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ELISA assay test protocols, methods & kits

What is an ELISA assay?

ELISA (Enzyme-Linked Immunosorbent Assay) is an antibody based technique to measure levels of proteins, peptides, hormones or chemicals in biological samples. In a sandwich ELISA assay a capture antibody is immobilised onto the surface of a 96 well plate, this is followed by the addition of a sample which contains the analyte of interest and a complex between the antibody and sample is formed. Following an incubation step the well is washed using a wash buffer to remove unbound analyte. Detection occurs following the addition of a detection antibody which is conjugated to enzyme. Following incubation another wash step is carried out to removed excess antibody and unspecific bound proteins. Substrate is then added and a colorimetric change occurs. The amount of analyte in the sample correlates to the increase intensity in colour change. Finally stop solution is added. Once the protocol is complete, samples are then analysed on an ELISA plate reader and a software program is used to plot and calculate results.

Figure : Cartoon of 96 well polystyrene ELISA plate

ELISA assay plates

ELISA assays are carried out in either 96 or 384 well polystyrene plates. Proteins and antibodies can immobilize to the 96 well ELISA plate following incubation. In a Sandwich ELISA assay a capture antibody is immobilized to the ELISA plate. However, in a Competitive ELISA assay, the analyte of interest is bound to the ELISA plate. The ELISA plate is then blocked with a BSA blocking solution to prevent the binding of non-specific proteins during the ELISA assay.

Binding of antibodies or analytes to the plates allows for wash steps with a wash buffer and thus the removal of non-specifically binding analytes.

ELISA plates are flat bottomed to allow the plate reader to read the absorbance in an ELISA plate reader at 450nm.

EIA vs ELISA

An immunoassay is an assay that uses antibodies to detect an antigen of interest. An EIA (Enzyme Immunoassay) is an assay that uses an enzyme conjugated to a detected antibody to allow for detection and quantification. Two examples of an EIA are a Western Blot and an ELISA. A Western Blot is an EIA that used Nitrocellulose or PVDF to immobolize proteins. This is following by the addition of a primary antibody to bind the protein of interest, followed by the incubation with a enzyme conjugated-secondary antibody to detect your analyte of interest. In an ELISA assay, the capture antibody is immobilized onto a polystyrene plate, followed by incubation a sample which contains the analyte of interested, followed by detection with a detection antibody and colorimetric change using TMB as a substrate.

Figure : A Human IL-6 ELISA kit with a range of 4.69-300pg/ml and sensitivity of <2.813pg/ml

ELISA assay sensitivity & range

An ELISA assay allows researchers to determine the amount of analyte they have in their sample (serum, plasma, supernatant, milk, urine) within a defined range using a set of known standards. When carrying our an ELISA assay a known concentration of analyte is use as a benchmark of the amount of analyte in a sample these are called the standards. During an ELISA assay, a stock of the standard is provided, usually in ng/mL or pg/mL amount, this stock is then diluted 6-7 fold to provide a range of known concentrations of an analyte in a volume. When plotting values a standard curve is created and unknown concentrations of samples are calculated versus these values.

High Sensitivity Human ELISA Kits

Analyte Range Sensitivity

1.87 pg/ml - 60 pg/ml

0.97 pg/ml

0.31 pg/ml - 10 pg/ml

0.31 pg/ml

1.56 pg/ml - 50 pg/ml

0.8 pg/ml

1.56 pg/ml - 50 pg/ml

1.30 pg/ml

0.78 pg/ml - 25 pg/ml

0.75 pg/ml

0.78 pg/ml - 25 pg/ml

0.69 pg/ml

Types of ELISA Assays

There are 6 main types of ELISA assays that researchers use in their day to day studies, the most common being the Sandwich & Competitive ELISA assays, followed by ELISpot and Indirect ELISA assays.

> Sandwich ELISA assay

Sandwich ELISA assays are the most common form of ELISA and are named after the formation of the capture and detection antibody sandwiching the analyte in between for detection. As mentioned above, Sandwich ELISA assays have their capture antibody immobilized onto a polystyrene ELISA plate. The sample is then incubated in the well of the ELISA plate, followed by a wash step. A enzyme linked detection antibody is then added followed by a further incubation and finally the addition of a substrate and stop solution to measure analyte levels.

Sandwich ELISA Assay Protocol

> Competitive ELISA assay

Competitive ELISA assays are mostly used to detect hormones. In a competitive ELISA assay the analyte of interest in immobilized onto the polystyrene ELISA plate. The competitive ELISA is named after the process the analyte in the sample competing with the immobilized analyte on the plate for the capture antibody. Therefore increased amounts of analyte in a sample will result in decreased signal and is inverse to the graph that you see in a Sandwich ELISA assay.

> ELISpot ELISA assay

An ELISpot assay is commonly used for vaccine development, veterinary research, monocytes/macrophages/dendritic cells characterization. The principle of an ELISpot is similar to a sandwich ELISA assay, whereby a plate is coated with capture antibodies. Cells are then incubated in the ELISA plate for up to 3 hours, this can depend on application. Cytokines produced by the cells are then bound by the capture antibody immobilized onto the ELISA plate. Cells are then washed from the ELISA plate and a detection antibody is added, followed by a substrate and stop solution to allow for the detection of cytokines.

> FluoroSpot ELISA assay

A FluoroSpot ELISA assay is identical to an ELISpot ELISA, however, instead of using a enzyme-linked detection antibody, the detection antibody is conjugated with a fluorophore to allow for detection and analysis.

> Indirect ELISA assay

In an indirect ELISA assay the analyte of interest is bound by the primary capture antibody. A secondary antibody is then used to bind the primary antibody. This is similar to the method of a Western Blot assay.

> Direct ELISA assay

The direct ELISA technique is a assay, whereby, an enzyme-labelled antibody is used to bind to an analyte in a solution. Once bound, the enzyme-labelled antibody can react with a substrate to provide a colour change, allowing for the quantification of the analyte. This removes the need for a secondary detection antibody as the capture antibody carries out both functions.

> Multiplex ELISA assay

A multiplex ELISA assay allows researchers to analyse multiple analytes in a single sample. This is very useful when researchers are looking to measure multiple cytokines in a sample simultaneously. Multiplex ELISA assays can be carried out through multiple formats, including flow cytometry, plate based multiplex or using PVDF or Nitrocellulose membranes.

Multiplex ELISA Panels

> CLIA assay

CLIA assays are similar in principle to a sandwich assay, however, instead of using a chromogenic substrate for the detection of samples, CLIA assays are chemiluminescent based. In a CLIA assay, the detection antibody converts a substrate to light. The amount of photons generated are proportional to the amount of analyte in a sample. To measure the amount of sample in an assay Luminescence is measured in Relative Light Units (RLU) by a luminometer.

CLIA assays are typically used for the detection of low concentration analytes (limit of detection = zeptomole 10-21 mol).

> In-Cell ELISA

An In-Cell ELISA is an indirect ELISA technique and is performed using cells that have been plated and cultured overnight onto a polystyrene ELISA plate. Cells are then fixed, permeabilized and blocked. Target proteins are then detected using a primary antibody which is either enzyme conjugated or fluorescently tagged. Fluorescently tagged antibodies allow for the detection by either fluorescent plate reader or microscope, whereas the enzyme conjugated secondary antibodies would allow for detection by a plate reader.

Detection strategies for ELISA

The detection step in an ELISA is last step to measure the amount of analyte that you have in your sample. The signal generated during the detection step is proportional to the amount of an. There are three options for the detection of analytes in an ELISA; radioactive (Radioimmunoassay) or fluorescent tags or using a chromogenic substrate.

Chromogenic is the most popular and most widely used technique for ELISA detection and involves the horse radish peroxidase (HRP) substrate TMB (3, 3’, 5, 5’-tetramethylbenzidine) which yields a blue colour when oxidized and turns to yellow following the addition of sulphuric acid. Which allows samples to be read at 450nm on an ELISA plate reader.

Other detections methods such as chemiluminescence can be used based on HRP using Luminol as a substrate which emits light at 425 nm.

Ready to use pre-coated ELISA Kits and development antibody pair ELISA kits

At ELISA Genie we provide a range of ready to use pre-coated ELISA kits and ELISA development kits for the detection of cytokines, chemokines, hormones, signalling proteins and a 1000’s of other biomarkers.

ELISA Genie ELISA Kits

ELISA Genie ELISA kits contain pre-coated ELISA plates, capture and detection antibodies, wash buffer, standard dilution buffer, TMB, stop solution. We also offer a kits for a range of species including Human, Mouse, Rat, Porcine, Bovine and many more.

High Sensitivity PharmaGenie ELISA Kits

PharmaGenie ELISA Kits from ELISA Genie are high quality ELISA Kits designed to meet the needs of pharma and biotech research. Focussing on high quality monoclonal antibody pairs & reagents that have been been validated according ISO 9001:2000 quality systems, PharmaGenie ELISA Kits are excellent assays to help discover our future.

  • Accuracy and reliability are guaranteed as all our reagents have been validated according ISO 9001:2000 quality systems
  • Recognises both Natural and Recombinant antigen Specificity
  • No cross reactivity with other human cytokines tested
  • Standard Calibration to NIBSC
  • Development ELISA Kits (Antibody Pairs)
  • Antibody pairs allow research

Development ELISA kits (antibody pairs)

Development ELISA kits allow researchers to create their own ELISA plates. Development ELISA kits come with antibody pairs (matched capture & detection antibodies) and buffers. This allows researchers to coat and plate their own ELISA kits. ELISA Genie provide a range of high quality development ELISA kits called SuperSet ELISA kits.

  • Highly optimised monoclonal antibody pair.
  • High quality purified recombinant protein to generate consistent standard curves.
  • Reagents have been validated according ISO 9001:2000 quality systems.
  • Natural and Recombinant antigen Specificity.
  • No cross reactivity with other cytokines tested.
  • Standard Calibration to NIBSC.
  • ELISA Kits developed with pharmaceutical and biotech research sectors in mind.
  • Different Kits Sizes for Efficient Research!

Key SuperSet ELISA Development Kits

SuperSet Development ELISA Protocol

Figure: Overview of the protocol steps for a SuperSet Development ELISA protocol using antibody pairs.

Benefits and features of Developement ELISA kits and pre-coated ELISA Kits

Features Development ELISA Kit Pre-coated ELISA Kit Multiplex ELISA

Read out

HRP-TMB

HRP-TMB

PE/APC

Incubation time

2-3 hours

2-3 hours

2 hours

Sensitivity

pg/ml

pg/ml

pg/ml

Number of targets per well

1

1

24

Instrumentation required

ELISA plate reader

ELISA plate reader

Flow Cytometer

Antibody types for ELISA

ELISA kits are made using either Monoclonal or Polyclonal antibodies for the detection and capture antibodies. Monoclonals provide the advantage of recognizing a single epitope, therefore provide a accurate analysis of a particular antigen. However, polyclonals have the advantage of capturing increased amounts of antigen. Lately, recombinant monoclonal antibodies have been used to create ELISA kits, thus providing increased specificity and consistency.

The ELISA Genie PharmaGenie range of ELISA kits are made using high quality monoclonal antibodies for key cytokine targets including IL-1 beta, IFN-gamma, IL-2, IL-4, IL-6, CXCL8/IL-8 and many more targets.

Blocking Buffers

> Blocking buffer optimization

In order to prevent non-specific binding of proteins to an ELISA assay plate, blocking buffers are used to coat a plate. The binding capacity of a ELISA plate is higher than the amount of protein coated (Capture Antibody/Antigen) coated onto the plate. Therefore the remaining area must be blocked to prevent non-specific binding of antibodies or other proteins during subsequent incubation steps. Therefore a blocking buffer is used using a protein that will not be bound or form complexes with other proteins or detection antibodies in subsequent steps. Therefore, the blocking buffer increases ELISA sensitivity as it prevents binding of non-specifics proteins, reduces background noise and therefore increases signal-to-noise ratio.

During ELISA development several different blocking buffers must be tested to optimize the assay and improve signal to noise ratio. Factors that can influence non-specific binding of proteins is the formation of protein:protein interactions. When optimizing a block buffer, the key is to optimize for an increase in signal to noise ratio, this is found by using a control well without the addition of the sample with target analyte.

When optimizing blocking buffer it is also important to not use excessive concentrations of blocker, which can inhibit antibody-antigen interactions or potentially inhibit enzyme activity, thus reducing the signal to noise ratio. When optimizing for blocking, a few buffers may be tested in order to optimize for the best signal.

> Coating buffer

An ELISA coating buffer is used to immobilize proteins/analytes or antibodies on microtitre plates. Key factors in immobilization of analytes/antibodies on to microtitre plates can be the pH of the coating buffer. Selecting a coating buffer between pH 7.4 and pH 9.6 can have an affect on the steric structure of protein/antibody/analyte binding and thus affect their immobilization. Testing of coating buffers can help increase mobility and performance of immobilized antibodies. In SuperSet Development ELISA kits for one plate, add 100µl of Capture Antibody into 10ml of Coating Buffer.

Recipe for Blocking Buffer

Phosphate buffered saline (PBS)

1% BSA

Coating Buffer Recipe (1L) Ingredient Amounts

Na₂CO₂

1.5g

NaHCO₃

2.93g

Distilled Water

1L (pH 9.6)

ELISA wash steps

Following incubation steps, wash steps are required to removed bound non-specific proteins and reagents in order to reduce background signal. With each kit, a specified amount of wash steps is required. When washing, an insufficient number of wash steps with result in a high background, however, on the contrary, over washing may result in the removal of antibody and/or antigen from the ELISA plate and thus decrease sensitivity and signal. Automated washing using plate washer may be more efficient than carrying out wash steps manually.

Wash steps are performed using a TBS (Tris-buffered saline) or PBS (Phosphate-buffered saline). In addition 0.5% Tween-20 may be added to remove non-specific bound proteins.  

Analysing ELISA data

Following the completion of your ELISA protocol the next step is to acquire and analyse the data using an ELISA plate reader.

When performing an ELISA it is recommended to run your samples in duplicate or triplicate to ensure statistical significance of your results. Further, positive controls such as standards, known positive controls and negative controls such as blanks or samples without the antigen of interest may be required.

  • Negative controls: Samples with no presence of your analyte
  • Positive controls: Samples with a known presence or quantity of your analyte

Depending on the type of ELISA used (qualitative, semi-quantitative or quantitative) data output will vary. Therefore you choose the specific ELISA you want to use based on the data that you want to analyse. Data is presented as a plot of optical density (OD) vs the log concentration of sample. Standards with known concentrations are used to generate a standard curve from which the concentration of an unknown analyte can be determined.

Coefficient of variation

The coefficient of variation helps identify any inconsistences and inaccuracies in the results. It is expressed as a percentage of variance to the mean. The larger the variance the greater the inconsistency and error. The coefficient variation (CV) is the ratio of the standard deviation σ to the mean µ:

Cv= σ/µ

  • A High CV can be attributed to some or all of the following:
  • Pipetting inaccuracies
  • Sample contamination with bacteria/fungi or other reagents
  • Temperature variation- plates should be incubated at a stable temperature away from drafts.
  • Drying out of wells – plates should be covered during all incubation steps