ELISA Development Guide

Developing an ELISA

An ELISA system consists of 4 technical elements:
1) ELISA plate-coating strategy
2) Antigen resources and Antibody pairs
3) Conjugating/labeling strategy
4) Enzyme and chromogen

1. ELISA Coating Strategy

When developing a new ELISA for a specific antigen, the first step is to determine an immobilizing strategy and optimize the plate-coating conditions for the antigen or capture antibody. Although generally we use Polyvinyl/ Polystyrene 96/ 384-well plate as the solid phase of ELISA, there are actually variety of solid phase materials can be used (see table 1).

Table 1. Types of Immunoassay Solid Phases

Materiala Binding Capacity Type of Interaction
Nitrocellulose High Hydrophobic, Hydrophilic
Polyvinylidene Difluoride(PVDF)
Nylon High Hydrophobic
Plate and Tubes
Polystyrene Low Hydrophobic
Polyvinyl Low Hydrophobic
Modified Plates Low Covalent, Hydrophobic, Hydrophilic
Polystyrene Moderate Hydrophobic
Modified Polystyrene High Covalent, Hydrophobic, Hydrophilic
Micro Particles High Covalent, Hydrophobic

Immobilization can be defined as the attachment of molecules (antigen and antibody) to a surface resulting in reduction or loss of mobility. The way in which proteins are immobilized will determine the properties of the ELISA. In some cases, immobilization may lead to partial or complete loss of protein activity, due to random orientation and structural deformation. In order to fully retain biological activity, proteins should be attached onto surfaces without affecting conformation and function. Generally, the choice of a suitable immobilization strategy is determined by the physicochemical and chemical properties of both surface and protein. Many immobilization techniques have been developed in the past years, which are mainly based on the following three mechanisms: physical, covalent, and bioaffinity immobilization (see Figure 1).

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Comparison of three different immobilizing strategy

Figure 1. Comparison of three different immobilizing strategies.

2. Antigen and Antibody

Antigen and antibody are two major factors determining the sensitivity and specificity of an assay. The purity and stability of antigen are key parameters that affect the performance of ELISA. High antigen purity can enhance the capability of capturing antibody so that increases the sensitivity of the assay.

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The three dimensional configuration of the antigen-binding site found in the Fab portion of the antibody controls the strength and specificity of the interaction with antigen. The stronger the interaction, the lower the concentration of antigen can be detected. A competing factor is the specificity of binding or the cross-reactivity of the antibody to serum proteins other than the target antigen. Depending on whether the antibodies being used are polyclonal or monoclonal, cross-reactivity will be caused by different forces. In either case, driving the assay to the limit of sensitivity may result in cross-reactivity, and one is faced with the conflicting needs of sensitivity versus specificity.

Either monoclonal or polyclonal antibodies can be used as the capture and detection antibodies in sandwich ELISA systems. Monoclonal antibodies have an inherent mono-specificity toward a single epitope that allows fine detection and quantification of small differences in the antigen. A polyclonal is often used as the capture antibody to pull down as much of the antigen as possible. Then a monoclonal is used as the detecting antibody in the sandwich assay to provide improved specificity.

An important consideration in designing a sandwich ELISA is that the capture and detection antibodies must recognize two different non-overlapping epitopes. When the antigen binds to the capture antibody, the epitope recognized by the detection antibody must not be obscured or altered. Capture and detection antibodies that do not interfere with one another and can bind simultaneously are called "matched antibody pairs" and are suitable for developing a sandwich ELISA.

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Creative Diagnostics can develop the most suitable matched pairs for your ELISA assay. Projects can be initiated in conjunction with our custom hybridoma development services or antibodies can be supplied by customers for evaluation. We utilize the expertise of our own internal ELISA development team which supports a variety of ELISA products.

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3. Conjugate Strategy

Enzyme labeled antibody is the key of the ELISA signal output. Conjugation of enzymes to antibodies involves the formation of a stable, covalent linkage between an enzyme and an antigen-specific monoclonal or polyclonal antibody in which neither the antigen-combining site of the antibody nor the active site of the enzyme is functionally altered.

Various reporter enzymes, such as horseradish peroxidase (HRP), alkaline phosphatase (AP) and many others, can be attached to antibodies and proteins through the use of different coupling chemistries to ensure the maximum retention of activity of both enzyme and protein.

Horse radish peroxidase (HRP) - can be visualized by chromogenic reactions such as diaminobenzidine (DAB) or chemiluminescence. HRP is a 44kDa glycoprotein enzyme label and is more stable than alkaline phosphatase.

Alkaline phosphatase (AP) - is a hydrolase enzyme and its signal is often measured through its colorimetric substrate pNNP.

Chemistry detail of conjugation sees Winston, S. E., Fuller, S. A., Evelegh, M. J. and Hurrell, J. G. 2001. Conjugation of Enzymes to Antibodies. Current Protocols in Molecular Biology. 50:I:11.1:11.1.1–11.1.7.

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Creative Diagnostics provides enzyme-antibody labeling conjugation services for signal generating molecules by using our proprietary enzyme-labeling strategy.

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4. Enzyme and Chromogen

The final stage of ELISA is dictation. The intensity of signal produced when the substrate is added will by directly proportional to the amount of antigen captured in the plate and bound by the detection reagents. The most common used enzyme-substrate system is performed below:

Table 2. The most commonly used enzymes and substrate/chromophore systems used in ELISA and the color changes with relevant stopping agents.

Enzyme label Substrate Dye Buffer Stopping solution Color change Reading wavelength
Unstopped Stopped Unstopped Stopped
Horseradish peroxidase H2O2 (0.004%) OPD Phosphate/ citrate pH 5.0 1.25 M H2SO4 Light orange Orange 450 nm 492 nm
H2O2 (0.004%) TMB Acetate buffer (0.1 M) pH 5.6 1% SDS Blue Yellow 650 nm 450 nm
H2O2 (0.002%) ABTS Phosphate/citrate, pH 4.2 No stop Green Green 414 nm 414 nm
H2O2 (0.006%) 5AS Phosphate (0.2M), pH 6.8 No stop Black/brown Black/brown 450 nm 450 nm
H2O2 (0.02%) DAB Tris or PBS, pH 7.4 No stop Brown Brown N/A N/A
Alkaline phosphatase pnpp pnpp Diethanolamine (10 mM) plus MgCl2(0.5 mM), pH 9.5. 2 M sodium carbonate Yellow/green Yellow/green 405 nm 405 nm

ELISA Trouble Shootings

Solve your ELISA problems with these troubleshooting tips, covering common causes of poor standard curve, no signal or weak signal, high background, large coefficient of variation and more. Detail sees CD ELISA TROUBLESHOOTING TIPS

Creative Diagnostics provides contract ELISA development kit services for the R&D and IVD community. We conduct ELISA kit development services to support regulatory approval submission. Creative Diagnostics will carry out the approval proposal and deliver the expected results and documents in a time and cost effective manner.

Detection Methods:
1) Light absorbance
2) Fluorescence intensity or polarization
3) Fluorescence resonance energy transfer
4) Luminescence

Assay Milestones:
1) Feasibility assessment and assay design
2) Assay optimization
3) Assay Validation
4) Manufacturing

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1. Lynes M A. Solid‐Phase Immunoassays[J]. Current Protocols in Toxicology, 2005: 18.7. 1-18.7. 19.
2. Hornbeck P. Enzyme‐Linked Immunosorbent Assays[J]. Current protocols in immunology, 1991: 2.1. 1-2.1. 22.
3. Crowther J. Enzyme linked immunosorbent assay (ELISA)[M]//Molecular biomethods handbook. Humana Press, 2008: 657-682.
4. Lequin R M. Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA)[J]. Clinical chemistry, 2005, 51(12): 2415-2418.
5. Hnasko R. ELISA: Methods and Protocols[J]. Methods in Molecular Biology, 2015, 1318.
6. Karaszkiewicz J W. Critical factors in immunoassay optimization[J]. Gaithersburg, MD: Kirkegaard & Perry Laboratories, Inc, 2005.

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