Factors Influencing the Signal Quality of ELISA Test


The ready-to-use ELISA kits have been optimized and validated to maximize the signal quality for specificity, sensitivity, coefficient of variation and other parameters. However, when a kit is unavailable for the specific target or is not optimized for the specific parameter required, labs may need to develop their own test. While ELISA is a powerful assay, it can be challenging to develop and optimize a specific test. It is necessary to consider the factors that influence the signal quality of ELISA.

Factors that may interfere with ELISA testing can occur at any stage of the analysis process, starting with sample collection. The quality and integrity of the assay plate, coating buffer, capture antibody, blocking buffer, target antigen, detection antibody, enzyme conjugate and other factors can all interfere with ELISA testing. Some of the factors that may interfere with the assay include:



Variable characteristics


material, shape and quality, pre-activation, even or uneven coating


composition, pH, contamination

Capture Antibody

incubation time, temperature, specificity, affinity

Detection Antibody

incubation time, temperature, specificity, affinity

Blocking Buffer

cross-reactivity, concentration, contamination

Target Antigen

conformation, stability, epitopes

Enzyme Conjugate

type, concentration, function, cross-reactivity, activity


contamination, frequency, volume, duration, composition


quality, manufacturer, sensitivity, manufacturer


filters, imaging instrument, exposure time, instrument dependent factors

Human Error

improper operation


In order to reduce the error of ELISA experimental operation, you can pay attention to the following details:

1. Use the calibrated pipette to eliminate natural errors. The accuracy of the pipette is very important for quantitative detection.

2. Read the instructions carefully and carry out standardized operations in strict accordance with instructions. Reagents of different batch numbers should not be mixed.

3. Strict control of reaction time. If the reaction time is too long, the enzyme is inactivated; if the reaction time is too short, the product structure is loose and weak, which may cause a false negative.

4. Adding samples should be accurate and fast.

5. Use a clean plastic container to configure the washing solution. Thoroughly mix all ingredients and samples in the kit before use. The washing solution should be fresh and ready to use. False positive results may occur with the old washing solution.

6. Reagents should be added in the same order to ensure that all reaction plates have the same incubation time.

Alpha Lifetech Inc. offers a variety of ELISA kits for customers throughout the world. Our sales representatives and technical experts will continue in providing advice throughout. In addition, we can offer customized services for your special requirements, providing protein expression, antibody production, ELISA construction, conduction of assays, results analysis and other one-stop technical services.

Please check our product list to choose the ELISA kit you want and feel free to contact us for more information.