearch Technologies (1 mentions) Isotype specific antibodies, by Southern Biotech (1 mentions) Tnp ficoll, by Biosearch Technologies (1 mentions) Igg2b, by Southern Biotech (1 mentions) Igg2a, by Southern Biotech (1 mentions) P nitrophenyl phosphate, by Merck Group (1 mentions)

6 protocols using ap conjugated secondary antibody

Quantifying Bacterial Capsule Using ELISA

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Whole-cell bacterial ELISA was performed as a quantitative measure of capsule on the bacterial surface. Cells were grown to mid-logarithmic phase (OD₆₂₀ = 0.4) in C+Y, pelleted, and diluted 1:5 in coating buffer (sodium carbonate). The bacteria were bound to the plate via centrifugation. After the plate dried overnight at room temperature and was subjected to blocking buffer (10% FBS), the cells were probed with serotype-specific antibodies (Statens Serum Institute) and AP conjugated secondary antibody (Southern Biotech), followed by detection using an AP yellow ELISA substrate (Sigma). Each strain was repeated in duplicate in each plate, which was repeated three times. Immunoreactivity was compared using unpaired parametric t test in Prism 6.

Echlin H., Frank M.W., Iverson A., Chang T.C., Johnson M.D., Rock C.O, & Rosch J.W. (2016). Pyruvate Oxidase as a Critical Link between Metabolism and Capsule Biosynthesis in Streptococcus pneumoniae. PLoS Pathogens, 12(10), e1005951.

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Quantifying Bacterial Capsule by ELISA

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Whole-cell bacterial ELISA was performed as a quantitative measure of capsule on the bacterial surface. TIGR4 and the S81F gyrA mutant variants were grown to OD₆₂₀ ~0.4 in C+Y, pelleted, and diluted 1:5 in coating buffer (sodium carbonate). Bacteria were bound to 96-well plates via centrifugation. Plates were dried and subjected to blocking buffer (10% FBS). Bound cells were probed with type-4 capsular antibody (Statens Serum Institute) and LytA antibody, followed by probing with AP-conjugated secondary antibody (Southern Biotech) and detection using an AP yellow ELISA substrate (Sigma). Capsule immunoreactivity was normalized to immunoreactivity to LytA.

Dao T.H., Echlin H., McKnight A., Marr E.S., Junker J., Jia Q., Hayden R., van Opijnen T., Isberg R.R., Cooper V.S, & Rosch J.W. (2024). Streptococcus pneumoniae favors tolerance via metabolic adaptation over resistance to circumvent fluoroquinolones. mBio, 15(2), e02828-23.

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Diverse ELISA Techniques for Virus-like Particles

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Intact binding ELISAs were performed by binding 500ng PsV to the wells of microtiter plates in PBS. The wells were blocked with blocking buffer (5% fat free dry milk in PBS/T). Hybridoma supernatants were added to the wells and binding was detected by an AP-conjugated secondary antibody (Southern Biotech). AP signal was developed with PNPP and the OD 405 450 was determined with a plate reader. For the denaturing ELISAs, PsV was added to the wells of the microtiter plate in 25 μl denaturing buffer (0.2M Na₂CO₃, 10mM DTT pH10.7). Plates were incubated overnight at 37°C or at 55°C for 1 hour allowing the protein to dry to the plate. Proteins were rehydrated for 15 minutes with 25μl PBS and further processed in the same manner as the intact ELISA. For sandwich ELISAs 10μg/ml purified mAb was bound to the microtiter plate in 25μl 50mM Na₂CO₃, pH 9.6 binding buffer overnight at 4°C. Wells were washed, blocked with blocking buffer and then 0.5μg PsV was added to the wells in blocking buffer for 1 hour. Wells were washed again and the detection mAb H16.V5 IgA was added followed by an IgA specific secondary antibody. Plates were further processed in the same manner as the intact ELISA.

Bywaters S., Brendle S., Biryukov J., Wang J., Walston J., Milici J., Roden R., Meyers C, & Christensen N. (2018). PRODUCTION AND CHARACTERIZATION OF A NOVEL HPV ANTI-L2 MONOCLONAL ANTIBODY PANEL. Virology, 524, 106-113.

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Mouse Immunization and Antibody Response Assay

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4–6-wk-old mice were immunized i.p. with 50 µg OVA with Alum (Sigma-Aldrich) in PBS at day 0, boosted at day 14, and bled at days 0 and 21. Mice were immunized i.p. with 25 µg TNP-LPS (Biosearch Technologies) in PBS or with 25 µg TNP–Ficoll (Biosearch Technologies) at day 0 and bled at days 0 and 14. Antigen-specific antibody responses were analyzed by OVA- or TNP-specific ELISA using 96-well plates coated with either OVA- or TNP-conjugated BSA (Biosearch Technologies) at 10 µg/ml in PBS. For serum Ig levels, plates were coated with isotype-specific antibodies (SouthernBiotech). AP-conjugated secondary antibodies (SouthernBiotech) and PNPP (Sigma-Aldrich) were used for ELISA.

Mooster J.L., Le Bras S., Massaad M.J., Jabara H., Yoon J., Galand C., Heesters B.A., Burton O.T., Mattoo H., Manis J, & Geha R.S. (2015). Defective lymphoid organogenesis underlies the immune deficiency caused by a heterozygous S32I mutation in IκBα. The Journal of Experimental Medicine, 212(2), 185-202.

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Western Blot Immunodetection Protocol

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Samples for immunodetection were transferred from SDS-PAGE gels to PVDF membranes and blocked with 5% (w/v) nonfat powdered milk in 0.14 M NaCl, 2.7 mM KCl, 6 mM P_i (pH 7.4), 0.1% Tween20, and 0.2% gelatin. The primary antibodies against GP and the α, β, and γ subunits of PhK have been previously characterized and were used as described [14 (link)-16 (link)]. Colorimetric detection of the immunoreactive bands was performed with AP-conjugated secondary antibodies from Southern Biotechnology.

Thompson J.A, & Carlson G.M. (2016). The regulatory α and β subunits of phosphorylase kinase directly interact with its substrate, glycogen phosphorylase. Biochemical and biophysical research communications, 482(2), 221-225.

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Quantitative ELISA for TRR1 Antibodies

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TRR1 proteins diluted to 10 μg/mL were used to coat polystyrene plates. After blocking with 1% BSA in PBS, dilutions of the sera from the immunized mice were incubated for 1 h at 37°C. In some of the experiments, we used as secondary antibody a combination of alkaline phosphatase-conjugated goat anti-mouse IgG, IgA, and IgM (Southern Biotech) in a 1:1000 dilution also for 1 h at 37°C. In other experiments, we used isotype-specific (IgA, IgG1, IgG2a, IgG2b, IgG3, and IgM) AP-conjugated secondary antibodies (Southern Biotech). Bound antibodies were detected using p-nitrophenyl phosphate (Sigma) as a substrate, with absorbance measured in a plate spectrophotometer at 405 nm. In some experiments we also included as negative control wells in which the TRR1 proteins were substituted for an unrelated his-tagged protein, to detect antibodies against the tag used for TRR1 purification.

de Oliveira F.F., Paredes V., de Sousa H.R., D’Áurea Moura Á.N., Riasco-Palacios J., Casadevall A., Felipe M.S, & Nicola A.M. (2020). Thioredoxin Reductase 1 Is a Highly Immunogenic Cell Surface Antigen in Paracoccidioides spp., Candida albicans, and Cryptococcus neoformans. Frontiers in Microbiology, 10, 2930.

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