Background

The human body has an elaborate defense mechanism, better known as the immune system, which functions akin to a fortress against incoming objects from alien regions that are likely to cause harm. This defense mechanism is largely constituted by a group of molecules collectively known as Human Leukocyte Antigens (HLAs). Among these molecules, HLA class II molecules play a crucial role in presenting the antigens derived from foreign entities to specific white blood cells.

HLA class II molecules are critical cell surface receptors operating within the immune system environment. They are expressed on the surface membranes of antigen-presenting cells such as macrophages, dendritic cells, and B lymphocytes. These molecules capture exogenous fragments and present them to CD4⁺ T helper cells, thereby triggering an augmented immune response through highly specific binding interactions.

Considering the sensitivity factors, in vitro assays focusing on the binding of HLA class II molecules represent a powerful and effective tool for understanding this mechanism experimentally. These assays effectively mimic in vivo conditions, enabling the measurement of peptide binding affinity to HLA class II molecules. By employing synthetic HLA class II molecules and a peptide of interest, scientists can assess the likelihood of peptide binding to the HLA molecules and the possibility of eliciting an immune response. This method holds importance in tumor immunity, immunogenicity risk assessment, and vaccine development, contributing to advancements in medical science.

Fig. 1 T cells recognize a complex of a peptide fragment and MHC (HLA in humans).¹

Fig. 2 The major histocompatibility complex (MHC) class II antigen presentation pathway.²

Application of HLA Class II Binding Assays

HLA binding assays serve as a crucial tool for studying the interactions of various complex proteins on their terms. They have the ability to attach to and interact with different protein fragments, thus finding applications in key biomedical research areas such as vaccine design, immunotherapy, and personalized medicine.

In the context of vaccine development, HLA binding assays aid in the identification of relevant antigenic epitopes capable of triggering an immune response. In the realm of immunotherapy, designing drugs that effectively bind to HLA class II molecules can potentially induce a beneficial immune response. These assays are also instrumental in studying the pathogenesis of autoimmune diseases, exploring the types of antigens that provoke undesired immune responses, and developing therapies to modulate such responses.

The competitive binding assay explains how the HLA class II molecules perform selective binding with the concerned peptides. This laboratory assay provides insights into the attachment of HLA class II molecules, offering potential applications in the treatment of immune diseases.

The Procedure of the Experiment

• Competition Binding Assay

In this assay, two or more different antigens are mixed and attempt to bind to the same site on the HLA class II molecule.

The labeled or reference antigen, along with various concentrations of unlabeled competing antigen, is incubated with a known quantity of HLA class II molecules per well. The mixture will then be allowed to react with a predetermined exposure to the unlabeled competing antigen. The situation of the competing antigen showing a higher affinity for the HLA class II molecules will flush out the labeled reference antigen.

• Fluorescence-based Assay:

The polypeptide mixture presented to the HLA class II molecules contains "competing" peptides known to bind to the HLA class II molecules and polypeptides to be determined.

Such "competing" peptides, which are known to bind to HLA class II molecules, are usually labeled with a fluorescent pigment to facilitate direct determination of the binding of the "competing" peptide to HLA class II molecules by detecting fluorescence intensity.

It is then observed whether the peptides under test can interfere with the binding of the "competing" peptides to HLA class II molecules. If the peptide under test is able to bind to the HLA class II molecules, it means that it can break the binding of the HLA class II molecules from the "competing" peptide, and the fluorescence intensity will be weakened.

By comparing the effect of different peptides on fluorescence intensity, the relative binding strength of these peptides with HLA class II molecules can be understood.

Through this experiment, researchers can understand the ability of HLA class II molecules to bind to different peptides, which will help understand the triggering mechanism of immune responses and design immunotherapies.

• Radiolabelling Binding Assay:

Before carrying out the testing, first prepare your test compound as well as the reference ligand that you are going to use during the testing. Both are necessary in order to identify HLA class II molecules that bind to the test compound.

The incubation assay of the HLA class II molecules consists of taking known quantities of the radioactively labeled standard ligand and varying concentrations of the unlabeled test compound.

The separation is usually done at the end of the incubation period. Technologies such as gel filtration and equilibrium dialysis can be used for the procedure. Other methods that can be used include antigen-antibody precipitation, among others.

The radioactivity left in the bound fraction corresponds to the amount of reference ligand remaining bound. By adding increasing amounts of the test compound, the binding of the reference ligand will decrease, resulting in lower radioactivity.

Calculate the IC50 value, which is defined as a compound's activity measurement reflecting a 50% inhibition of receptor-ligand binding activity by the compound in question.

• Direct Binding Assay

First, the HLA class II molecules are typically incubated with a specific antigen peptide. Then, a known competitor peptide is introduced to the mixture to examine its ability to inhibit the interaction between the HLA class II molecules and the specific antigen peptides.

The samples are analyzed using techniques such as enzyme-linked immunosorbent assay (ELISA) or fluorescence polarization immunoassay (FPIA), where the amount of HLA class II molecules bound to the antigen is quantified. Subsequent comparison of these quantities allows for the determination of the competitor peptide's relative affinity for binding to the HLA class II molecules.

• Real-time Kinetic Binding Assay

First, the HLA class II molecules are usually isolated and purified. After purification, the molecules are typically labeled or conjugated with a marker such as a fluorescent dye for detection. Next, the target proteins or peptides are also prepared. They may also be labeled with a different marker. The HLA class II molecules and the target proteins or peptides are then mixed together in a suitable binding buffer. The mixture is typically incubated at a specific temperature and for a timescale relevant to the binding kinetics. Throughout the incubation, measurements are taken at regular intervals to monitor the binding process. This may entail measuring the intensity of the fluorescent signal, which changes as the HLA class II molecules bind to the target proteins or peptides. The data are then collected and analyzed to determine the affinity between HLA class II molecules and the target protein.

Creative Biolabs

Creative Biolabs offers SIAT^® in vitro class II HLA/MHC binding assays that effectively measure the binding affinity of hypothetical epitope peptides to HLA alleles. On this basis, we can rule out false positives or negatives from the computer model. The identification of immunogenic peptides through SIAT^® in vitro class II HLA/MHC binding assay is tremendously beneficial for the development of new biomedicines. Based on the outcomes of the assay, manipulations of the protein structure to remove these T cell epitopes can be done, helping to avoid any adverse effects and production of anti-drug antibodies (ADAs).

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.