Ligand Binding Assay

 

Radioactive and non-radioactive ligand binding assays

There are numerous types of ligand binding assays, both radioactive ligand binding assays and non-radioactive ligand binding assays.  As such, ligand binding assays are a superset of radio-binding assays, which are the conceptual inverse of radioimmunoassays (RIA). Some newer types are called “mix-and-measure” assays because they do not require separation of bound ligands.

Ligand binding assays are used primarily in pharmacology for various demands. Specifically, despite the human body’s endogenous receptors, hormones and other neurotransmitters, pharmacologists utilize assays in order to create drugs that are selective or mimic, the endogenously found cellular components.  On the other hand, such techniques are also available to create receptor antagonists in order to prevent further cascades.  Such advances provide researchers with the ability not only to quantify hormones and hormone receptors, but also to contribute important pharmacological information in drug development and treatment plans.

Our Services Comprise:

– Assay Development and Validation
– Customizing de novo ELISA Kits in Different Formats
– Development of Immunogenicity Assays for Therapeutic Proteins, Monoclonal Antibodies or Polypeptides (Preclinical & Clinical)
– Method Transfer, Optimization & Development
– Quantitative ELISA Assay Development
– Single or Multiplexed Biomarkers

Medicilon has expanded its bioanalytical services and now offers ligand binding assays (LBA) for macromolecular drug therapeutics, immunogenicity screening, and biomarkers in biological matrices.  Enzyme immunoassays (EIA) or enzyme-linked immunosorbent assay (ELISA)-based applications are the primary tools for these services.

The Ligand Binding Assay

A ligand is a substance that binds with a target biomolecule. In the Good Laboratory Practice (GLP) facility, a ligand serves as a signal triggering molecule that binds to a protein, revealing specific test results. Various ligand binding assay applications may utilize ligands that are substrates, inhibitors, activators, or neurotransmitters. Their binding property, or the strength of the bond formed, is called its affinity.

A ligand binding assay, carried out under the strict standards of Good Laboratory Practice (GLP), can determine the binding affinity between a ligand and its receptor molecule. The strength of the affinity becomes relevant when the energy created by a high affinity ligand bond can be directed toward causing a change in reception conformation, which alters the behavior of an associated enzyme.

A ligand that behaves in this way, changing its receptor and triggering a behavioral response, is called an agonist. By helping researchers to determine the level of affinity between a ligand and its receptor, a Good Laboratory Practice (GLP) compliant ligand binding assay reveals the optimum concentration of the agonist for triggering the intended physiological response, thus providing data important to determining dosages of microRNA drugs.

A ligand binding assay can also determine ligand efficacy — the biological effect and the magnitude of the effect when the ligand binds to its receptor. Efficacy is another critical factor in dosage determination. Only a Good Laboratory Practice (GLP) compliant CRO, such as Accelerō® Bioanalytics, is fully qualified to perform a ligand binding assay that provides the most accurate, reproducible, and meticulously tracked results.

By carrying out ligand binding assay support in an FDA certified, Good Laboratory Practice (GLP) environment, Accelerō® Bioanalytics produces indispensable, reproducible data for pharmacokinetic assessment purposes in the development of biomarkers and detection of immunogenicity, accelerating the progress of essential new drug therapies towards FDA approval.

Ligand binding assays (LBA) are analytical procedures to quantify an analyte (macromolecule) based on its affinity for a ligand, including but not limited to antibodies, antigens, receptors, oligonucleotides and peptides. Typical LBA platforms include traditional enzyme-linked immunosorbent assay (ELISA), Meso Scale Discovery (MSD), Gyros, and Radioimmunoassay (RIA). Ligand binding assays performed by biopharmaceutical CROs are specially designed to support analyses such as in vitro screening, pharmacokinetics (PK), immunogenicity and biomarker studies. With expert assay development scientists at the core of our ligand-binding assay group, let Medicilon use our expertise in ligand-binding assays to provide you with valuable PK/TK, PD and immunogenicity information to support your non-clinical studies.

Types of Ligand Binding Assays

Ligand binding test method Incubate the radiolabeled antibody, overshooting unlabeled ligand and the receptor to be tested at the same time, determine the amount of labeled ligand in the bound and free state, and calculate the maximum number of binding sites and dissociation constant for the receptor to be tested Methods of analysis. Medicilon Bioanalysis Department can provide FDA/CFDA GLP-compliant macromolecular drug bioanalysis services to support the screening and development of protein drugs, antibody drugs, vaccines and biomarkers, as well as preclinical and clinical research.

(1) ELISA

Enzyme-linked immunosorbent assay, also called ELISA, can detect the content of antigen or antibody in the sample. The basic method is to adsorb the known antigen or antibody on the surface of a solid-phase carrier (polystyrene microreaction plate) to make the enzyme-labeled antigen The antibody reaction is carried out on the surface of the solid phase, and the free components in the liquid phase are washed away by the washing method. In the research and development of biotechnology drugs, this technology can be used to detect macromolecular antigens and specific antibodies, etc. It has the advantages of rapid, sensitive, simple, and easy to standardize the carrier. According to the connection and application form of the immunosorbent, conjugate and the test substance during the experiment, ELISA mainly includes sandwich method, indirect method, competition method and other types.

(2) RIA

The immune response caused by drugs is an important indicator of drug safety and effectiveness, which is also the common concern of regulatory agencies, manufacturers, clinicians and patients. Radioimmunoassay is also a method to analyze the immunogenicity of antibody drugs. This method uses the sensitivity and accuracy of radionuclide detection and the specificity of antigen-antibody reaction combined with an immunological technique, including the principle of competitive binding reaction. Two methods of radioimmunoassay (RIA) and non-competitive combined immunoradioassay (IRMA). Radioimmunoassay technology is often used in the determination of trace substances such as various hormones, trace proteins, tumor markers and drugs.

(3) TRFIA

Time-resolved fluorescence immunoassay (TRFIA) is a non-isotopic immunoassay technique developed on the basis of fluorescence analysis (FIA). This method uses lanthanide elements to label antigens or antibodies. According to the luminescence characteristics of lanthanide chelates, time-resolved technology is used to measure fluorescence. The two parameters of wavelength and time are simultaneously detected for signal resolution, which can effectively eliminate the interference of non-specific fluorescence. . It can be used to analyze hormones, viral hepatitis markers, tumor-associated antigens, pepsinogen (PG), drugs and peptides.

Advantages and Disadvantages of LBA Quantitative Analysis Method

LBA test methods have various advantages in the quantitative analysis of various biomolecules; they are usually low cost on most platforms (such as colorimeters or planar electrochemiluminescence). When high-affinity MAbs are used in LBAs, the LBA method has high sensitivity and specificity in detecting and quantifying target analytes that exist in a heterogeneous matrix environment. For research purposes, the sensitivity of this type of method can be as low as femtograms per milliliter (femtogram/mL). Most LBAs operating procedures do not involve the steps of sample separation, while quantitative analysis methods based on LC-MS/MS are required. Of course, LBA also has several disadvantages. Compared with the LC-MS/MS method, the dynamic range of the LBA method is narrower. Although the methods used in basic research can reach a dynamic range of 4-6 exponential levels, the validated methods used in regulated studies often have a quantitative range of only 2-3 exponential levels. This is because the robustness of the method and better reproducibility must be maintained.

The most important difference is that the performance of LBA depends on the quality and specificity of the reagents used. Therefore, reagent generation/selection (MAbs or PAbs) is a critical step in the method development process; this can be very time-consuming, ranging from 3 to 9 months. When using analyte-specific reagents to capture the target analyte, this disadvantage also exists for the LC-MS/MS method. From the perspective of biomarker methods, cross-reactivity of reagents can lead to non-specificity of the method; therefore, it is strongly recommended to perform additional tests to clarify the cross-reactivity and non-specificity of reagents.

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