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Choose Your ELISA Format
Use this ELISA protocol guide to choose and run a plate-based immunoassay workflow. Select sandwich, indirect, direct, or competitive ELISA to view detailed benchtop starting conditions, including coating, blocking, washing, antibody incubation, detection, plate reading, controls, and troubleshooting.
Sandwich ELISA protocol
Use this workflow when a target is captured by one antibody and detected by a second antibody that recognizes a different epitope.
- Use this format when: Quantifying low-abundance soluble antigens in complex biological matrices where a compatible matched antibody pair is available.
- Optimize first: Capture antibody concentration, detection antibody concentration, sample dilution, blocking buffer, wash stringency, standard curve range, and matrix effects.
Sample types
- Best sample types: Serum, plasma, saliva, culture supernatants, and tissue homogenates.
- Best for: Quantifying low-abundance antigens, such as cytokines, hormones, and biomarkers, in complex biological matrices.
- Accuracy note: Sandwich ELISA is usually not the best choice for very small or single-epitope analytes because two non-overlapping antibody binding sites are needed.
1
Prepare plate map and reagents
Plan standards, blanks, negative controls, positive controls, and samples before starting. Bring coated plate, standards, samples, wash buffer, blocking buffer, detection antibody, enzyme conjugate, substrate, and stop solution to room temperature unless the kit manual says otherwise. For manual coating, use a high-binding 96-well ELISA plate.
2
Coat capture antibody
Dilute capture antibody in carbonate-bicarbonate coating buffer, pH 9.6, or the buffer specified for the antibody pair. Add 100 uL per well. Cover the plate and incubate overnight at 4 degrees C, or 2 hours at room temperature as a faster starting condition. Aspirate the coating solution after incubation.
3
Wash and block
Wash 3 times with 250-300 uL per well 1x PBST or TBST. Add 200 uL per well blocking buffer, such as 1-5 percent BSA, casein, or nonfat dry milk in PBS/TBS. Incubate 1 hour at room temperature. Wash 3 times.
4
Add standards and samples
Prepare standards in duplicate or triplicate using the recommended diluent. Dilute samples so readings fall within the linear range. For cell lysates or tissue homogenates, prepare clarified samples and include fresh protease inhibitor during upstream extraction when compatible with the assay, such as R-1101 Protease Inhibitor Cocktail. Add 100 uL per well and incubate 1-2 hours at room temperature, or overnight at 4 degrees C for low-abundance targets. Wash 4-5 times.
5
Add detection antibody
Dilute detection antibody in assay diluent. If this product is used as the detection antibody, add primary antibody at the datasheet-recommended dilution or start at 0.25-2 ug/mL for optimization. Add 100 uL per well and incubate 1 hour at room temperature. Wash 4-5 times.
6
Add enzyme conjugate and substrate
Add 100 uL per well HRP-conjugated secondary antibody, or R-1098 Streptavidin: HRP Conjugate when the detection antibody or binding reagent is biotin-labeled. Incubate 30-60 minutes at room temperature protected from light when needed. Wash 5 times, then add 100 uL TMB substrate per well.
7
Stop and read
Develop color for 5-20 minutes until the standard curve is visible but not saturated. Add 50-100 uL stop solution per well and read absorbance at 450 nm, with 570 or 620 nm correction if available. Analyze standards with a 4-parameter logistic curve when appropriate.
ControlsInclude blank wells, zero standard, standard curve, matrix control, no-sample control, and positive sample when available.
Wash consistencyMost high background in sandwich ELISA comes from insufficient washing, over-concentrated detection antibody, or incompatible blocking buffer.
Direct ELISA protocol
Use this workflow when the target is immobilized or captured and detected directly by a labeled antibody, direct detection reagent, or kit-specific detection system.
- Use this format when: A direct detection kit, directly labeled antibody, purified antigen, semi-purified antigen, or target-specific direct detection reagent is available.
- Optimize first: Coating or sample input, target purity, detection reagent amount, incubation time, wash stringency, and substrate development time.
Sample types
- Best sample types: Purified or semi-purified antigens, recombinant proteins, and clarified cell or tissue lysates when target coating and background are acceptable.
- Best for: Screening antibody specificity or confirming the presence of an antigen in an isolated, high-purity state.
- Accuracy note: Direct ELISA is simple and fast, but it is less ideal for crude complex matrices because non-target proteins can compete for plate binding and increase background.
1
Prepare plate and samples
Plan standards, blanks, negative controls, positive controls, and sample dilutions. For manual direct ELISA, use purified or semi-purified antigen, recombinant protein, or clarified lysate only when plate coating and background are acceptable. Add 100 uL per well of standards or samples according to the kit format or coating requirement.
2
Immobilize target if required
For coated-plate direct ELISA, incubate antigen or sample in the well overnight at 4 degrees C or 2 hours at room temperature. For direct target-detection kits, follow the kit manual for binding, capture, or sample incubation conditions.
3
Wash and block
Wash 3 times with 250-300 uL per well wash buffer. Add 200 uL per well blocking buffer for 30-60 minutes if the assay format requires blocking. Some direct detection kits include a proprietary blocking or assay buffer that should be used instead.
4
Add direct detection reagent
Add 100 uL per well of the directly labeled antibody, direct detection reagent, or enzyme-conjugated target-specific reagent. If using a directly conjugated antibody, start with the datasheet-recommended dilution or test 0.1-1 ug/mL. Incubate 30-90 minutes at room temperature protected from light when appropriate.
5
Wash and develop
Wash 4-5 times to reduce background. Add 100 uL per well substrate. Develop until standards or positive controls show clear separation without saturation.
6
Stop and read
Add 50-100 uL stop solution when using TMB and read at 450 nm. Use the correction wavelength recommended for the plate reader if available. Compare results to the standard curve or kit-specific calculation method.
Direct detection kitsSome EpigenTek assay kits use direct target-detection formats for small molecules, DNA/RNA damage markers, or epigenetic modifications. In those cases, the kit manual overrides generic antibody-only workflow assumptions.
Background controlInclude blank, no-target, no-detection-reagent, and positive control wells when compatible with the kit or assay design.
Indirect ELISA protocol
Use this workflow to detect antibody binding to an immobilized antigen or target molecule using an unconjugated primary antibody and labeled secondary antibody.
- Use this format when: Measuring antibody binding, total antibody concentration, titer, or immune response to a coated antigen.
- Optimize first: Antigen coating concentration, sample or antibody dilution, blocking buffer, secondary antibody dilution, and wash stringency.
Sample types
- Best sample types: Serum, plasma, saliva, and other body fluids.
- Best for: Measuring total antibody concentrations, titers, or screening immune responses against specific pathogens or antigens.
- Accuracy note: Match the secondary antibody to the sample antibody species and isotype, and validate saliva or other non-serum matrices before relying on quantitative comparisons.
1
Coat antigen or target
Dilute purified antigen, peptide, protein, histone, modified target, cell lysate, or other coating material in carbonate-bicarbonate buffer or PBS. Add 100 uL per well. A common starting range is 0.5-5 ug/mL for purified protein or peptide. If the antigen or capture reagent is biotinylated, a streptavidin-coated plate such as R-1102 Streptavidin-Coated Strip Microwell Plate may be used instead of passive coating. Incubate overnight at 4 degrees C or 2 hours at room temperature.
2
Wash and block
Aspirate coating solution and wash 3 times with 250-300 uL per well PBST or TBST. Add 200 uL per well blocking buffer. Incubate 1 hour at room temperature, then wash 3 times.
3
Add primary antibody
Dilute primary antibody in assay diluent. Start with the datasheet-recommended dilution when available; otherwise test a dilution series such as 1:250, 1:500, 1:1000, and 1:2000. Add 100 uL per well and incubate 1 hour at room temperature or overnight at 4 degrees C for weak targets.
4
Wash after primary antibody
Wash 4-5 times with 250-300 uL per well wash buffer. Tap the inverted plate on clean absorbent material after the final wash to remove residual liquid without drying the wells.
5
Add enzyme-conjugated secondary antibody
Dilute HRP- or AP-conjugated secondary antibody in assay diluent, commonly 1:2000-1:10000 depending on supplier and signal strength. For common mouse or rabbit primary antibodies, host-matched options include A12003 HRP-Goat Anti-Mouse Secondary Antibody or A12004 HRP-Goat Anti-Rabbit Secondary Antibody. Add 100 uL per well and incubate 30-60 minutes at room temperature.
6
Develop signal
Wash 5 times. Add 100 uL substrate per well. For HRP/TMB, develop 5-20 minutes protected from strong light, then stop with 50-100 uL stop solution and read at 450 nm.
7
Interpret results
Subtract blank background and compare signal across antigen-coated, uncoated, no-primary, and no-secondary controls. High signal in no-primary wells suggests secondary antibody or blocking-related background.
Antigen coatingToo much coated antigen can increase background. If the signal is high but nonspecific, reduce coating concentration before changing everything else.
Primary antibody variableThe product page can replace the primary antibody variable in this protocol with the selected antibody name.
Competitive ELISA protocol
Use this workflow when sample target competes with immobilized or labeled target for antibody or binding reagent.
- Use this format when: The analyte is small, has one dominant epitope, or is measured by competition rather than two-antibody sandwich capture.
- Optimize first: Sample dilution, competitor concentration, antibody concentration, incubation time, standard curve range, and matrix interference.
Sample types
- Best sample types: Serum, plasma, saliva, culture supernatants, and crude biological fluids.
- Best for: Detecting small molecules, hormones, or targets with only a single antigenic epitope.
- Accuracy note: Competitive ELISA produces an inverse signal, so higher analyte levels usually give lower absorbance or fluorescence.
1
Prepare standards and samples
Prepare a full standard curve using the assay diluent recommended for the target. For serum, plasma, saliva, culture supernatants, or crude biological fluids, dilute samples into the same matrix when possible. Competitive assays often need careful sample dilution because high target concentration produces lower signal.
2
Add standards, samples, and competitor
Add 50-100 uL per well of standards or samples according to the assay design. Add competitor, tracer, or labeled target reagent if the protocol requires a pre-mix. Mix gently without splashing.
3
Add antibody or binding reagent
Add the antibody or target-specific binding reagent at the optimized concentration. When using an antibody-based format, add primary antibody only if the product is the intended competitive binding antibody and follow the datasheet dilution first.
4
Incubate competition reaction
Incubate 1-2 hours at room temperature, or as specified by the kit manual. Keep incubation times consistent across the plate because competitive assays are especially sensitive to timing differences.
5
Wash thoroughly
Wash 4-5 times with 250-300 uL per well wash buffer. Avoid drying the wells. Inconsistent washing can distort the inverse standard curve.
6
Develop and read
Add 100 uL substrate per well and develop until the low-analyte standards produce strong but unsaturated signal. Stop and read at the appropriate wavelength. Fit the data using the curve model recommended by the kit or assay format.
Signal directionIn competitive ELISA, stronger sample target usually means lower signal. Label graphs and reports clearly to avoid reversed interpretation.
TimingUse a multichannel pipette and consistent timing between rows when possible.
Plan your 96-well ELISA plate layout
Before loading standards and samples, map blanks, standards, controls, replicates, and sample dilutions. A plate map reduces pipetting mistakes and makes downstream analysis easier.
Open 96-well plate templateELISA reference notes
Format selectionUse sandwich ELISA for low-abundance antigens in complex matrices when matched antibody pairs are available. Use direct ELISA for purified or semi-purified antigen checks. Use indirect ELISA for antibody titers or immune-response screening. Use competitive ELISA for small molecules, hormones, modified nucleosides, peptides, or single-epitope targets.
Sample matrixSerum, plasma, saliva, culture supernatants, tissue homogenates, lysates, and crude biological fluids can behave differently. Matrix-match standards and controls when possible, and follow kit-specific sample preparation instructions.
Plate setupMap blanks, standards, controls, samples, and replicates before pipetting. Keep standard curve wells, sample dilutions, incubation times, and wash cycles consistent across the plate.
ControlsInclude blank wells, zero standard, standard curve, negative control, positive control, replicate wells, no-detection controls, and matrix-matched samples when possible.
Antibody fitFor ELISA antibodies, review application validation, target specificity, epitope availability, host species, conjugation status, capture/detection pair compatibility, and whether the target has enough epitopes for sandwich detection.
Common problemsHigh background often comes from insufficient washing, too much antibody, poor blocking, edge effects, or matrix interference. Weak signal can result from low coating, inactive antibody, incompatible antibody pair, insufficient target, or over-diluted detection reagent.
Use these conditions as practical starting points. Follow the product datasheet, kit manual, safety data sheet, and institutional safety requirements when they provide application-specific instructions.
