Immunogenicity Testing: ELISA and MSD Assays

Cell-Based Immunogenicity Testing

Immunogenicity is the ability of a drug or biologic to induce an immune response in the human body, which may include the formation of anti-drug antibodies (ADA). Immunogenicity testing is one of the core techniques in biomedical research and clinical trials, for the detection of immune response and side effects caused by vaccines or drugs. There are many different types of immunogenicity testing methods, among which ELISA (Enzyme-Linked Immunosorbent Assay) and MSD (Multiplexed Electrochemiluminescence Detection) are two of the most commonly used methods.

ELISA in Immunogenicity Testing

ELISA is an immunoassay technique that relies on the specific binding between antigen and antibody. The ELISA method relies on binding target antigens or antibodies to a solid-phase carrier followed by interaction with sample antibodies or antigens and a quantitative signal produced through an enzymatic reaction-induced color change or fluorescence.

Immunogenicity testing for drugs typically employs Bridging-ELISA as the standard screening method to detect anti-drug antibodies (ADA). Bridging-ELISA pre-coats the drug or its fragments, and adds a positive control anti-drug antibody (ADA) and biotin-labeled drug to test for the presence of ADA in the sample. Bridging-ELISA provides high sensitivity and specificity and eliminates the interference of the drug itself to enhance detection accuracy.

Workflow:

1. Coating: Dilute the drug (e.g., monoclonal antibody) to an appropriate concentration and add it to a microplate to adsorb onto the surface.
2. Blocking: Use a blocking solution to cover unoccupied sites on the microplate to prevent non-specific binding.
3. Sample Addition: Add patient serum samples; if ADA is present, it will bind to the drug fixed on the plate.
4. Detection Antibody: Add a labeled secondary antibody (e.g., HRP-labeled anti-drug antibody) to detect the bound ADA.
5. Color Development: Use a substrate color reaction to determine the presence and concentration of ADA based on color intensity.

Optimization of bridging ELISA

The Bridging ELISA is advantageous for its simplicity, low cost, and provision of quantitative results. However, traditional Bridging ELISA faces some limitations, such as low sensitivity, false negatives, or matrix effects. To address these issues, various improved Bridging ELISA methods have been developed, such as:

• Acid-Dissociation Bridging ELISA: Enhances detection sensitivity by dissociating ADA from the drug through an acid dissociation step. It demonstrates high sensitivity and specificity in detecting GQ1001 antibodies in monkey serum samples.
• Drug Tolerance Testing: Maintains accuracy in the presence of high drug concentrations by dilution or using drug-tolerance reagents.
• NanoLuc-Based Bridging Immunoassay: Uses NanoLuc enzyme-labeled antibodies, eliminating the need for secondary antibodies, simplifying the workflow, and increasing sensitivity and dynamic range.

MSD in Immunogenicity Testing

MSD technology is a multiplexed detection method based on electrochemiluminescence. Its core principle utilizes electrochemiluminescent-labeled antibodies or antigens binding to target substances, detecting the content of multiple factors in a sample through luminescence intensity.

The basic principle of MSD in drug immunogenicity testing relies on bridging reactions and acid dissociation techniques. During the assay, the biological therapeutic drug or its biotinylated version is immobilized on an MSD plate, and the diluted sample is mixed with labeled Protein A/G detection reagents. The response of the electroluminescent signal allows for the quantitative detection of antibodies binding to the drug in the sample. This method effectively identifies antibodies bound to the drug, thereby determining if the drug has triggered an immune response.

Workflow:

1. Capture Antibody Coating: Dilute capture antibodies to an appropriate concentration and add them to MSD electrode plates for adsorption onto the electrode surface. Typically incubate at room temperature for 1-2 hours or overnight at 4°C.
2. Blocking: Use a blocking solution to cover unoccupied sites on the electrode surface, preventing non-specific binding. Incubate at room temperature for 30 minutes to 1 hour.
3. Sample Addition: Add the test sample to the electrode plate, allowing specific binding between target factors in the sample and capture antibodies. Incubation time varies from 30 minutes to several hours.
4. Washing: Wash the electrode plate thoroughly with a wash buffer to remove unbound substances. Typically wash 3-5 times, drying the plate after each wash.
5. Add Detection Antibody: Add electrochemiluminescent-labeled detection antibodies to form a sandwich complex with target factors. Incubate for 30-60 minutes.
6. Detection: Place the electrode plate in the MSD detector, apply voltage to excite electrochemiluminescence, and the detector automatically measures the light signal intensity.
7. Result Analysis: Calculate the concentration of each factor in the sample based on a standard curve. Analyze the levels of multiple factors in different samples by comparing light signal intensities.

Comparison of ELISA and MSD