Question

Asked 8 January 2023

University of São Paulo

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%

Amino Acids, Peptides, and Proteins

Serum

Gabriela Rodrigues e Fonseca

University of São Paulo

Hello Shezan,

Thank you so much for your consideration. I'll look into it. Luckily I can have some answers!

Cite

Chinaza Godswill Awuchi

University of Central Lancashire

Sridharani Biochem

Global Biotech Research Centre

Adam B Shapiro

Arun Manakkal Sebastian

Jordan Bio-Research Centre

Rob Keller

Charlemagne College, Nijmegen, The Netherlands

Juan Carlos Apaza Paucara

Jorge Basadre Grohmann National University

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Gabriela Rodrigues e Fonseca

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Thank you Juan!

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Shezan Ahmed

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.

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2 Recommendations

Gabriela Rodrigues e Fonseca

University of São Paulo

Hello Shezan,

Thank you so much for your consideration. I'll look into it. Luckily I can have some answers!

Cite

Peptide ELISA with low reproducibility - can you help us??

Question

2 answers

Asked 25 May 2023

Gabriela Rodrigues e Fonseca

We are developing a peptide ELISA for IgM and IgG detection.

We infected mice with parasite 1; for cross-reactivity analyses, we infected mice with two parasites (groups 2 and 3) and included a negative control group. Peptides were synthesized by solid phase peptide synthesis (Fmoc), with ~80% purity, from a native antigenic protein from parasite 1. Sera from different periods of infection were collected and frozen.

We coated the plates with BSA (40μg/mL) for 1 hour, and treated with glutaraldehyde 5% for 1 hour. Then, we added the peptide and incubated it overnight. Finally, we added glycine for 30min. We blocked the plate with PBS-BSA 5% for 1 hour, added the diluted samples and incubated for 1 hour under mild agitation. We added anti-biotinylated IgM or anti-IgG-HRP for 1 hour. For IgM, we added avidin-HRP for 30 minutes. For both, we added TMB for 10min, stopped with sulfuric acid and read at 450nm.

The results are not consistent inter-assays (low reproducibility). The ODs from the negative control group are higher than blank wells and similar to those from parasite 1. Sera from groups 2 and 3 resulted in higher ODs than those of the parasite 1 group. We did not expect this cross-reactivity, given the peptides appeared specific to parasite 1 in certain periods of infection and showed no homology to other parasites or microorganisms on Protein BLAST.

Does anyone have any experience with peptide ELISA that could help us?

We washed the plates after every step, as shown below:

•BSA: washed once with carbonate-bicarbonate buffer (pH 9,6)

•Glutaraldehyde: washed twice with carbonate-bicarbonate buffer (pH 9,6)

•Peptides: washed twice with carbonate-bicarbonate buffer (pH 9,6)

Glycine: washed once with PBS

•IgM

Blocking and sera: washed 3 times with PBS-Tween 20 0,05%

Anti-biotinylated IgM: washed 5 times with PBS-Tween 20 0,05%

Avidin-HRP: washed 7 times, 5 minutes each, with PBS-Tween 20 0,05%

•IgG

Blocking: washed 3 times with PBS-Tween 20 0,05%

Sera: washed 3 times with PBS-Tween 20 0,05%

Secondary antibody: washed 3 times, 5 min each, with PBS-Tween 20 0,05%

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