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ELISA Kit Types

There are several technical methods employed in ELISA type immunoassays. Each type of assay method has specific advantages and may be particularly suited to a sample or target protein type. Slight variations exist for each method. Below are primary methods for ELISA.

 

 

Sandwich ELISA

An immobilized capture antibody first binds the target protein analyte. Subsequently a detector antibody in liquid phase will bind ("sandwich") the capture antibody immobilized target analyte. Detection of the analyte is achieved with catalysis of a substrate mediated by an enzyme conjugated to the detector antibody or a secondary detector complex. Detection output is proportional to the quantity of analyte in the test sample.

AdvantagesDisadvantages
  • Highly specific, requires binding of two antibodies
  • Capable of accurate quantitative measurements
  • Broad dynamic range of detection
  • Very sensitive - secondary detection reagents provide detection signal amplification
  • Specificity - High sensitivity facilitates larger sample dilution, mitigating interference factors
  • Good reproducibility and assay recovery (Provided good technique is employed)
  • Common for, but not limited to, typical protein antigen measurements
  • Complex and longer duration protocol (Shortened protocols exist, but only with highly developed assays due to additional non-specific interactions)
  • More expensive due to additional reagents and complex assay development
  • Assay technique can greatly influence, poor technique yields less adequate date, exacerbated by numerous assay processing steps
  • May not work for small molecules or antigens without sufficient binding epitopes for two antibodies

 

 

  1. Micro-plate wells are pre-coated with capture antibody and blocked.
  2. Sample is incubated in the wells, where the target sample analyte is bound by the immobilized capture antibody.
  3. Wells are washed to remove unbound non-specific sample matrix.
  4. A detector antibody, typically conjugated to an enzymatic reporter molecule (ex. HRP) or an affinity molecule (ex. Biotin, where a secondary detector complex is used) is incubated in the wells where it binds to the captured analyte.
  5. Wells are again washed to remove unbound detector antibody.
  6. Colorimetric, fluorescent or luminescent detection is then performed. The enzymatic reporter (conjugated to the detector antibody or to a secondary detector complex added in an additional step) catalyzes a detection substrate producing a signal which is proportional to the quantity of analyte.

 

Indirect (Reverse-Phase) ELISA

Specific target antigen is coated in wells. The immobilized antigen acts as "bait" where the assay target analyte then binds and is subsequently detected. Enzymatic detection output is proportional to the quantity of analyte in the test sample.

AdvantagesDisadvantages
  • Shorter assay protocol, simple method provides a robust assay, less dependent on technique
  • Less reagent cost
  • Good for quantitative measurements of samples containing relatively abundant analyte
  • Good reproducibility from small number of assay processing steps
  • Flexibility from common detection reagent set
  • Common for, but not limited to, immune response or other infectious disease measurements
  • Specificity dependent on immobilized "bait" antigen
  • Shorter dynamic range, more suited to qualitative measurements
  • Avergage sensitivity, although novel amplification methods are available with extra cost and development

 

 

  1. Micro-plate wells are pre-coated with target specific antigen ("bait") and blocked.
  2. Samples are incubated in the wells, where target analyte in the sample binds the immobilized target antigen.
  3. Wells are washed to remove unbound non-specific sample matrix.
  4. An analyte specific detector antibody conjugated to a reporter (ex. HRP) or affinity molecule (ex. Biotin, where a secondary detector is used) is incubated and binds the target analyte.
  5. Wells are washed to remove unbound detector antibody.
  6. Colorimetric, fluorescent or luminescent detection is then performed. The enzymatic reporter (conjugated to the detector antibody or to a secondary detector complex added in an additional step) catalyzes a detection substrate producing a signal which is proportional to the quantity of analyte.

 

Competitive ELISA

Typically a variation of indirect (or reverse-phase) type assays wherein target analyte in samples competes for detector antibody binding with immobilized target analyte. In some cases a target analyte competes for binding in solution with a labeled standard for immobilized capture antibody binding sites. In either orientation, the method produces an inhibition of the detector and the output signal is inversely proportional to the quantity of analyte in the test sample.

AdvantagesDisadvantages
  • Typically shortest and easiest assay protocol, simple method provides a very robust assay
  • Very low reagent costs
  • Good reproducibility from small number of assay processing steps
  • Common for, but not limited to, small molecule and biochemical measurements
  • Development can be difficult to optimize competitive assay components
  • Shortest dynamic range and provides primarily qualitative measurements
  • Lowest level of sensitivity, although novel amplification methods are available with extra cost and development

 

 

In-Cell ELISA

Also termed, In-Cell-Western or Cell Based ELISA, this novel method uses whole cells in-situ and highly specific detection antibodies for the relative measurement of protein expression levels.

AdvantagesDisadvantages
  • Greater through-put than western blot
  • More quantitative and reproducible than western blot
  • Reduced handling time for sample preparation compared to western blot, no lysate prep
  • Can easily be Duo-plexed if using flourescent detection
  • Relative or absolute protein quantity measurements can be obtained
  • Specificity completely dependent on primary detection antibody (same as western blot)
  • Slightly more costly than western blot

 

  1. Micro-plate wells are pre-coated with target analyte and blocked.
  2. Sample is added to the micro-wells.
  3. Detector antibody is added and mixes with the sample. Detector antibody can be conjugated to a reporter enzyme (ex. HRP) or affinity molecule (ex. Biotin, where a secondary detector is used) or detected using a secondary detection conjugate. The analyte in the sample competes for binding of the detector antibody with the immobilized analyte.
  4. Wells are washed to remove unbound detector antibody and sample matrix.
  5. Colorimetric, fluorescent or luminescent detection is then performed. The enzymatic reporter (conjugated to the detector antibody or to a secondary detector complex added in an additional step) catalyzes a detection substrate producing a signal which is proportional to the quantity of analyte.