Analysis of the role of Biacore assay in drug development

 SPR biosensors have a wide range of applications in life sciences, drug residues, food detection, disease mechanisms, etc. due to their advantages of no labeling, online detection, reproducibility, no sample pre-treatment, etc.

In the fields of protein network regulation and cellular signal transduction, it is often necessary to search for the unknown factors interacting with a known protein to discover the whole regulatory network. Surface plasmon resonance (SPR) technology is a biosensing analysis technique for analyzing interactions between biomolecules and is now commonly used in the Biacore, a biomolecular interaction analysis system.

The Biacore assay can be used for the analysis of binding specificity between biomolecules, concentration quantification, binding kinetics and affinity analysis, and thermodynamic analysis, enabling real-time detection of biomolecular interactions between DNA and proteins, between protein molecules, between drugs and proteins, between nucleic acids and nucleic acids, between antigens and antibodies, and between receptors and ligands.

Principle of Surface Plasmon Resonance SPR Technology

Biacore technology is a new biosensing analysis technique based on a physical optical phenomenon called surface plasmon resonance (SPR).The key to Biacore technology is the use of a biosensor developed based on the SPR phenomenon as a detection system. The three core components of the technology are the sensor chip, the SPR optical detection system and the micro jet chuck.

For the experiment, a biomolecule is now immobilized on the dextran surface of the sensor, and the molecule interacting with it is dissolved on the surface of the chip through which the solution flows. The SPR detector is able to record the molecule in solution into a sensing map with kinetic and affinity data based on tracking the molecule in solution as it binds and dissociates from the molecule on the chip surface throughout the process.

Biacore system has different models, such as intelligent high-performance molecular interaction system, Biacore X100; full-featured molecular interaction analysis system, Biacore T200; the latest generation of ultra-high-sensitivity molecular interaction analysis system, Biacore S200; high-throughput and high-quality interactions data-cum-Biacore 8K, etc.

1. Measuring antibody-antigen interactions.

Protein-antibody interaction is an extremely important research area in the pharmaceutical industry and protein research.Antibody-based biotechnology drugs have presented effective treatments for some chronic diseases, such as bevacizumab for cancer treatment and adalimumab for rheumatoid arthritis.

The SPR technique was applied to measure the interaction of human serum serum albumin antibodies (anti-HSA) with human serum albumin (HSA) immobilized on a microchip.As hypothesized, commercial anti-HSA binds strongly to HSA as well as dissociates extremely slowly from HAS, and steady-state affinity as well as kinetic parameters of binding are calculated from the interactions.

2. Measuring drug-monolayer cell interactions

In vitro cellular assays are widely used in drug discovery, traditionally these assays require labeled material and such analysis is based on post-detection using UV, fluorescence or mass spectrometry. Monolayers of cells are deposited onto the chip surface of the SPR, and the MP-SPR is used to measure the interaction of drug molecules (takayasu and D-mannitol) with monolayers of cells under controlled flow conditions and in real time.

Based on the above measurements, it is possible to distinguish and discriminate between two different drug uptake pathways in paracellular and transcellular.

3. Role in cancer treatment

Using biosensors, the viability of biological agents and antibody titers in patient serum can be detected and quantified, and animal models and human clinical trials can be followed.Using SPR technology to study the signaling pathways controlling gene transcription, cell cycle, cell division and apoptosis in the extracellular environment, so that antagonists of these biochemically acting catalysts can be accurately designed and applied to cancer treatment.

4. Measuring the interaction of small molecule drugs with human serum serum albumin (HSA)

Human serum serum albumin (HSA) is the most important protein in plasma due to its abundance. Its main function is to transport fatty acids and maintain the colloidal osmotic pressure of blood, and it is an important carrier for many hormones and drugs, especially hydrophobic ones.

Binding of drug molecules to HSA increases the half-life of the drug and decreases the concentration of free drug molecules in the blood, which makes it extremely important for clinical care.

Early in the drug discovery process, it is important to determine the binding of plasma proteins, as it is used to evaluate the dose required and the clearance of the drug from the body. Human serum serum albumin (HSA) was immobilized on the chip surface by amino coupling, and the interaction between small molecule drugs and HSA was studied by SPR technique, and steady-state affinity values and kinetic data of binding were determined by measurement.

5. Study the binding kinetics of single chain antibodies with certain specificity

PKCε (protein kinase ε) plays an essential role in a variety of signaling systems and dysregulation of PKCε is associated with several fatal diseases, such as cancer, type II diabetes and Alzheimer's disease. Therefore, specific activators and inhibitors of PKC ε are promising as research tools and future drugs./

Single-chain antibodies (VHHs) to American camel are a new class of monoclonal antibodies that specifically activate or inhibit human PKCε (protein kinase ε). A recent publication reports the analysis of the dynamics of VHHs and PKCε by applying surface plasmon resonance techniques.

VHHs were likewise tested in kinase activity assays to determine the kinetics of activation or inhibition of PKCε.In addition, since VHHs have different effects on PKCε migration in Hela cells, they have PKCε activity in vivo.

SPR sensor can detect a variety of biochemical indicators rapidly, sensitively, accurately and easily, and monitor the interaction between biomolecules and the action link of influencing factors in real time, which can be used as an effective tool for disease diagnosis and mechanism research.

6. Biacore in the field of angling research

In the research of natural products such as Chinese medicine, the core is to identify its effective single components.The process of discovery of these unknown factors is collectively referred to as ligand fishing, which is the direct screening of lysates or cell extracts to find and identify molecules that may interact with "bait proteins".

The SPR-MS-based angling technique can identify new interaction factors at the molecular level by immobilizing "decoy molecules" on the surface of the microarray. When the sample is flowed across the surface of the microarray, the unknown interaction factors can be combined with the "decoy molecules" to achieve the capture of the unknown interaction factors, and then automate the recovery of these interaction factors and identify the unknown factor structures by mass spectrometry.

Biacore research in the field of angling covers many aspects of life activities, such as: (1) identification of drug loci; (2) discovery of new targets; (3) plant signaling and gene regulation; (3) aquatic bio-optimization breeding and disease resistance research; (4) isolation and identification of natural herbal active ingredients, etc.

In recent years, several important proteins based on Biacore pendant technology have been discovered and reported one after another.

The group of Prof. Junping Zhang at the Second Military Medical University used the tumor necrosis factor receptor (TNFR) as the "bait protein" and carried out the screening and isolation of five Chinese herbal medicines by Biacore angling technology. The instrument can automate all the processes of angling experiments, such as sample feeding, binding, washing, incubation, dissociation, recovery and neutralization, and finally the ligand of TNFR was isolated and recovered from rhubarb.

This is the first time for human to discover the PMG component in rhubarb as the ligand of TNFR, which provides a new direction for the development of anti-inflammatory drugs and a new method and idea for the isolation and identification of active ingredients in traditional Chinese medicine, and the related results were published in Anal Bioanal Chem.

Compared with other angling methods, such as cell membrane chromatography, network pharmacology, etc., SPR-MS method has high sensitivity, high resolution, high specificity, simple operation and high automation, which provides great accuracy and convenience for finding unknown molecules.

7. Biacore detection in addition to the medical field is more widely used in drug residues and food detection is also more widely used.

According to online reports, such as the use of SPR biochemical analyzer for rapid detection of medroxyprogesterone acetate (medroxy progesterone acetate, MPA) residues in food of animal origin that are potentially harmful to human health, and the fixation conditions of the antigen, the concentration of antibody and chip regeneration conditions were optimized, while the stability and specificity of the antibody was analyzed.

Due to the short detection time and easy portability, it is suitable for rapid detection in the field and has a wide application prospect.

The SPR bioanalyzer was used to detect sulfadimethoxine, the main component of veterinary drug residues in milk, and the method was found to be simple, rapid, sensitive and reproducible, and suitable for the detection of veterinary drug residues in large quantities of unlabeled milk./

Other researchers used the SPR biosensor system to achieve rapid online detection of E. coli O157:BH7.This is because E. coli O157:H7 is one of the internationally recognized food-borne pathogenic bacteria, and its rapid detection helps to rapidly screen the source of infection and minimize the hazard.

The biosensor's online analysis capability and high sensitivity with micro sample requirements make this instrument an ideal tool for food and environmental safety monitoring.

In conclusion, as a non-labeling technology based on SPR, Biacore assay can reveal the deeper biological properties of biomolecules, and its application areas involve scientific research, drug development and production, quality control and biotechnology industry, etc. It can serve as a powerful means of discovery and confirmation, and accelerate the process of transforming scientific research results into biomarkers with medical value.