Go through our list of tips and you will be prepared for a successful ELISA experiment.
1. Double-check the protocol
I know, this might sound like the most obvious advice ever. But trust me, it’s worth emphasizing! Take a moment to double-check the protocol that comes with our kits. Why? Because our protocols aren’t one-size-fits-all. They differ not just between applications (like ELISA, ELISpot, and FluoroSpot) but might be specific to the analyte. For instance, the recommended concentrations of capture and coating antibodies can vary between kits.
Sometimes, we tweak a protocol to accommodate batch-specific updates (like the ELISA standards) or introduce improvements, such as our updated sample dilution recommendation for our Human ApoB ELISA.
By following the provided protocol, you’ll reduce the risk of troubleshooting. Speaking of which, check out our ELISA troubleshooting guide for extra tips. Most importantly, sticking to the protocol ensures reproducibility – central to reliable, consistent results across experiments.
Looking for a more in-depth breakdown of our ELISA protocol? Explore our ELISA step-by-step guide for all the details!
2. Pick the right plates
Your choice of plate can make or break your ELISA results, so don’t grab just any plate off the shelf! ELISA plates are specially designed to bind high amounts of protein.
Here’s what to look for:
- Flat-bottomed (for optimal readings)
- High protein-binding polystyrene (the classic material for ELISA plates)
- 96-well format (that’s what our protocols are designed for)
And one more thing – cell culture plates are a no-go! They’re great for growing cells, but not for ELISA experiments. Stick with plates made for the job, and you’ll set yourself up for success.
If you prefer to skip plate coating altogether, have a look at our ELISA Pro kits which include pre-coated plates.
3. Check your reagents before start
Before diving into your ELISA experiment, take a moment to ensure you have all the necessary reagents and consumables on hand. Running out mid-assay? Not fun.
Pay extra attention to your enzyme conjugate and substrate: they need to be compatible with your ELISA setup. If you’re using our ELISA Flex kits, remember that substrates are sold separately. But if you’re working with our ELISA Pro or ELISA PathRF kits, good news, these reagents are already included.
Your substrate choice matters. It directly impacts assay sensitivity, so do a quick quality check:
- Expiration date? Still good.
- Proper storage? Check.
- Color unchanged? Perfect.
Need help choosing between an ALP- or HRP-based ELISA? Click here for guidance.
| Buffer/reagents | Product | When to use | Protocol |
| Incubation buffer | Not available on our website | Sample, standard, and detection antibody dilution, for example, when analyzing cell supernatants | PBS (pH 7.4) + 0.05% Tween 20 + 0.1% BSA |
| ELISA diluent | Available as separate product 3652-D2, component of our ELISA Pro kits | Sample, standard, and detection antibody dilution, recommended when analyzing serum/plasma | Ready to use |
| RF-block diluent | Component of our ELISA PathRF kits | Sample, standard, and detection antibody dilution, recommended when analyzing serum/plasma containing rheumatoid factor | Ready to use |
| Apo ELISA buffer (5x) | Available as separate product 3652-M2, component of our Apo ELISA Pro kits. | Sample, standard, and detection antibody dilution when using our Apo ELISA kits | Dilute 5x with distilled or deionized water |
| Assay buffer | Available as separate product 3652-J2, component of certain ELISA Pro kits | Sample, standard, and detection antibody dilution when using certain ELISA kits | Ready to use |
| Sample solvent | Component of our Human ApoB ELISA Pro kit | Sample preperation of plasma and serum samples | Ready to use |
| Standard reconstitution buffer A5 | Component of selected ELISA kits | ELISA standard reconstitution of selected standards | Ready to use: PBS (pH 7.4) |
| Standard reconstitution buffer A7 | Component of selected ELISA kits | ELISA standard reconstitution of selected standards | Ready to use: PBS (pH 7.4) + 0.1%BSA + 0.5 mM DTT |
| Standard reconstitution buffer A8 | Component of selected ELISA kits | ELISA standard reconstitution of selected standards | Ready to use: PBS (pH 7.4) + 1%BSA |
| Wash buffer | Not available in our webshop | Wash steps | PBS (pH 7.4) + 0.05% Tween 20 |
| Wash buffer concentrate (20x) | Component of our ELISA Pro kits and ELISA PathRF kits | Wash steps |
(If crystals have formed, bring to room temperature and mix gently to dissolve) |
| Streptavidin-HRP diluent | Component of our ELISA Pro kits and ELISA PathRF kits | Detection step with HRP conjugate |
Ready to use. Alternative: incubation buffer |
| ELISA substrate: TMB for HRP | 3652-F10, Component of our ELISA Pro kits and ELISA PathRF kits | Substrate to be used in HRP ELISA kits | Ready to use |
| ELISA substrate: pNPP for ALP | 3652-P10 | Substrate to be used in ALP ELISA kits | Ready to use |
| Stop solution | Component of our ELISA Pro kits and ELISA PathRF kits | Stop reaction when you are using TMB substrate | 0.2M H2SO4 |
4. Choose a validated antibody pair
The antibody pair in your assay isn’t just another reagent – it’s the key player determining your ELISA’s sensitivity, specificity, and dynamic range. Choose wisely!
At Mabtech, we validate our antibody pairs for each application, ensuring they bind to the native protein (and yes, that’s important – find out why here).
Another crucial step? Finding the optimal antibody concentrations – this can vary depending on your assay or setup. But if you’re using a Mabtech ELISA kit, good news, we’ve already done the titration work for you. Just follow the recommended concentrations in your ELISA protocol, and you’re good to go.
5. Prepare the ELISA standard
The ELISA standard sets the stage for accurate quantification, so preparing it correctly is non-negotiable! Follow the instructions carefully, as protocols can differ between kits, and even between batches.
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First things first: Store the standard vial at the recommended storage temperature. (Please check the respective datasheet for details. Many of our standards are kept at -20°C, which is different from other kit components.)
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Follow our protocol for the reconstitution of the standard. In most of our kits, this involves adding the appropriate reconstitution buffer to the lyophilized (freeze-dried) protein. You may gently tap the vial if needed. Allow the mixture to sit for the recommended time as specified in the protocol, then briefly vortex the vial to complete the reconstitution.
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Standard curve setup: Our ELISA protocols include optimized dilution recommendations to match the assay’s dynamic range. For best results, accurate pipetting is essential. Small errors can have a big impact!
- Avoid freeze-thaw cycles: If needed for future experiments, prepare aliquots of the reconstituted standard to avoid repeated freeze-thaw cycles, which can degrade the standard.
- And to make your life easier, all Mabtech ELISA kits include our recommended ELISA standard reconstitution buffer – but keep in mind that exact volumes for the resuspension may be batch-specific.
Standard reconstitution is followed by a serial dilution to obtain standard curve at indicated concentrations. This example was taken from ELISA Flex: Human IFN-α (HRP).
6. Sample dilution
Proper dilutions help with accurate results. We never add undiluted samples directly to the ELISA plate. Instead, we recommend at least a 2x dilution. For example, mix 180 µl of sample with 180 µl of dilution buffer.
- Why is dilution important? Proper dilution ensures that sample measurements fall within the standard range of the kit, leading to accurate and reliable results.
- Not sure what dilution to use? Run a small pilot experiment with a few samples to determine the ideal dilution for your study. Find a detailed suggestion in the figure below.
- Choosing the right dilution buffer: Your buffer isn’t just a filler – the optimal buffer blocks interference from heterophilic antibodies, especially in blood or serum samples. This is particularly important when analyzing patient samples. Find out more in the next paragraph.
Example of ELISA sample dilutions, giving 2x, 20x, and 200x dilution of the original sample. Volumes will be sufficient for duplicates.
7. Block heterophilic interference
Heterophilic antibodies can disrupt your ELISA by crosslinking capture and detection antibodies, ultimately leading to false-positive results. The outcome? Inaccurate analyte quantification – nobody wants that.
What are heterophilic antibodies?
Heterophilic antibodies bind to other antibodies. In human samples, these can be:
- Autoantibodies that bind human immunoglobulins
- Human anti-animal immunoglobulin antibodies (HAIA), including human anti-mouse antibodies (HAMA)
How do heterophilic antibodies affect ELISA?
- False positives: Heterophilic antibodies can crosslink the assay antibodies in a sandwich ELISA, generating a positive signal even when no analyte is present.
- False negatives: They may also block assay antibodies, preventing proper detection.
Heterophilic interference is commonly found in plasma and serum samples across species. Prevalence estimates for HAIA in the general population vary widely, between 1% to 80% (Kricka 1999). Particularly high concentrations of heterophilic antibodies and rheumatoid factor (RF) are prevalent in samples from patients with conditions such as rheumatoid arthritis, cancer, or chronic infections.
How to reduce interference?
We recommend you trying our ELISA diluent when analyzing plasma and serum samples. Our ELISA PathRF kits have specifically been developed to eliminate heterophilic interference. These kits feature a recombinant backbone in the detection antibody and a specialized RF-block diluent. In validation experiments with RF-containing plasma, ELISA PathRF kits eliminated false-positive results. Click to learn more.
A) Principle of a sandwich ELISA.
B) Interference by heterophilic antibodies and rheumatoid factor (RF) causing a false-positive signal.
C) Circumventing interference with an ELISA PathRF kit.
8. Include useful controls
Controls help you judge whether you can trust the ELISA results.
Buffer only
- This control consists of sample dilution buffer only. This is your negative control, also called assay background control.
- Use the same dilution buffer for this control, the standard, and the samples.
(Plate) Blank
- Assign a few wells per plate to the plate blank. These wells get blocked and washed as the remaining plate, however, we don’t add any sample or detection reagent, we only add the substrate (and the stop solution, if we are using HRP+TMB) during the final steps of the protocol. The mean value from these wells is subtracted from all measurements during analysis. Typically, the OD of the blank is low, but still, we, at Mabtech, think this is an important control.
Duplicates
- We recommend running all samples and standards in duplicates. This allows for the calculation of CV values and could help uncover potential pipetting errors.
Standard
- Well, the standard is not a classical control, the standard allows for quantification. However, if the standard curve looks like in previous experiments, this is a good indicator of a functional ELISA setup.
- When running more than one plate, preferably include the standard curve on every plate.
Experimental controls
- Which internal controls to include will depend on your specific study setup. However, including a sample with a known concentration (for example, from a previous experiment) is such an extra control that might be nice to have.
A typical ELISA plate layout includes the standard curve and samples in duplicates as well as blank and assay background control (buffer).
9. ELISA analysis
Once your ELISA plate has been read, it’s time to analyze the data. Luckily, most ELISA readers simplify this process, but understanding the basics will help you make the most of your results.
Get to know your reader software
Before diving into the data, take a moment to explore your ELISA reader’s capabilities. Many software programs allow you to create templates, set up automatic calculations, and adjust analysis settings. These features can save you time in future experiments.
Processing OD values
The optical density (OD) values need a bit of cleanup before they’re useful:
- Subtract the reference wavelength (if applicable). Most ELISA readers measure at two wavelengths to reduce background noise. Typically, OD values are measured at the main absorbance wavelength (e.g., 450 nm for TMB, 405 nm for pNPP) and a reference wavelength (e.g., 650 nm). If your reader supports this, subtract the reference wavelength first.
- Subtract the plate blank to account for background absorbance. The plate blank typically consists of wells containing only the substrate (and stop solution).
Check the raw data
Before trusting your results, take a moment to review the raw OD values. Look for inconsistencies like unusually high or low readings (which may indicate pipetting errors), discrepancies between duplicates (CV values >10%), edge effects, or contamination. If something looks off, consider repeating the assay or excluding problematic data points.
Choose a good standard curve model
The accuracy of your sample concentration calculations depends on the curve fit. Avoid using a simple linear model unless your data truly follows a straight line. Instead, choose a 4-parameter logistic (4PL) or 5-parameter logistic (5PL) regression. These provide better curve fits for ELISA’s typical sigmoidal response curves, and finally more reliable results.
Representative 5-parameter ELISA standard curve
10. Practical advice
- ELISA plates
- Do not let plates dry out.
- Strip plates are included in our ELISA Pro kits. They allow you to save unused strips (but pre-coated with the capture antibody) for another experiment.
- Label the strips. (In case they fall out, you can still put them back in the correct order.)
- Use adhesive covers
- They prevent evaporation and contamination during incubation times.
- Washing
- Using an automated ELISA washer is preferable.
- After a final wash step, remove the wash buffer by tapping the plate against absorbent paper before adding the next reagent.
- Pipetting
- Avoid creating bubbles.
- Increase accuracy by using multi-channel, multi-step pipettes, and master mixes whenever possible.
- Change tips between samples and standard dilutions.
- Substrate developing
- Use cold TMB (directly from the fridge).
- Develop the plate in the dark.
- ELISA plate reading
- Make sure that the plate bottom is dry before placing it in the reader.
11. Why we prefer sandwich ELISA with two-step detection
The perfect ELISA setup depends on several factors – your experimental goal, antibody/antigen availability, and analyte size, to name a few. But when in doubt, we always reach for a sandwich ELISA first!
Why sandwich ELISA?
If you're working with low-abundance targets in complex samples (loaded with other proteins), a sandwich ELISA is your best bet for high sensitivity. In contrast, direct and indirect ELISAs may lack the sensitivity needed – or fail entirely – under these conditions.
Why two-step detection?
If sensitivity is key, go for an ELISA with two-step detection – it enhances the signal for more precise measurements. (That said, if you're measuring an abundant analyte like IgG, a one-step detection kit might be the better fit.)
Comparison of a sandwich ELISA with A) one-step versus B) two-step detection.