Indirect ELISA detects plate-bound antigen with an unlabelled primary antibody followed by an enzyme-conjugated secondary antibody. The additional secondary-antibody step can increase signal and makes the format flexible because one labelled secondary can be used with many compatible primary antibodies.
The following is a general colourimetric workflow. Antigen coating, antibody dilution, incubation time and substrate development must be optimised for the selected antigen–antibody pair.
Protocol at a glance
| Best suited to | Detecting antibodies or plate-bound antigen when an unlabelled primary antibody and labelled secondary system are available. |
|---|---|
| Detection format | Primary antibody binds antigen; enzyme-labelled secondary antibody amplifies the signal. |
| Strength | Flexible and sensitive because one secondary reagent can support many primary antibodies. |
| Critical controls | No-primary, blank, positive antigen and negative/irrelevant antigen controls. |
| Critical optimisation | Antigen coating, primary dilution, secondary dilution, blocking and wash stringency. |
Equipment and reagents
- High-binding 96-well microplate
- Coating buffer
- Purified antigen or validated sample preparation
- Blocking buffer
- Wash buffer such as PBS with approximately 0.05% Tween-20
- Unconjugated primary antibody
- Species- and isotype-appropriate enzyme-conjugated secondary antibody
- Substrate such as TMB for HRP
- Stop solution where required
- Microplate reader
Indirect ELISA procedure
- Coat the plate. Dilute antigen in coating buffer. A common starting range for purified antigen is 1–10 µg/mL. Add approximately 100 µL per well and incubate overnight at 4°C or under another validated coating condition.
- Wash three times.
- Block. Add approximately 200 µL blocking buffer per well and incubate for about 1 hour at room temperature.
- Wash three times.
- Add primary antibody. Dilute the antibody according to its datasheet or a titration series. Add approximately 100 µL per well and incubate for about 1–2 hours at room temperature or overnight at 4°C.
- Wash three times.
- Add enzyme-conjugated secondary antibody. Dilute the secondary antibody in the recommended diluent and add approximately 100 µL per well. Incubate for about 1 hour at room temperature.
- Wash thoroughly. Perform at least three washes; increase wash number or duration where background remains high.
- Add substrate. Add the recommended substrate volume. For HRP/TMB systems, develop the colour for a consistent, validated period while avoiding saturation.
- Stop and read. Add stop solution if required. Stopped TMB is commonly read at 450 nm.
Controls
- Blank wells without antigen
- Known positive antigen/sample
- Negative or irrelevant antigen/sample
- No-primary control to estimate secondary-antibody background
- Where comparing antibodies, include the same antigen coating and secondary-antibody conditions across the plate
Interpretation
- Higher absorbance generally reflects greater binding of the primary antibody to plate-bound antigen within the assay's linear range.
- For quantitative work, generate a standard curve and report the validated working range.
- For antibody-screening applications, compare signal-to-background rather than raw OD alone.
Recommendations and warnings
- Match the secondary antibody to primary host and isotype. An otherwise good assay can fail through a host/isotype mismatch.
- Use highly cross-adsorbed secondaries when samples contain immunoglobulins from other species. This can substantially reduce cross-reactive background.
- Do not use raw OD as a concentration unless the assay has a validated standard curve. Screening and quantitative ELISA are different analytical tasks.
- Use an antigen concentration that preserves dynamic range. Excessive coating can hide differences between primary-antibody dilutions.
- Control wash technique. Residual secondary antibody is one of the most common causes of elevated background.
Antibody-titre applications
Indirect ELISA is commonly used to compare antigen-specific antibody responses. For titre work, prepare serial sample dilutions across a range broad enough to capture both high- and low-response samples. Define the endpoint rule before comparing groups and include the same reference or control sample across plates when experiments span multiple runs.
Advantages and limitations
- Advantages: signal amplification, flexible secondary detection and no need to conjugate every primary antibody.
- Limitations: additional incubation and wash steps, potential secondary-antibody cross-reactivity and species/isotype constraints.
Troubleshooting
| Problem | Possible cause | What to try |
|---|---|---|
| Weak signal | Primary antibody too dilute, low antigen coating or inactive secondary | Titrate antigen and both antibodies and verify positive controls. |
| High background | Secondary cross-reactivity, insufficient blocking or washing | Use a highly cross-adsorbed secondary, optimise blocker and increase wash stringency. |
| Signal in no-primary control | Secondary antibody binding directly to plate components or antigen | Reduce secondary concentration or change secondary/blocking system. |
| Poor replicate agreement | Pipetting or timing variation | Use multichannel pipetting and consistent incubation/start-stop timing. |
| Curve saturation | Too much antigen/antibody or overdeveloped substrate | Reduce concentrations or shorten substrate development. |
Frequently asked questions
What is the main advantage of indirect ELISA?
The labelled secondary antibody provides amplification and allows one detection reagent to be used with many unlabelled primary antibodies from the same host/isotype.
Why does the no-primary control matter?
It shows how much signal comes from direct secondary-antibody binding to the antigen, blocker or plate rather than from the primary antibody.
Can I compare OD values between different plates?
Only cautiously. Plate-to-plate reagent timing and development vary, so use shared controls or a standard/reference curve if data from separate plates must be compared.
How can I reduce background?
Optimise blocking, reduce primary/secondary concentrations, increase washing, and check whether the secondary antibody cross-reacts with sample components.
Related ABMIUM protocols
References and primary methods
- Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry. 1971;8:871–874.
- Indirect ELISA. Methods Mol Biol. 2015. PMID: 26160563.
- Butler JE, McGivern PL, Swanson P. Amplification of ELISA in the detection of class-specific antibodies. J Immunol Methods. 1978;20:365–383.
Relevant ABMIUM products
For mouse-primary assays, ABM2976 Goat anti-Mouse IgG(H+L)-HRP is listed for ELISA and related immunodetection workflows. For rabbit-primary assays, ABM2983 Goat anti-Rabbit IgG(H+L)-HRP is the corresponding catalogue option. Always confirm host, isotype, species cross-reactivity and required adsorption profile before use. Browse primary antibodies or use Find your MATCH for a target-specific pairing.
Explore more with ABMIUM
Need a target-specific antigen/antibody pairing? Use Find your MATCH, explore HRP secondary antibodies, or compare the Direct ELISA, Competitive ELISA and Sandwich ELISA workflows.