ELISA Troubleshooting

If you are having trouble in your ELISA data results, check out this table for DLdevelop’s possible solutions to your problem:

1. No Signal

Standard		Samples
0.072	0.073	0.063	0.058
0.064	0.041	0.054	0.032
0.062	0.071	0.053	0.041
0.044	0.049	0.055	0.062
0.072	0.053	0.063	0.048
0.064	0.059	0.057	0.072
0.052	0.070	0.053	0.071
0.074	0.061	0.066	0.067

Possible causes:

1.Failure to store kit at temperature specified in the manual;

2.Expired or degraded substrate (TMB);

3.Reagent addition order error: biotinylated antibody and enzyme conjugate added in wrong sequence;

4.Reagent mixing: used with reagents from other manufacturers;

5.Missing key reagents (e.g., detection antibody, substrate).

2.Poor Standard Curve Fitting

Standard
2.590	2.598
1.560	1.619
1.232	1.240
0.611	0.581
0.661	0.618
0.197	0.183
0.150	0.155
0.089	0.086

Possible causes:	Suggested Solution
1.Inaccurate pipetting	Calibrate pipettes
2.Inaccurate standard dilutions	Mix thoroughly
3.Incomplete washing	Increase wash cycles and ensure thorough aspiration
4.Cross-contamination	Increase wash cycles and ensure thorough aspiration
5.Inadequate incubation (time or temperature)	Incubate in a 37℃ incubator
6.Large bubbles present during sample loading	Mix gently to minimize large bubble formation

3.Standard Curve too Low

Standard
0.585	0.706
0.341	0.307
0.219	0.226
0.112	0.122
0.082	0.091
0.078	0.066
0.071	0.062
0.054	0.073

Possible causes:	Suggested Solution
1.Failure to keep reagents according to the labels on the vials	All the reagents should be kept according to the labels on the vials.
2.Failure to warm reagents to room temperature before use	Bring all kit components and samples to room temperature (18-25℃) before use.
3.Inadequate incubation (time or temperature)	Incubate in a 37℃ incubator
4.Short substrate incubation time resulting in insufficient color development	Extend color development time, typically ≤25 min
5.Working solution prepared too far in advance	Prepare 15 min before use
6.Inaccurate standard dilutions	Dilute 100x working solution to 1x
7.Insufficient reagent volume or missed addition	Check washing and loading processes; ensure all reagents are added in order and in sufficient volume

4.Standard Curve and all Samples Positive

Standard		Samples
2.527	2.706	3.527	3.706
2.341	2.307	3.341	3.307
2.211	2.226	3.211	3.226
2.112	2.122	3.112	3.122
1.827	1.791	2.827	2.791
2.075	2.066	3.075	3.066
2.607	2.652	3.607	3.662
2.453	2.773	3 .459	3.863

Possible causes:

1.Inter-well cross-contamination

2.Inadequate mixing in serial dilution: Recommendation to pipette up/down 20 times per dilution step before loading

3.Wash buffer contamination: Reused wash solution or contaminated preparation container

5.High Background in Standard Curve

Standard
2.442	2.439
1.801	1.799
1.165	1.164
0.639	0.641
0.411	0.409
0.347	0.341
0.258	0.275
0.184	0.202

It is recommended that the OD value of background wells be ≤0.1 for the sandwich type.

Possible causes:

1.Incomplete washing, leaving non-specific binding;

2.Improper storage of strips: Storing at high temperatures may elevate background (e.g., remaining strips not sealed in self-sealing bags promptly after use, leading to prolonged exposure to ambient conditions).

3.Contamination at the bottom of plate wells

4.Excessive HRP enzyme conjugate addition; Over-incubation or high incubation temperature

5.Wash buffer preparation issues: Contaminated wash buffer

6.Failure to change tips when dispensing into wells

6.High Replicate Variation (High Intra-Assay CV%)

Standard		Samples
2.458	2.306	0.765	0.423
1.566	1 327	0.341	0.307
0.953	0.945	0.211	0.426
0.46	0.852	0.412	0.422
0.277	0.264	0.827	0.791
0.168	0.174	0.675	0.366
0.12	0.137	0.607	0.662
0.046	0.053	0.453	0.473