Optimizing ELISA

ELISA – Enzyme-Linked Immunosorbent Assay – is a commonly used biochemical technique for the evaluation of an antigen or antibody in a sample. This technique is used in a wide range of applications, including: clinical diagnostics, plant pathology, the detection of food allergens and drug screening. Many laboratories rely on ELISA for qualitative and quantitative day-to-day analysis of distinct proteins in complex sample mixtures. ELISA testing is performed in two forms: direct and indirect.

Direct vs indirect ELISA

Both forms of ELISA testing are quite simple. Samples containing an unknown amount of antigen are immobilized by absorption on a solid support, typically a 96-well microplate. A non-specific protein is then added to block any remaining binding sites. Then a primary antibody is added, which will bind to any recognized antigens. If the primary antibody is directly linked to an enzyme used for detection the antigen can be quantified in a process known as direct ELISA. If the primary antibody is not linked to an enzyme used for detection, then indirect ELISA must be used. In indirect ELISA an enzyme-linked secondary antibody is added which will bind to all available primary antibodies. This binding will only occur if the primary antibody is linked to a suspected antigen: this is known as indirect ELISA. Following each antigen or antibody addition step – in both direct and indirect ELISA tests – the plate wells are washed with buffer to remove any unbound antigen or antibodies. After the final wash step, a chromogenic enzyme substrate is added. If the enzyme linked to the antibody has not been washed away due to direct or indirect binding to antigen molecules, then the addition of the chromogenic enzyme substrate will cause the solution in the wells to change color. This colorimetric response, due to the enzyme reacting with its substrate, is proportional to the amount of antigen in the sample.

Environmental conditions

Scientists will often prepare ELISA experimental buffers and sample solutions in advance, storing them in a refrigerator. All material used in ELISA testing should be equilibrated to room temperature prior to use. Solution temperatures can affect binding, washing, enzyme reaction and even pipetting accuracy.

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Choosing the right tools

ELISA testing involves multiple pipetting steps. Selecting the correct lab tools, using good laboratory procedures, and precise timing are all essential for accurate results. There are numerous practices to help improve pipetting performance: liquid viscosity properties, sample throughput requirements, and the required experimental accuracy and precision. Before performing ELISA experiments, careful consideration should be given to the sample properties. Sticky and viscous samples – such as bodily fluids or highly concentrated protein samples – can be difficult to pipette. Often, residue will remain on the interior of the pipette tip, leading to variable measurements. Using specialty tips such as low retention or wide orifice tips will alleviate such problems. Users of air displacement pipettes may also find reverse pipetting to be advantageous. Positive displacement pipettes are also recommended for use with difficult solutions. Reproducibility can be further enhanced by preparing solutions using an electronic single channel pipette with an automatic mixing function.

Being cautious

It is essential to avoid distractions while performing ELISA. Paying attention to detail is very important, especially when pipetting multiple micro samples into 96 wells. Non-uniform or inadequate plate washing increases the risk of variability and the chances of false positives and negatives.

Cross-contamination, which is often caused by incorrect sample labeling or poor pipetting practice, can also increase the risk of false positive results. Cross-contamination can be virtually eliminated by careful labeling and handling of samples. Pipettes should always be cleaned regularly – especially whenever contamination is known to have occurred – and tips should be changed between sample and buffer additions.

For consistency, multi-channel pipettes should be used to simultaneously dispense samples into an entire column of a microplate, taking care not to introduce contamination when using the same tips for repeat reagent additions. Proper pipette timing should be used here to ensure that samples and buffers interact with the plate and samples for a consistent amount of time. For peace of mind, 96-well pipetting systems can dispense into every well of the microplate simultaneously, increasing speed, ensuring consistent sample/buffer additions, and eliminating the risk of missed wells.

Timing is everything

The final stage of ELISA – digestion of the enzyme substrate – is time critical and has a big impact on quantitative assays. If the duration of the reaction is not consistent from row to row across the microplate, the linearity of the standard curve generated and the accuracy of the concentrations reported will be affected. A 96-well pipetting system provides the best possible consistency, enabling simultaneous dispensing of the enzyme quenching solution into every well. Alternatively, an 8- or 12-channel pipette can be used in single dispense mode; multi-dispensing should be avoided as it is less precise. When using an 8- or 12-channel pipette, making use of a timer to ensure the duration of the enzymatic reaction is the same from well-to-well is very important. Timing is not quite so critical with qualitative ELISA testing – which simply determines if an antigen is present. For these qualitative assays, manual or electronic multichannel pipettes with or without the multi-dispense function are ideal for these assays.

Ergonomic pipetting

With so many pipetting stages involved, it is also important to consider ergonomics. Uncomfortable pipetting can lead to repetitive strain injuries and affect pipetting performance. Scientists should use modern pipettes with lower pipetting forces and when possible minimize repetitive pipetting actions. The use of multichannel pipettes or 96-well pipetting systems, where appropriate, decreases the number of pipetting actions performed, lessening hand fatigue and improving reproducibility.