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[GUIDELINES]
<strong>Solutions and Buffers: </strong><br /><strong>Note:</strong> Do not use sodium azide in any buffers or solutions, as sodium azide inactivates the horseradish-peroxidase enzyme. <br /><br /><strong>Carbonate Coating Buffer </strong><br />BioLegend Cat. No. 421701 or… <br />8.4 g NaHCO3 <br />3.56 g Na2CO3 <br />Add ddH2O up to 1.0 L, pH to 9.5 <br /><br /><strong>Phosphate Buffered Saline (PBS): </strong><br />80.0 g NaCl <br />14.4 g Na2HPO4 <br />2.4 g KH2PO4 <br />2.0 g KCl <br />Add ddH2O up to 10 L, pH to 7.2 with HCl <br /><br /><strong>PBS/Tween: </strong><br />0.5 ml of Tween-20 in 1 L PBS<br /><br /> <strong>Blocking Solution: </strong><br />10% fetal bovine serum or 1% BSA in PBS. Filter before use to remove particulates.<br /><br /><strong>ABTS Substrate Solution: </strong><br />150 mg 2,2’-Azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (Sigma, Cat. No. A-1888) <br />Add to 500 ml of 0.1M citric acid in ddH2O <br />Adjust pH to 4.35 with NaOH <br />Aliquot 11 ml per vial and store at -20˚ C. <br />Avoid light exposure during preparation and storage. <br /><br /><strong>ABTS Stop Solution: </strong><br />Combine 50 ml dimethylformamide (DMF; Pierce, Cat. No. 20672) with 50 ml ddH2O <br />Add 20 g sodium dodecyl sulfate <br /><br /><strong>TMB (tetramethylbenzidine) Substrate Reagent Set: </strong><br />BioLegend Cat. No. 421101 <br /><br /><strong>TMB Stop Solution: </strong><br />BioLegend Cat. No. 423001 or 1M H3PO4 or 2N H2SO4 <br /><br /><strong>General References: </strong><br />1. Davies, C. 1994. The Immunoassay Handbook. D. Wild, Ed. Stockton Press, New York. <br />2. Abrams, J.S. 1995. Immunoenzymetric assay of mouse and human cytokines using NIP-labeled anti-cytokine antibodies. Current Protocols in Immunology (J. Coligan, A. Kruisbeek, D. Margulies, E. Shevach, W. Strober, Eds). John Wiley and Sons, New York. Unit 6.20. <br />3. Sander, B., et al. 1993. J. Immunol. Meth. 166:201. <br />4. Abrams, J.S., et al. 1992. Immunol. Rev. 127:5. <br /><br /><strong>Cytokine ELISA Troubleshooting Tips <br /><br />Poor signal-to-noise ratio </strong><br />• Try Capture Antibody at 1 – 10 µg/ml (generally 2 µg/ml). <br />• Try Detection Antibody at 0.25 – 2 µg/ml (generally 1 µg/ml). <br />• Titrate against each other to obtain optimal dilutions. <br /><br /><strong>Low Sensitivity </strong><br />• Try overnight incubations of standards and samples at 4°C.<br /><br /><strong>Poor Signal </strong><br />• If using HRP, avoid sodium azide in wash buffers and diluents, as sodium azide inhibits HRP. <br />• Verify that appropriate antibody pairs were used and the activity of the samples and/or standards. <br />• Check the activity of enzyme and substrate by coating with Detection Antibody (1 µg/ml), adding biotinylated avidin and revealing with the appropriate substrate. If the enzyme/substrate is active, a strong signal should be observed. <br /><br /><strong>Poor Standard Curve </strong><br />• Handling Instructions for standards are lot-specific. Refer to product information for proper handling. <br />• Recombinant protein vials should be quick-spun for maximum recovery. <br />• BioLegend suggests that cytokines be stored in a concentrated format (&gt;100 ng/ml) and in the presence of a protein carrier. <br /><br /><strong>High Background </strong><br />• Increase stringency of washing steps by soaking plates for ~1 minute during washes. <br />• Determine optimum Capture and Detection Antibody dilutions. <br />• Increase the dilution of Detection Antibody and/or increase the number of washes after Av-HRP incubation.

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