Poor or Non-Linear Standard Curve

Symptom

Standard curve shows irregular shape, poor linearity, or inconsistent serial dilution response. R-squared value below 0.95 or curve does not follow expected sigmoidal or linear pattern across dynamic range.

1. 1 Pipetting errors or calculation mistakes in preparing serial dilution series
2. 2 Poor or variable adsorption of coating reagents to plate surface
3. 3 Capture antibody did not properly bind to plate due to incorrect coating buffer or presence of interfering proteins
4. 4 Reagents poorly mixed before dispensing into wells
5. 5 Inhomogeneous samples or improper ELISA plate selection

1. 1 Verify pipetting technique and dilution calculations; use calibrated pipettes; prepare fresh dilution series
2. 2 Use proper coating buffer: PBS pH 7.4 or carbonate-bicarbonate buffer pH 9.6; extend coating incubation time if needed
3. 3 Dilute capture antibody in PBS without other proteins; verify proper high-binding ELISA plate is used
4. 4 Thoroughly mix reagents before use; vortex or invert multiple times
5. 5 Check sample homogeneity; consider different plate types (high-binding vs medium-binding); use appropriate plates for fluorescence detection if applicable

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