Troubleshooting - JONLNBIO

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ELISA Troubleshooting

**High Background**
Possible Causes	Solutions
Insufficient washing	Add wash buffer to the wells to ensure sufficient washing
Insufficient washing	Ensure to decant all residual antibody solutions before washing
Interference in samples or standards	Setting a blank control
Buffer contaminated	Reformulation of buffer

**No Signal**
Possible Causes	Solutions
Incorrect order of addition of reagents or inaccurate preparation of reagents	Repeat assay
	Check preparation methods
	Review reagent addition process
HRP contaminated	Use freshly prepared reagents
Insufficient amount of antibody	Use the recommended amount ofantibody

**Regular Blue in Color**
Possible Causes	Solutions
Insufficient washing or skipping plate washing step	Strictly follow the instructions for the washing process
Plate cover or reagent reservoir reuse leads to HRP residual contamination	Each step requires to use new plate cover or reagent reservoir
Buffer contaminated with metal orHRP	Preparation of fresh buffer

**Poor Standard Curve**
Possible Causes	Solutions
Incorrect assay procedure	Strictly follow the procedures in the manual
Incorrect calculation of standard curve dilution	Recalculate the standard curve

**Poor Intra-plate Repeatability**
Possible Causes	Solutions
Insufficient washing	Add wash buffer to the wells to ensure sufficient washing
Reuse plate cover	Use new plate cover for each step
No plate cover used	Use plate cover

**Poor Inter-plate Repeatability**
Possible Causes	Solutions
Insufficient washing	Add wash buffer to the wells to ensure sufficient washing
Insufficient washing	Ensure to decant all residual solution before washing
Incubation temperature changed	Strictly follow the recommended temperature incubation
Incubation temperature changed	Avoid incubation in areas with large changes in ambient temperature
Assay procedure changed	Strictly follow the same procedures
Reuse plate cover leads to residual HRP contamination	Use new plate cover for each step

**Good Standard Curve but No Signal in Sample Wells**
Possible Causes	Solutions
Samples with no or very low level expression of detectives	Set up a positive control
Samples with no or very low level expression of detectives	Repeat assay

**Low OD Value**
Possible Causes	Solutions
Incorrect wavelength setting	Check Microplate reader settings
Expired pre-coated plate	Use new kit

**Edge Effect**
Possible Causes	Solutions
Incubation temperature imbalance	Incubation with plate cover
Incubation temperature imbalance	Avoid incubation in areas with large changes in ambient temperature

**Slow Color Development**
Possible Causes	Solutions
Incorrect temperature with plate	Ensure plate and reagents are at room temperature before use
Solution Contaminated	Avoid reagents containing NaN3