Enzyme-linked immunosorbent assay (ELISA) is routinely used for qualitative and quantitative protein analysis. Reliable results depend on standardized sample preparation, quality control at critical procedural steps and appropriate data analysis. This guide covers sample collection and storage, pipetting and plate washing, reader settings and dual-wavelength measurements, standard curve fitting with an emphasis on four-parameter models, and concentration calculations. It also outlines causes and troubleshooting approaches for eight common types of abnormal results.
Introduction
ELISA signal intensity does not vary linearly with analyte concentration; it typically follows a sigmoidal dose–response curve. Systematic errors introduced during sample preparation, incubation, washing or measurement can be amplified through this relationship and produce substantial bias in the calculated concentration.
I. Sample collection and preparation
1. General principles
1). Collection containers
Collect samples in disposable, pyrogen-free and endotoxin-free tubes to avoid interference from exogenous pyrogens or endotoxins.
2). Avoid hemolysis
Lysis of red blood cells releases substances with peroxidase activity. In HRP (horseradish peroxidase)-based ELISA systems, hemolyzed samples can produce additional nonspecific color and increase background. Minimize mechanical hemolysis during serum collection and separation.
3). Mixing after thawing
Freezing and thawing can produce localized protein concentration gradients and uneven distribution. Mix thawed samples thoroughly by gentle inversion. Avoid vigorous vortexing and bubble formation.
4). Clarification
Centrifuge or filter turbid samples and samples with visible precipitates before testing. Remove visible precipitates before analysis. Severely hemolyzed or highly lipemic samples are not recommended.
2. Collection and preparation by sample type
|
Sample type |
Collection and preparation |
|
Cell culture supernatant |
Centrifuge at 1,000 × g for 20 min to remove sediment. Test immediately or aliquot and store below −20°C. |
|
Serum |
Allow whole blood to stand for 2 h at room temperature or overnight at 4°C. Centrifuge at 1,000 × g for 20 min and collect the supernatant. Use disposable, pyrogen-free and endotoxin-free collection tubes. |
|
Plasma |
Collect using EDTA, sodium citrate or heparin as anticoagulant. Within 30 min, centrifuge at 2,000 × g for 20 min at 2–8°C. Test immediately or aliquot and store below −20°C. |
|
Cell lysate |
For adherent cells, remove medium and wash with PBS, saline or serum-free medium. Add an appropriate amount of lysis buffer and pipette up and down; cells typically lyse within about 10 s. For suspension cells, centrifuge to collect cells, add lysis buffer and mix thoroughly. Centrifuge at 10,000–14,000 × g for 3–5 min and collect the supernatant. |
|
Urine |
Collect aseptically and centrifuge at 2,000–3,000 rpm for approximately 20 min. Carefully collect the supernatant; centrifuge again if precipitate forms. Test immediately or store as appropriate. Standing at room temperature may produce phosphate crystals that interfere with the assay. |
|
Tissue homogenate |
Wash a suitable tissue sample in prechilled PBS (0.01 M, pH 7.0–7.2) to remove blood; lysed red blood cells can affect results. Mince the tissue and add PBS at a 1:9 tissue mass-to-buffer volume ratio; 1% protease inhibitor is recommended. Homogenize thoroughly on ice. Further disrupt cells by sonication on ice or repeated freeze–thaw cycles (twice if needed). Centrifuge at 5,000 × g for 5–10 min and test the supernatant. |
|
Saliva |
Collect between meals. Amylase levels rise briefly after eating and may interfere with the assay. |
For less common sample types, consult the relevant literature or confirm suitability in a preliminary experiment.
3. Storage and stability
Storage periods: 48 h at 2–8°C; 1 month at −20°C; 6 months at −80°C.
Avoid repeated freeze–thaw cycles. For samples collected over time, prepare single-use aliquots in advance and store at −20°C or −80°C. Samples tested on the day of collection may be held temporarily at 2–8°C. Before testing, allow frozen samples to equilibrate gradually to room temperature (25°C ± 3°C), mix gently and then load the plate.
II. Critical procedural controls
1). Pipetting
Pipetting accuracy directly affects consistency between wells and is a major source of repeatability error. Observe the following controls.
• Check that each new tip matches the pipette and is securely attached.
• Select a pipette with an appropriate working range; avoid volumes near either extreme.
• Tips may be retained when loading replicate wells of the same sample, but must be changed between samples and between standard concentrations to avoid cross-contamination.
• Expel air before aspirating reaction solution with a new tip to prevent bubbles during dispensing.
• For viscous samples, allow the liquid to equilibrate in the tip before withdrawing it.
• Keep the tip below the liquid surface during dispensing and at an angle to the well wall. Avoid touching the wall or bottom: liquid retained outside the coated area can reduce the effective volume delivered.
• Use a multichannel pipette for samples and standards where possible to improve speed and consistency.
• Control incubation timing carefully; keep timing differences within ±5 min per hour of incubation.
2). Plate washing
Washing is critical to ELISA background and precision. Incomplete washing increases background, while excessive washing or allowing wells to dry reduces reproducibility.
a. Wash buffer preparation
Dilute concentrated wash buffer with deionized or distilled water to avoid interference from water impurities.
b. Manual washing
Discard the reaction solution → add the specified volume of 1× wash buffer → soak for the time stated in the instructions → invert and empty the plate → tap dry on clean absorbent paper → repeat for the specified number of cycles. After the final wash, empty the wells completely and tap the plate thoroughly dry.
c. Automated washing
A plate washer is preferred for more consistent washing between wells. Confirm that aspiration works correctly and each well is fully aspirated. Once liquid has been removed, do not leave the aspiration head in the well to continue aspirating. Avoid excessive or insufficient wash speed, incomplete washing and over-aspiration. After the final wash, invert the plate and tap dry on clean absorbent paper. Wash thoroughly without allowing the wells to dry, and proceed promptly to the next step.
3). Incubation and contamination control
• The 96-well plate has detachable strips. Remove only the strips required, reseal the remainder and store at 4°C to prevent contamination.
• Reagent containers usually contain more than the labeled volume. Withdraw the amount required rather than pouring out the reagent.
• Prepare working solutions immediately before use. Use reconstituted protein within 12 h. It may be stored at −20°C for up to 2 days, but must not undergo repeated freezing and thawing.
• Remove covers and sealing films gently to avoid splashing and cross-contamination between wells.
4). Color development and stopping the reaction
• TMB substrate should be clear and colorless. Withdraw only what is required and protect it from light throughout handling.
• Stop solution is corrosive. Wear appropriate protection and avoid skin contact.
• Add stop solution in the same sequence used to add TMB substrate to minimize systematic differences in reaction time.
• Sample loading time, incubation temperature and duration, and washing all affect results. Become familiar with the workflow before starting and control these conditions carefully.
III. Results and data analysis
Standard curves can be generated with different plotting programs. The source recommends ELISACalc and illustrates the following workflow with an example dataset.
1). Organize the data
Prepare an Excel table containing the standard curve and sample data.
2). Import into ELISACalc
Open ELISACalc, available as a free download from www.boster.com, copy the table data and click Paste in the program.
3). Fit the standard curve
Select a four-parameter fitting model in the upper-left panel, then click Regression/Fit to generate the curve.
4). Review the fit and copy the equation
The curve should rise smoothly. An r² value closer to 1 indicates a better fit. If the four-parameter model is unsuitable, try another model. Click Regression Equation, then Copy, to paste the equation into the spreadsheet.
5). Calculate sample concentrations
Click Calculate X from Y. Copy the sample absorbance readings from the spreadsheet—the yellow cells in the source example—into the program and click Paste, then Calculate. The program returns the corresponding sample protein concentrations.
6). Apply the dilution factor
Click Copy to paste the calculated concentrations into the spreadsheet. Organize the results and multiply by the dilution factor to obtain the final protein concentration in each sample.
IV. Troubleshooting common abnormal results
|
Observation |
Possible cause |
Corrective action |
|
Weak signal |
Insufficient reagent in wells |
Check and calibrate the pipette; ensure a tight connection between tip and pipette. |
|
Incomplete antigen–antibody reaction |
Extend reaction time and maintain the optimal temperature. |
|
|
Improper reagent storage |
Store the kit strictly as instructed. |
|
|
Incorrect reader filter |
Check the filter wavelength; TMB is read at 450 nm. |
|
|
Kit not equilibrated to room temperature |
Allow the kit to reach room temperature before use. |
|
|
Insufficient color development time |
Extend color development appropriately. |
|
|
Insufficient antibody/ABC reagent |
Check the antibody dilution factor and calibrate the pipette. |
|
|
Standard incompletely dissolved or used too late |
Dissolve thoroughly after adding diluent and use within the specified time; use a vortex mixer if necessary. |
|
|
High background |
Excessive color development |
Control development time and stop the reaction promptly. |
|
Insufficient washing |
Increase wash cycles and soaking time; remove residual liquid completely. |
|
|
Incorrect reagent order or omitted addition |
Review the workflow and repeat the assay. |
|
|
Standards give a signal but samples do not |
Low or absent analyte |
Include a positive control and repeat. |
|
Sample matrix interference |
Choose an appropriate dilution factor and dilute the sample again. |
|
|
Isolated abnormal wells |
Evaporation during incubation |
Seal or cover the plate during incubation. |
|
Uneven washing |
Confirm washer function; use an automated washer where possible. |
|
|
Debris or droplets on the plate bottom |
Clean the plate bottom before reading and keep the work area clean. |
|
|
No signal |
Reagents or plates from different kits mixed |
Label materials clearly and avoid mixing kits. |
|
Plate stacked on another plate |
Do not stack assay plates. |
|
|
Sample signal higher than standards |
Analyte exceeds the standard curve range |
Select a suitable dilution factor and dilute the sample again. |
|
Poor reproducibility |
Reagents not mixed in wells |
Do not allow wells to dry; mix thoroughly. |
|
Unequal loading volumes |
Check and calibrate the pipette. |
|
|
Sample dispensed onto the uncoated well wall |
Avoid touching the well wall during loading. |
|
|
Tips or containers reused |
Change tips for each addition and use a new container when preparing another solution. |
|
|
Sealing film reused |
Use a fresh seal for each incubation. |
|
|
Incubation temperature outside specification |
Calibrate the incubator or water bath. |
|
|
Inconsistent addition volumes or timing |
Keep conditions and operators consistent in repeat measurements. |
|
|
Edge effects |
Evaporation or stacked plates |
Seal or cover during incubation and avoid stacking plates. |
|
Uneven incubation temperature |
Calibrate the incubator; avoid vents and locations near doors or windows. |
|
|
Incomplete washing |
Ensure correct washer operation; do not reduce wash volume or cycle count. |
Please note
An ELISA kit promotion is coming in November. Stay tuned.
Related products
|
Cat. No. |
Product name |
Assay range |
Sensitivity |
|
97109ES |
Human IL18 ELISA Kit |
125–8000 pg/mL |
37.2 pg/mL |
|
97171ES |
Human IL23 ELISA Kit |
7.82–500 pg/mL |
2.7 pg/mL |
|
97080ES |
Human IL6 RapidStep ELISA Kit |
1.56–100 pg/mL |
0.1 pg/mL |
|
97081ES |
Human cTnI RapidStep ELISA Kit |
0.39–25 ng/mL |
19.6 pg/mL |
|
97082ES |
Human hs-CRP RapidStep ELISA Kit |
78.13–5000 pg/mL |
21 pg/mL |
|
97083ES |
Human IL5 ELISA RapidStep Kit |
3.13–200 pg/mL |
0.15 pg/mL |
|
97084ES |
Human VEGFA RapidStep ELISA Kit |
78.2–5000 pg/mL |
31 pg/mL |
|
97024ES |
Human IFN-γ ELISA Kit |
15.63–1000 pg/mL |
9.64 pg/mL |
|
97072ES |
Human TNF-α ELISA Kit |
15.63–1000 pg/mL |
5.06 pg/mL |
|
97029ES |
Human IL-2 ELISA Kit |
15.63–1000 pg/mL |
3.06 pg/mL |
|
97036ES |
Human IL-10 ELISA Kit |
62.5–4000 pg/mL |
27.5 pg/mL |
|
97093ES |
Human PDL1 RapidStep ELISA Kit |
15.63–1000 pg/mL |
1.23 pg/mL |
|
97094ES |
Human CALR RapidStep ELISA Kit |
1.56–100 ng/mL |
0.57 ng/mL |
|
97095ES |
Human GDF15 RapidStep ELISA Kit |
46.88–3000 pg/mL |
5.22 pg/mL |