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 soluble proteins, cytokines, hormones, histones, modified proteins, or other targets where a matched antibody pair is available.Optimize first: Capture antibody concentration, detection antibody concentration, sample dilution, blocking buffer, wash stringency, and standard curve range. 1 Prepare plate map and reagentsPlan 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 antibodyDilute 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 blockWash 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 samplesPrepare standards in duplicate or triplicate using the recommended diluent. Dilute samples so readings fall within the linear range. 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 antibodyDilute detection antibody in assay diluent. If this product is used as the detection antibody, add RPH3AL Polyclonal Antibody, Biotin Conjugated 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 substrateAdd 100 uL per well HRP-conjugated secondary antibody or streptavidin-HRP as required by the detection antibody system. 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 readDevelop 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.
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: Testing antigen recognition, screening antibody reactivity, comparing sample antibody levels, or using an unconjugated primary antibody.Optimize first: Coating concentration, sample or antibody dilution, blocking buffer, secondary antibody dilution, and wash stringency. 1 Coat antigen or targetDilute 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. Incubate overnight at 4 degrees C or 2 hours at room temperature. 2 Wash and blockAspirate 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 antibodyDilute RPH3AL Polyclonal Antibody, Biotin Conjugated 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 antibodyWash 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 antibodyDilute HRP- or AP-conjugated secondary antibody in assay diluent, commonly 1:2000-1:10000 depending on supplier and signal strength. Add 100 uL per well and incubate 30-60 minutes at room temperature. 6 Develop signalWash 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 resultsSubtract 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.
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, or target-specific direct detection reagent is available.Optimize first: Coating or sample input, detection reagent amount, incubation time, wash stringency, and substrate development time. 1 Prepare plate and samplesPlan standards, blanks, negative controls, positive controls, and sample dilutions. Add 100 uL per well of standards or samples to the assay plate according to the kit format or coating requirement. 2 Immobilize target if requiredFor 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 blockWash 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 reagentAdd 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 developWash 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 readAdd 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.
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, and standard curve range. 1 Prepare standards and samplesPrepare a full standard curve using the assay diluent recommended for the target. 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 competitorAdd 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 reagentAdd the antibody or target-specific binding reagent at the optimized concentration. When using an antibody-based format, add RPH3AL Polyclonal Antibody, Biotin Conjugated only if the product is the intended competitive binding antibody and follow the datasheet dilution first. 4 Incubate competition reactionIncubate 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 thoroughlyWash 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 readAdd 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.
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