Improving Reproducibility between your ELISA experiments:

Sandwich ELISA – Is the most common ELISA used for complex protein samples as only the specific antigen becomes immobilized rather than the entire sample of proteins. Two antibodies are required for sandwich ELISA which bind different epitopes. The first antibody is bound to the plate and is known as the capture antibody. The secondary antibody commonly referred to as the detection antibody, this detects the immobilized antigen which is bound to the capture antibody. Both monoclonal and polyclonal antibodies can be used for sandwich ELISA.

2. Biotin Signal Amplification:

Streptavidin-based amplification systems are typically applied in fluorescence imaging to detect Biotinylated biomolecules such primary and secondary antibodies, ligands and toxins, or DNA probes for in situ hybridization for improved detection sensitivity. With a simplified process, streptavidin-based detection provides signal amplification for medium- and low-abundance targets.

3. Enzymatic Detection:

 Horseradish peroxidase (HRP) and alkaline phosphate are the most widely used enzymes for ELISA detection (AP). HRP is the most often used because of its compact 40kDa size, which makes it easy to couple to antibodies and interact with a variety of substrates. Although AP is utilized, its size of 140 kDa makes conjugation problematic. It also has a number of concerns with stability.

4. Substrate Selection:

Enzymatic detection involves the catalysis of a substrate to form a colored signal that may be evaluated with a spectrophotometer. Colorimetric substrates produce a soluble colored product that builds up over time in proportion to the amount of enzyme in each well. TMB, OPD and ABTS are colorimetric substrates for HRP. BCIP/NBT and pNPP are examples of AP substrates.

1. Pipette reagents and samples into the center of each well but do not touch the bottom or the well.
2. Tap the plate gently after pipetting to ensure thorough mixing of reagents.
3. If your sample is viscous, please pre-rinse the pipette tip with the reagent to compensate for surface tension. Then wait a moment to allow the volume to reach equilibrium before removing the pipette tip from the reservoir.
4. Inadequate or uneven fill volumes in the wells is a clear indication that the pipette needs to be calibrated. Check the pipette function and recalibrate if necessary.
5. Always be sure to change pipette tips between reagents, standards, and samples to avoid carryover or cross-contamination.
6. Check to be sure that the pipette tips are fitted properly. If a pipette tip that is not fitted properly, it may not draw or dispense the volume accurately.

6. Washing Technique:

1. If using an automated washer, aspiration apparatus, or a multi-channel pipette, always be sure that each tip is dispensing and aspirating properly.

2. After the last washing, decant any remaining Wash Buffer by inverting the plate and blotting it dry by tapping it firmly against clean paper towel on a hard surface. Washing the wells too rapidly or too slowly may result in poor precision. Do not allow the wells standing to dry. Proceed immediately to the next step in the kit insert.
3. Using less Wash Buffer might often lead to poor CVs. Be sure to wash the wells with the volume specified in the kit manual/IFU to ensure that each well has been thoroughly washed.
4. If the Wash Buffer has run out, please don’t use Wash Buffer from another kit because not all Wash Buffers are general. You can contact the Technical Service of the manufacturer for assistance.
5. Don’t skip or shorten the soak time in the washing procedure, otherwise it may lead to poor precision. Always follow the directions in the manual for optimal performance.
6. It is recommended that the number of washing steps and cycles specified in the kit manual be adhered to. Changing the number of washing steps or cycles can affect the precision of the assay.

7. Reservoir:

 Use separate reservoirs for each reagent to avoid contamination. Some analytes are highly susceptible to contamination (e.g., saliva, oxidizing reagents, etc.). Refer to the manual. Take necessary precautions to protect the reagents.

8. Plate Sealer:

 A reused plate sealer may contain residual from the previous step which can contaminate the current component in the well, thus leading to poor precision. Using a new plate sealer for each incubation is recommended.

9. Incubation:

 To achieve precision in your assay, be sure to adhere to strictly follow the incubation guidelines indicated in the assay manual/IFU. Temperature, time, and shaking (if required) can all greatly affect the status of the assay if they are not followed. Reducing incubation time can prevent everything from getting to equilibrium and may result in different absorbance values. Increased incubation may lead to abnormally high noise and signals. Large temperature changes can also affect binding kinetics. If shaking is suggested, it is always best to follow that guideline. Accuracy and consistency of handling during plate incubation really can help enhance assay performance. Things like incubation at room temperature can also lead to poor precision. Ambient room temperature should be consistent to achieve greater degree of reproducibility.