ELISA

ELISA is an enzyme-linked immunosorbent assay, widely used in biology as a research method for quantifying proteins or other antigens in an unknown solution. The sensitive and straightforward nature of the assay makes it useful for an array of applications, such as testing blood samples for the presence of infectious disease antibodies, testing the binding between a newly isolated antibody and its target protein, or a variety of other tests that rely on antibody-antigen interactions.

ELISAs are sandwich-type immunoassays carried out in 96-well plates. A specific antibody for the antigen of interest is bound to the bottom of the well. The sample is then placed in the well and if the antigen of interest is in the sample, it will bind to the immobilized antibody. The bound antigen is then detected using a second, labelled antibody. The detection antibody is conjugated to an enzyme that can cause a color change when a substrate is added, allowing detection with a plate reader like the Ao Absorbance Microplate Reader. For detection, HRP-secondary antibodies like those for chemiluminescent Western blots can also be used.

What are ELISAs used to detect?

ELISAs can be used to look for the presence or absence of a protein within a mixture, to compare relative amounts of a protein, or for other experiments that rely on native protein-antibody interactions.

How to use ELISAs to look for proteins

The technique starts with a 96-well plate where the material of the wells allows proteins to adhere to the surface. Immobilization allows the antigen to be bound by its detector-antibody and then washed of any unbound antibodies, followed by measurement of the antigen. An enzyme linked to the detection antibody will interact with a substrate added in the final step to produce a signal proportional to the antigen (Figure 1).

Four ways ELISAs can be set up in terms of antibodies and antigens:

  1. Direct ELISA: the unknown sample containing the antigen of interest is adhered directly to the well, followed by detection with a primary antibody.
  2. Indirect ELISA: begins the same way as a Direct ELISA, but the primary antibody is not linked with the enzyme and a secondary antibody must be used for detection.
  3. Sandwich ELISA: depicted in Figure 1. This starts with an antibody that is adsorbed to the plate first; then, the antigen-containing sample is added and the antigen
    is captured by the immobilized antibody and detected similar to the direct or indirect methods.
  4. Competitive (or inhibition) ELISA: can be setup similarly to the other three, but instead of the antibody being labeled, the antigen is labeled. The labeled antigen is added after the unknown samples to see how much unlabeled antigen in the samples competes with the labeled antigen. Unlike the other three types, higher signal from the competitive ELISA means that there is less competition and thus less antigen in the unknown.

ELISAs are usually performed in 96-well plates, where many conditions and repeat samples can be tested at once, and then measured using a micro plate reader.

An absorbance-based plate reader, like the Azure Ao Absorbance Microplate Reader can measure the absorbance of light at chosen wavelengths relevant to whatever is produced from the enzyme reaction, providing quantitative differences between positive and negative samples.

The Ao Absorbance Microplate Reader is compatible with numerous 96-well plate assays including nucleic acid quantification, NAD/NADP conversion, colorimetric protein assays, such as Bradford and Lowry.

Multi-well plates can also be imaged in the Azure Digital imagers and the Azure Sapphire Biomolecular imager, facilitating in-cell Westerns and other applications. AlphaSpot Pro software includes an analysis module that facilitates the analysis of plate-based assays including presence/absence detection and normalization.

In the Application note below, we describe use of the Azure Ao Absorbance Microplate Reader for measuring and analyzing results from an ELISA.

Materials and Methods

The Ao Absorbance Microplate Reader was used for all measurement and analysis.

To demonstrate the ELISA protocol, a Histamine EIA Kit (Oxford Biomedical Research) was used to measure histamine levels in biological samples. The kit provided PBS, wash buffer, substrate, histamine-HRP conjugate, histamine standards, and a 96-well plate pre-coated
with monoclonal anti-histamine antibody. Unknown samples were prepared by diluting food samples in the kit provided PBS.

The experiment was a competitive indirect ELISA. The plates were pre-coated with anti-histamine antibody and the samples were incubated in the wells to allow binding of the histamine to the antibody. Horseradish peroxidase (HRP) labeled histamine was added with the samples, creating competition between binding of the labeled histamine conjugate and any unlabeled histamine occurring in the samples. After incubation and washing, the substrate was added to the wells to allow the reaction of the HRP enzyme with the substrate to produce a colored substance. The reaction was stopped using 1N Hydrochloric Acid and the change in color produced a change in absorbance that was read at 450nm with a substrate reference read at 630nm using the Azure Ao Microplate Reader.

Results

How to perform ELISAs with the Ao plate reader

The purpose of this experiment was to demonstrate the ease of performing and reading ELISA assays using the Azure Ao Absorbance Microplate Reader. To demonstrate the ELISA protocol, the Histamine EIA Kit (FS35) from Oxford Biomedical Research was used to measure histamine levels in food samples using a competitive indirect ELISA.

ELISA figure 2
ELISA figure 3 a and b

Taking measurements and retrieving both raw and analyzed data is simple and intuitive with the Azure Ao Microplate Reader. Just perform the ELISA according to the manufacturer’s protocol, adjust the pre-programmed ELISA protocol as needed, load the plate in the
microplate reader and start the protocol. After reading the plate, the software opens the REPORT tab with raw data measurements. The standard curve can be viewed and printed by selecting “CURVE” in the REPORT tab (Figure 5).

The curve-fit and axes can be changed through the PROGRAM tab or the button beside the curve plot, even after the plate is read. The antigen concentrations are automatically calculated using the standard curve (set to point-to-point for this assay). Results can be viewed and exported by selecting “ANALYSIS” or “RAW DATA” in the REPORT tab. Figure 4 shows both raw data and analyzed results interpolated from the standard curve.

ELISA figure 4 a and b
ELISA figure 5

Conclusion

The Ao is a convenient plate reader for the classic ELISA

ELISAs are valuable in the scientific research community, and the Ao plate reader provides performance and convenience that allows for the best quality research. ELISAs can also use a variety of enzyme and substrate combinations other than horseradish peroxidase, and the
Ao plate reader has the flexibility to measure the majority of commonly used colorimetric reagents.

Data analysis is automatically performed by the Azure Ao plate reader software, and results are easily viewable and exportable using the large touch screen interface, minimizing the need for outside analysis or calculation software.

REFERENCES

  1. Goldsby RA, Kindt TJ, Osborne BA, Kuby J. ELISA. In: Immunology, Fifth Edition. Freeman, W.H. & Company. 2002. pp 148-149
  2. William J. Antigen Measurement Using ELISA. In: The Protein Protocols Handbook. Humana Press. 2009. pp 1827-1833
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Documents

Document TypeDescription
Application NoteMeasuring Histamine Levels in Food SamplesDOWNLOAD
Application NotePerforming an ELISA with the Ao Absorbance Microplate ReaderDOWNLOAD

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