ELISAs are used to detect and quantitate peptides, proteins, antibodies and hormones in biological specimens.
Antibodies (Ab) or antigens (Ag) are adsorbed to a solid surface and bind with high affinity their complementary reagents that are typically conjugated to enzymes. The enzymatic reaction triggered by appropriate substrates allows the quantitation of the analyte of interest.
ELISAs are useful tools because they allow for the rapid screening or quantitation of a large number of samples. Other techniques have been developed, but ELISA remains popular because of the ease of performance of the assay, accuracy, and the low cost.
This guide is divided in eight sections that follow the traditional development of an ELISA.
|Figure 1. A schematic description of the most common type of ELISA, the sandwich ELISA.
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1. ELISA Design
This section introduces the most common ELISA formats, presenting their advantages and disadvantages.
Suggestions for optimizing the coating of the solid phase are listed in this section.
Wash out excess reagent to reduce background signal and increase the signal-to noise ratio (SNR). Guidelines for optimizing the washing buffer and the washing procedures are suggested.
4. Blocking and stabilizing ELISA plates
The critical aspects to consider for preventing non-specific-binding (NSB) are discussed. Kem-En-Tec Diagnostics’ Synthetic Blocking Buffer and WellChampion are ideal reagents for blocking and stabilizing ELISA plates.
5. Dilution of samples, standards & controls
Specimens, standards and controls need to be diluted in specific buffers before adding them in the microwells. Kem-En-Tec Diagnostics’ SamplePLUS2, Effect Diluents, and Protein-StabilPLUS are the ideal diluents for reducing cross reactions and interferences. The use of Ab’s is also described in this section.
6. Dilution and stabilization of the conjugate
Specific diluents are used for achieving the optimal concentration of conjugated Ab’s or Ag’s to be added in the assay. Most of Kem-En-Tec Diagnostics’ diluents also have stabilizing properties to prevent loss of activity. The Alkaline Phosphatase (AP) and Horseradish Peroxidase (HRP) labels are also described.
7. Enhanced conjugates: amplification systems
Kem-En-Tec Diagnostics has developed an amplification system based on the biotin-streptavidin binding, which enables 100 fold signal enhancement.
Adding the substrate is the last step in an ELISA. This guide focuses on chromogenic substrates for AP and HRP. The broad range of Kem-En-Tec Diagnostics’ pNPP and TMB substrates is described in details for the highest level of optimization.