I am working to develop a Mac Elisa protocol that is able to capture the level of Immunoglobulin M (IgM) in mice infected with fungus T. marneffei. I've been mainly running into trouble with false positives from unspecific binding, but I've also had the opposite problem where I got a negative signal from a known positive control. As such, I've been tinkering with the concentration and incubation time of the reagents, including running a couple of concentration grids, but haven't found much success. I am wondering if anyone on here has much experience running or troubleshooting Mac Elisas, and has anything to suggest trying out! Thanks!
Current protocol:
Step 1 (Coating): Anti-mouse IgM polyclonal antibody (5ug/ml), 100ul overnight @ 4 C
Step 2 (Blocking): Blocking buffer, 200ul for 2 hours at room temperature
Step 3 (Sample incubation): Mouse plasma (diluted 1:20), 100ul for 1 hour at 37 C
Step 4 (Antigen): Detect anti-MP1P mouse-IgM with Recombinant MP1P (0.1 ug/ml), 100ul for 1 hour at 37 C
Step 5: Detect immune-complex with Biotinylated anti-MP1P mouse monoclonal antibody (MAb) (1:1000), 100 ul for 1 hour at 37 C
Step 6: Detect biotinylated anti-Mp1p mouse MAb with Stretavidin-HRP (1:2000), 100ul for 30 min at room temperature in the dark
Step 7: Detect HRP with TMB, 10 min at room temperature in the dark
Step 8: Stop activity of TMB with HCL 0.3M
Here are some suggestions based on my experience with ELISAs:
1. Check the specificity of your reagents: Make sure that all of your reagents, especially the primary and secondary antibodies, are specifically reacting with the intended target (i.e., IgM) and not cross-reacting with other proteins in the sample. Cross-reactivity can cause false positives or negatives.
2. Optimize the concentration and incubation time of reagents: As you've already tried, optimizing the concentration and incubation time of reagents can help improve the assay's sensitivity and specificity. You might want to try different concentrations of the reagents and compare their performance. Also, consider using appropriate controls (e.g., negative controls, positive controls) to monitor the assay's performance.
3. Use appropriate blocking agents: Non-specific binding can be a common issue in ELISAs. In addition to blocking buffers, you can try adding milk or bovine serum albumin (BSA) to the blocking solution to reduce non-specific binding.
4. Consider using a different detection method: Instead of using streptavidin-HRP, which can sometimes give rise to non-specific signals, you could switch to a different detection method like alkaline phosphatase (ALP) or horseradish peroxidase (HRP)-conjugated secondary antibodies. ALP tends to produce less background than HRP and can be a good alternative.
5. Ensure proper washing steps: Washing steps are crucial in removing unbound material and reducing non-specific signals. Be sure to perform thorough washes after each step, and use appropriate wash buffers (e.g., PBS-Tween).
6. Validate your protocol with known samples: Test your optimized protocol with a set of known samples (both positive and negative controls) to ensure it's performing correctly. This will help you identify any issues before proceeding with unknown samples.
7. Consider consulting experts or literature: If you continue to encounter problems, reach out to experts in the field or consult relevant literature to gain insights into successful Mac ELISA protocols for detecting IgM in mice infected with T. marneffei.
8. Evaluate the possibility of sample contamination: Sample contamination can introduce false positives or negatives into your results. Make sure to handle samples properly, and consider testing your samples for potential contaminants.
9. Assess the impact of dilution factors: Diluting samples can affect the accuracy of the assay. Try comparing the results of undiluted and diluted samples to determine whether dilution is causing issues.
10. Monitor assay performance over time: ELISA performance can degrade over time due to reagent degradation or changes in experimental conditions. Regularly assess the assay's performance and make adjustments as needed.
By addressing these points, you may be able to improve the reliability and accuracy of your Mac ELISA protocol for detecting IgM in mice infected with T. marneffei. Good luck with your research!
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