ELISA Analyzer

Comprehensive ELISA data analysis following industry best practices.

Quick Start

1. Try Example Data: Click one of the 4 example data buttons in the Upload tab to see the analyzer in action.
2. Upload Your Data: Or upload your own Excel/CSV file with ELISA results.
3. Configure: Verify column mappings and adjust settings (assay type, normalization, etc.).
4. Analyze: Click the Analyze Data button to process your data.
5. Review: Check Results, Plate View, Quality Control, and Data Tables tabs.

Types of ELISA

ELISA (Enzyme-Linked Immunosorbent Assay) is a plate-based technique for detecting and quantifying proteins, hormones, antibodies, and other analytes. There are four main types:

1. Direct ELISA

The simplest format. Antigen is immobilized on the plate surface, and an enzyme-labeled primary antibody binds directly to the target.

     ▼ Enzyme-labeled 1° Ab
     |-----Y-----|
     ▲ ▲ ▲ ▲ ▲  Antigen
  ========================  Well surface

  Signal: Higher [antigen] → Higher OD

Use case: Fast screening, fewer steps. Example: IgG detection.

Curve direction: Positive (higher concentration = higher signal).

2. Indirect ELISA

Antigen is immobilized on the plate. An unlabeled primary antibody binds the antigen, then an enzyme-labeled secondary antibody detects the primary antibody.

  ▼ Enzyme-labeled 2° Ab
  |-----Y-----|
     |--Y--|  Unlabeled 1° Ab
     ▲ ▲ ▲ ▲ ▲  Antigen
  ========================  Well surface

  Signal: Higher [antigen] → Higher OD

Use case: Signal amplification via secondary Ab. Example: TNF-α, antibody screening.

Curve direction: Positive (higher concentration = higher signal).

3. Sandwich ELISA

A capture antibody is coated on the plate. The antigen is "sandwiched" between the capture antibody and an enzyme-labeled detection antibody. Most quantitative format.

  ▼ Enzyme-labeled detection Ab
  |-----Y-----|
     ▲ ▲ ▲ ▲ ▲  Antigen (analyte)
  |-----Y-----|  Capture Ab
  ========================  Well surface

  Signal: Higher [antigen] → Higher OD

Use case: Highest specificity and sensitivity. Example: Cytokines (IL-6, IL-8), hormones.

Curve direction: Positive (higher concentration = higher signal).

4. Competitive ELISA

Sample antigen competes with enzyme-labeled antigen for binding to a limited number of antibody sites. More sample antigen means less labeled antigen binds, producing lower signal.

  ▼ Enzyme-labeled Ag (competing)
  ▲   ▲  ▲  ▲ ▲  Labeled + Sample Ag
  |--Y--|--Y--|--Y--|--Y--|  Antibodies
  ========================  Well surface

  Signal: Higher [sample Ag] → Lower OD
  (sample antigen displaces labeled antigen)

Use case: Small molecules, haptens. Example: Melatonin, cortisol, drug metabolites.

Curve direction: Negative/inverse (higher concentration = lower signal).

Comparison Summary

Curve Fitting Models

• 4-Parameter Log-Logistic (4PL): Standard dose-response curve. Best for most ELISA assays. Parameters: upper/lower asymptotes, slope, and EC50.
• 5-Parameter Log-Logistic (5PL): Adds an asymmetry parameter for curves with hook effects or asymmetric responses.
• Linear: Simple fallback model for limited dynamic range.
• Weighted Regression: Optional 1/Y, 1/Y², 1/X² weighting for heteroscedastic data.

Quality Control Features

• LOD/LOQ/ULOQ: Automatic calculation of assay detection and quantitation limits.
• Back-Calculation Accuracy: Each standard should recover 80–120% of nominal concentration.
• CV Monitoring: Replicate precision checks (< 15% CV for duplicates).
• Outlier Detection: Grubb’s and Dixon’s Q tests for identifying outliers.

Expected Data Format

Your data file should contain the following columns:

You can also load raw plate reader data and use the Plate View tab to visualize your layout.

Troubleshooting

• Unexpected curve shape: Try toggling ‘Skip B0/NSB Normalization’ — direct, indirect, and sandwich ELISAs typically don’t need it.
• Poor model fit: Check that standards have concentration > 0 (zero concentration is excluded from curve fitting).
• Samples out of range: Values are color-coded when outside the standard curve range.
• High CV warnings: Replicate CV > 15% indicates inconsistent measurements.
• Standards failing accuracy: Recovery should be 80–120%; check pipetting or reagents if many fail.
• No standards detected: Check that your Standard Label matches the value in your Sample Type column (case-sensitive).

ELISA Analyzer (Beta) — Uses log-logistic 4PL/5PL models from the drc package for accurate dose-response curve fitting.