Ensure that you are using an ELISA plate, not a tissue culture plate. Ensure that you are using an ELISA plate, not a tissue culture plate. Dilute antibody in PBS. Ensure correct preparation and incubation time for both coating and blocking steps.

Three most common ELISA Challenges

• Weak or Low Signal Intensity. Are you repeatedly getting readings below the lower limit of absorbance?
• High Background.
• High Well-to-Well Variation.

Pipetting technique

1. Use the correct pipette that is within the range suggested by manufacturer.
2. Confirm tip is firmly seated on the pipette.
3. Confirm there are no air bubbles while pipetting.
4. Change tips between each standard, sample, or reagent.
5. Use different reservoirs for each reagent.

ELISAs have the potential for high background which hurts the sensitivity of the assay. This could come from TMB substrate contamination, poor washing steps or cross reactivity. High background can lead to data loss or false negative/positive data.

Causes of false-negative EIA results include the following:

• Technical error.
• Testing during the window period.
• Decreased host immunoglobulin production such as in a common variable immunodeficiency and advanced AIDS.
• HIV-2 if tests to detect HIV-1 only are used.
• Non–clade-B HIV-1 or type N or O strains of HIV-1.

Common causes of a false positive ELISA include: administration of flu vaccine, presence of HLA-DR antibodies in multigravada women, presence of rheumatoid factor, positive RPR test, hypergammaglobulinemia (e.g. multiple myeloma) and autoimmune hepatitis.

There tends to be two main reasons for high background developing: plate washing and plate blocking.

How to optimize your ELISA experiment

1. Capture Antibody Concentration. Prepare different concentrations of the capture antibody in coating buffer.
2. The Blocking Buffer.
3. The Standard Diluent.
4. Sample Concentration.
5. The Detection Antibody Concentration.
6. The Enzyme Conjugate Concentration.
7. Signal Detection.

An ELISA blocking buffer is a solution of irrelevant protein, mixture of proteins, or other compound that passively adsorbs to all remaining binding surfaces of the plate that are not occupied by the coated protein.

One method of reducing this problem is to use affinity purified or cross-absorbed polyclonal antibodies. To increase assay sensitivity, the detection method for an ELISA can be switched from direct to indirect detection using a polyclonal antibody.

There are some variations in plate coating protocols, but they typically involve the following steps: Aspirate (suck-off blocking solution). Residual volume is critical in this step! At the most basic level, a multichannel pipette is enough to coat a single plate.

Plate coating is a demanding process. Most demands come from the fact that it is a production process, thus involving the preparation of many plates per day. The most important demands are: Throughput: Can range from a few dozen plates per day for homebrew ELISA assays to hundreds or even thousands of plates per day for kith manufacturers.

In a traditional (direct coating) ELISA, antigens are directly attached to the plate by passive adsorption, usually using a carbonate/bicarbonate buffer at pH >9. Most but not all proteins bind tightly to the polystyrene surface of microplates in alkaline conditions.

However, ELISA coating is typically applied to many plates at a time and is normally a production process. Therefore, automation is highly desired. Plate coating is a demanding process. Most demands come from the fact that it is a production process, thus involving the preparation of many plates per day.