ELISA (PART 1)

Enzyme-linked immunosorbent assay (ELISA)

- Specific interactions between antigens and antibodies
- Replicating the interaction allows to detect or measure the presence of specific biological molecules.

Antigens

- Molecular markers
- Found on the surfaces of viruses, bacteria, allergens, parasites, and body cells.

Antibodies

- Immune system proteins
- Recognize and eliminate foreign or harmful substance.

Enzyme Immunoassay (EIA)

- Uses enzymes as labels
- Identification and quantification of target molecules.

Enzyme-linked immunosorbent assay (ELISA)

- One component is immobilized onto a solid phase
microtiter plate
magnetic particle
plastic bead
- Separates bound and unbound reactants Enhance sensitivity and specificity

Gold standard among immunoassays
- Accurate, versatile, easy to use
- Detects antigens, antibodies, proteins, hormones in fluids (blood, plasma, urine, saliva, CSF)
Safer Alternative to Radioimmunoassay 
- Developed in 1970s replacing radioactive isotopes
- From colorimetric to fluorogenic, electrochemiluminescent, nanoparticle-based

Benefits of ELISA

Engvall & Perlman
Van Weeman & Schuurs

Pioneers of ELISA

IgG (rabbit serum)
hCG (urine, HRP enzyme)

First Applications of ELISA

Coating 
- Antigen or Antibody is immobilized on the plate
Blocking
- Nonspecific binding sites are blocked
Detection 
- DNA is transcribed into RNA and then translated into proteins which control cellular activities 
Final Read 
- Substrate is added, enzyme reaction produces color change 

Methodology of ELISA 

Direct
Indirect
Sandwich
Competitive

Types of ELISA

Direct ELISA

Types of ELISA
- Binds antigens, including the desired target, in a simple directly to the plate. An enzyme-conjugated antibody in then added as a probe for the desired analyte.

Indirect ELISA

Types of ELISA
- Binds antigens, including the desired target in the sample to the plate.
- However, it involves two antibodies; a primary and a secondary conjugated antibody.

Sandwich ELISA 

Types of ELISA
The target is bound between a captured antibody (for antigen detection) or capture protein (for antibody detection) and the conjugated detecting antibody, creating a “sandwich” .

Competitive ELISA 

Types of ELISA
- Involves competition between the binding of the sample antigen and conjugated antigen to a specific amount of antibody.
- The more antigen in the sample, the less conjugated antigen binds and the lower assay signal.

- Pharmacokinetics
- Pharmacodynamics (PD) / Biomarker Analysis
- Immunogenicity (Anti-Drug Antibody, ADA Monitoring)
- Drug Stability and Tissue Distribution
- Toxicology / Safety Biomarkers

APPLICATIONS IN DRUG DEVELOPMENT/PRE-CLINCIAL STUDIES

Pharmacokinetics (PK)

APPLICATIONS IN DRUG DEVELOPMENT/PRE-CLINCIAL STUDIES
- ELISA quantifies therapeutic proteins (e.g., monoclonal antibodies, fusion proteins) in animal serum to calculate PK parameters like half-life, Cmax, AUC.
- Example:
Meng et al. (2024) developed and validated an ELISA for bispecific antibody Q-1802 in mouse serum (LLOQ 50 ng/mL; linear range 50–3200 ng/mL).

Pharmacodynamics (PD) / Biomarker Analysis

APPLICATIONS IN DRUG DEVELOPMENT/PRE-CLINCIAL STUDIES
- ELISA measures biomarkers such as cytokines or signaling proteins that reflect drug action in animal models.
- Provides a link between drug exposure (PK) and drug effect (PD).

Immunogenicity (Anti-Drug Antibody, ADA Monitoring)

APPLICATIONS IN DRUG DEVELOPMENT/PRE-CLINCIAL STUDIES
- ELISA detects antibodies raised against biologic drugs in animal studies.

Example:
- Chowdhury et al. (2010) validated ELISAs for both pharmacokinetics of a chimeric anti-CD40 mAb and human anti-chimeric antibodies (HACA).
- Kim et al. (2021) used bridging ELISA assays to detect anti-GX-G3 antibodies (a G-CSF Fc fusion protein) in rats and monkeys.

Drug Stability and Tissue Distribution

APPLICATIONS IN DRUG DEVELOPMENT/PRE-CLINCIAL STUDIES
ELISA can measure drug levels in tissue homogenates (skin, organs) and assess drug stability during preclinical experiments.

Toxicology / Safety Biomarkers

APPLICATIONS IN DRUG DEVELOPMENT/PRE-CLINCIAL STUDIES
ELISA assays quantify proteins indicative of tissue damage, inflammation, or immune response during preclinical safety studies.

- Straightforward and easy to perform
- Relies on specific antigen–antibody interactions for high sensitivity and accuracy
- Highly effective with the ability to analyze multiple samples simultaneously
- Requires no complex sample preparation
- Environmentally friendly and safe, avoiding hazardous radioactive materials and excess organic solvents
- Cost-effective due to low reagent costs

Advantages of ELISA 

- Antibody production is time-consuming and expensive, needing advanced techniques and costly culture media
- Risk of false positive or negative results, especially if immobilized antigen blocking is insufficient
- Antibodies can be unstable, requiring refrigerated storage and transport because they are protein-based

Limitations of ELISA

- Informed consent and autonomy require that patients receive full information about the test's implications and limitations before agreeing to ELISA antibody testing
- Handling of personal data and test samples must ensure strict privacy and data security to avoid unauthorized use or breaches
- There is a risk of false positives, which may cause individuals who are not immune to wrongly assume they are protected and consequently take unsafe risks, raising health concerns for themselves and others.
- Tests must meet reasonable standards of accuracy and reliability to prevent false results that could mislead people about their immunity status.

Ethical considerations of ELISA 

- Standardize procedures and quality controls to ensure reliability.
- Adopt advanced detection systems to improve sensitivity.
- Invest in recombinant antibody development to reduce costs and ethical concerns.
- Ensure ethical compliance through informed consent, data security, and accurate reporting.

Recommendations for ELISA

Horseradish Peroxidase (HRP)
Alkaline Phosphatase (AP) 

Detection Systems ( with chromogenic subtrates )