How to Operate an ELISA Microplate for Absorbance Measurement

Описание: Measuring absorbance is the final, critical step in a colorimetric ELISA, determining the quantitative result. Proper operation ensures accuracy and reproducibility. Below is a concise guide to the standard operating procedure.

I. Pre-Read Preparation

Reaction Termination: Add the stop solution (e.g., sulfuric acid for TMB substrate) as per protocol. The color will change (e.g., blue to yellow) and stabilize.

Bubble Removal: Gently tap the plate or briefly centrifuge it (300 x g, 1 min) to dislodge bubbles at the well bottom, which scatter light and cause errors.

Bottom Cleaning: Wipe the underside of the plate with a lint-free tissue moistened with ethanol or water to remove fingerprints, dust, or droplets.

II. Microplate Reader Setup

Instrument Warm-up: Turn on the reader and software. Allow the lamp to warm up for the time specified in the manual (typically 15-30 min).

Method Selection: Open a new absorbance/endpoint assay protocol.

Wavelength Setting:

Primary Wavelength: Set to the optimal absorbance peak of your chromogen. For stopped TMB, this is 450 nm.

Reference Wavelength (Optional but Recommended): Set to a wavelength where the chromogen does not absorb (e.g., 620 nm or 650 nm). The reader will subtract this background, correcting for plate imperfections. The reported value is A450 – A620.

III. Plate Reading & Data Acquisition

Plate Loading: Place the ELISA plate correctly in the carriage, aligning well A1 with the reader's designated position.

Plate Layout Definition: In the software, label the wells according to your plate map (e.g., Standard 1-7, Samples, Blank, Controls). This is essential for automated analysis.

Initiate Read: Start the reading process. The reader will scan each well.

Raw Data Output: The software generates a table of absorbance (Optical Density, OD) values for each well.

IV. Essential Data Processing Steps

Blank Correction: Subtract the average OD of your blank wells (containing only substrate + stop solution) from all other OD values. Most software performs this automatically during setup.

Standard Curve Generation:

Plot the corrected OD values of the standard wells against their known concentrations.

Apply the appropriate curve fit (e.g., linear regression for a linear range, or a 4-parameter logistic/sigmoidal curve for a typical binding assay).

Sample Calculation: Use the standard curve equation to interpolate the concentration of unknown samples from their blank-corrected OD values.

V. Key Considerations & Troubleshooting

Dynamic Range: Ensure sample ODs fall within the linear portion of the standard curve. Samples with ODs above the highest standard (Hook Effect) or below the lowest standard must be re-assayed at an appropriate dilution.

Timing: Read the plate promptly after stopping the reaction (usually within 30 minutes) to prevent potential signal drift.

Consistency: Use the same reading parameters (wavelengths, integration time) for all plates within an experiment.

Conclusion
Proper absorbance measurement is a systematic process. Careful pre-read preparation, correct instrument settings, and mandatory data processing (blank subtraction and curve fitting) are non-negotiable for transforming raw optical signals into reliable, quantitative biological data. Always consult your specific ELISA kit protocol for any deviations from this general guideline.