Technical Interview Preparation - How To Answer ELISA Testing Troubleshooting Questions

We have already looked at other chromatography technical questions for an interview with HPLC here and GC here. Make sure to check those out if they are areas of interest for you. Today we are going to look at ELISA troubleshooting and how to answer an interview question based on this. I will also be showing some soft skills that you will be displaying when talking about specific steps, important for you to realise so that you know what to talk about when it comes to your soft skills in an interview.

Troubleshooting in assay testing, particularly Enzyme-Linked Immunosorbent Assay (ELISA), in the pharmaceutical industry involves identifying and resolving issues or errors that may occur during the testing of pharmaceutical samples. Here's a step-by-step guide to explaining troubleshooting in ELISA testing in pharmaceuticals:

Define the Problem with your ELISA Testing

Clearly define the specific issue or error encountered in ELISA testing. This could include low signal or signal-to-noise ratio, high background noise, inconsistent or inaccurate results, or variations in replicates. It’s important to identify the problem with certainty before diving head first into the issue, you could make it worse. What is exactly wrong? What were you expecting and how was this deviating from what you were expecting?

Gather Information

Collect all relevant information related to the problem. This includes the ELISA protocol, reagents used (antibodies, buffers, substrates), sample preparation methods, incubation times and temperatures, instrument parameters (reader settings), and any observations or symptoms associated with the issue.

Review the Assay Protocol

Carefully review the assay protocol and compare it with the recommended guidelines or established standards. Ensure that all steps are followed correctly, including sample dilutions, plate coating, incubation times, and washing steps. This is the foundation of the scientific method and if you can show your methodical approach, you are showing that you are conscientious.

Inspect Reagents for your ELISA Testing

Check the integrity and quality of the reagents used in the assay. Examine the antibodies for degradation or contamination, confirm the expiration dates of the reagents, and verify proper storage conditions. Consider preparing fresh reagent solutions if needed.

Sample Preparation

Evaluate the sample preparation method to ensure it is appropriate for the pharmaceutical analyte being tested. Verify that the samples are properly collected, stored, and prepared according to the assay requirements. Address any potential issues such as sample degradation or contamination. This can be a potential area of conflict that needs to be addressed. If a client or customer has given you a sample to test that is contaminated, you will need to address this and obtain a fresh sample and this can be an excellent scenario for you to talk about having difficult conversations with your manager or clients.

Optimise Incubation Conditions

Assess the incubation times and temperatures used during the assay. Optimise the conditions to ensure optimal binding of the analyte and antibodies. Consider performing time course experiments to identify the optimal incubation duration. By isolating variables, you are showcasing your problem solving skills and the next few steps will also showcase this too.

Troubleshoot Washing Steps

Pay attention to the washing steps between each incubation to minimise non-specific binding and reduce background noise. Ensure that the wash buffer is properly prepared and that the washing procedure is thorough but gentle. Consider adjusting the number of washes or the washing technique.

Verify Reader Settings

Check the reader settings and calibration to ensure they are correctly configured for the assay. Verify the filter or wavelength settings for absorbance or fluorescence detection. Calibrate the reader using appropriate standards or controls to ensure accurate measurements.

Troubleshoot Background Noise

If encountering high background noise, identify the potential sources such as non-specific binding, plate contamination, or improper blocking. Optimise the blocking step with suitable blocking agents or adjust the concentration of blocking reagents to minimise background noise.

Verify Standards and Controls

Review the standards and controls used in the assay. Ensure that the concentration or activity of the standards is accurate and within the validated range. Verify that the controls are working properly and provide the expected results.

Check Plate Coating

Assess the plate coating procedure and verify that the appropriate coating conditions, such as coating buffer, incubation time, and temperature, are followed. Confirm that the analyte or capture antibody is coated evenly and uniformly across the wells.

Troubleshoot Signal Issues

If experiencing low signal or signal-to-noise ratio, consider optimising the concentration of the detection antibody or adjusting the detection system (substrate or fluorophore) used. Ensure that the detection system is appropriate for the assay format and the reader settings.

Data Analysis for your ELISA Testing

Analyse the assay results and look for patterns, trends, or anomalies that could help identify the root cause of the problem. Compare the obtained data with previous or expected results to identify any discrepancies or outliers. This is important for a number of reasons. One of the reasons that you underwent this process is because you saw that there was an anomaly in the data. This will further solidify your process, it will also mean that if you or your team come across this specific issue in the future, you will be in a position to fix it swiftly and showcases your teamworking skills.

Implement Corrective Actions

Based on the analysis of the troubleshooting data, select appropriate corrective actions. This may involve modifying assay parameters, optimizing reagent concentrations, adjusting incubation conditions, or improving sample preparation techniques.

Verify and Validate

After implementing the corrective actions, verify the effectiveness by retesting samples, standards, and controls. Ensure that the problem has been resolved and the assay results are accurate and reliable. Document the troubleshooting process, including the identified problem, steps taken, and the outcome.

Preventive Measures

Take preventive measures to minimise the recurrence of similar issues in future assays. This may include conducting regular quality control checks, implementing proper reagent storage and handling practices, ensuring adequate training for personnel, and updating standard operating procedures.

Remember, troubleshooting in ELISA testing requires a systematic approach and attention to detail. It is crucial to document each step of the troubleshooting process for future reference and to ensure reliable and consistent assay results in the pharmaceutical industry.

If you are someone who is recently graduated and wondering how to best talk about technical skills in a CV/Resume when most of your experience, make sure to check out my article how to do just that here.