ELISA Development Guide - OriGene

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ev022912 Parameter Modification Possible Effects Composition of the biotinylated detection antibody diluent Detection antibody incubation time Composition of the streptavidin-HRP conjugate diluent Streptavidin-HRP conjugate concentration Streptavidin-HRP incubation time Number of washes after Streptavidin-HRP incubation Addition of blocking agents such as proteins, polymers, or additional detergents. Increase or decrease in the salt (NaCl) concentration Increase or decrease in the buffer pH Alternate buffers: Tris, borate, MES Increased recovery Increased linearity Increased sensitivity Reduced sample interference Alternate buffer concentrations Increase or decrease. Increased or decreased signal Increased or decreased sensitivity Altered dynamic range Addition of blocking agents such as proteins, polymers, or additional detergents Increase or decrease in the salt (NaCl) concentration. Increase or decrease in the buffer pH Alternate buffers: Tris, borate, MES Increased or decreased signal Increased or decreased sensitivity Increased or decreased dynamic range Increased or decreased detection time Alternate buffer concentrations Increase or decrease Increased or decreased signal Increased or decreased sensitivity Alternate dynamic range Increase or decrease Increased or decreased signal Increased or decreased sensitivity Increase or decrease Increased or decreased signal and/or background. OriGene Technologies, Inc. 9620 Medical Center Drive, Suite 200, Rockville, MD 20850 www.origene.com techsupport@origene.com 1-301-340-3188 (P) 1-301-340-9254 (F) Page 14
ev022912 ELISA Assay Troubleshooting Problem Possible Causes Solutions High Background No Signal or Low Signal Insufficient washing Increase number of washes. Add 1-5 minute soak step between washes. Increase the volume of wash buffer per well. If using a plate washer, check to ensure that all ports are clean and that the target volume is delivered to all wells. Check that the residual volume after aspiration is less than 5 l per well. Detection antibody concentration is too high Streptavidin-HRP conjugate concentration is too high Check dilution. Titrate to find the optimal concentration. Use a medium-binding plate from a reliable source (Costar, Nunc, Greiner). Check dilution. Titrate to find the optimal concentration. Non-specific interactions Add additional protein to assay buffers and wash buffer. Contaminated buffers Remake buffers. Non-binding (eg, tissue culture) microplate used for antibody coating Diluted capture antibody stored in coating buffer for extended period before coating. Reagents omitted or added in incorrect order Incorrect standard was used or standard dilutions were made incorrectly Use a high-binding plate from a reliable source (Costar, Nunc, Greiner) Dilute antibody into coating buffer immediately before use. Do not store diluted capture antibody. Check calculations and protocol. Re-make solutions. Repeat assay. Verify that the correct standard was used. Check calculations for standard dilutions. Re-dilute standards. Inhibition of HRP by azide Make sure that the SA-HRP dilution buffer or final wash buffer does not contain azide. Concentration of Detection Antibody or Streptavidin-HRP is too low. Check dilution. Titrate to find the optimal concentration. OriGene Technologies, Inc. 9620 Medical Center Drive, Suite 200, Rockville, MD 20850 www.origene.com techsupport@origene.com 1-301-340-3188 (P) 1-301-340-9254 (F) Page 15
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Page 7 and 8: ev022912 Recommended Plate Layout B
Page 9 and 10: ev022912 d. Assay uses 48 wells. e.
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Page 19 and 20: ev022912 Preparing Antibody-Coupled
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Page 25 and 26: ev022912 Note : To achieve optimal
Page 27 and 28: ev022912 7. For accurate quantitati
Page 29 and 30: ev022912 7. Ten to fifteen minutes
Page 31 and 32: ev022912 30. Optional: shake plate
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Page 39 and 40: ev022912 Post-Coat/Blocking Buffer
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ELISA Development Guide - OriGene

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