University of São Paulo
Question
Asked 8 January 2023
Why the reproducibility of my ELISA with peptides is poor?
Dear students, researchers, and anyone who can help
I'm attempting to optimize and validate an indirect ELISA test with peptides for a parasite, but it's lacking reproducibility. The test works sometimes and then does not at others. I tried changing different variables, but the reproducibility is still poor. I cannot find what variable I need to change.
To standardize, I used a pool of sera of known infected animals as a positive control; individual sera from healthy animals as a healthy control, and individual sera of animals infected with other parasites. The test worked perfectly, differentiating the positive control from the healthy control and animals infected with other parasites.
However, when I performed the ELISA with individual samples of infected animals (not the positive control, but the sera of infected animals with the same parasite), the group results were similar to healthy control animals. The O.D. values of healthy control and blank samples are the only ones constant on every test. Also, I used a native antigen as the reagent control, which worked every time.
I tried several modifications: different antibody dilutions, peptide concentrations, substrate dilution, blocking buffer, washing buffer, the water used on the washing buffer, the type of washing (manual vs automatic), different volumes added to the well, and the microplate brand.
I appreciate your suggestions!
Buffers used:
For coating the wells: carbonate-bicarbonate, pH 9.6
Blocking and diluting samples and substrate: BSA 1%
Washing: PBS-Tween 0,05%
Most recent answer
Hello Shezan,
Thank you so much for your consideration. I'll look into it. Luckily I can have some answers!
All Answers (4)
Jorge Basadre Grohmann National University
Algunos artículos científicos
un péptido sintético de la glucoproteína 36 del VIH 2...
There can be several reasons why the reproducibility of your ELISA with peptides is poor. Some possible causes include:
- Variations in peptide preparation: Peptides can be prone to degradation and oxidation, which can affect their activity and stability. Variations in the preparation of the peptides, such as the method of synthesis or purification, can lead to differences in the final product and affect the reproducibility of the ELISA.
- Antibody variability: The antibodies used in the ELISA can also affect reproducibility. Variations in the source, purity, and storage conditions of the antibodies can lead to different binding affinities and specificity, which can affect the results of the ELISA.
- Buffer and reagent variability: The buffers and reagents used in the ELISA can also affect reproducibility. The pH, ionic strength, and composition of the buffers can affect the stability and activity of the peptides and antibodies, leading to variations in the results of the ELISA.
- Inconsistency in experimental conditions: Inconsistency in experimental conditions, such as variations in incubation time, temperature, and washing protocols, can also affect reproducibility.
- Human error: Finally, human error, such as variations in the way the samples are handled or measured, can also affect reproducibility.
It's important to note that reproducibility is a critical aspect of scientific research, and it is essential to identify and address any issues that may affect reproducibility. A careful examination of the experimental conditions and protocols, as well as a thorough characterization of the peptides and antibodies used in the assay, may help to identify the cause of poor reproducibility and develop strategies to improve it.
2 Recommendations
Similar questions and discussions
Recommendations
Background:
We previously reported a novel tumor associated antigen with a molecular weight around 48 kDa that was a fragment derived from human DNA-topoiomerase I. The aim of this study is to further investigate the clinical significance of the autoantibody in patients with non-small cell lung cancer (NSCLC).
Methods:
We determined serum levels...