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One. Measuring antibody-antigen interactions.

Protein-antibody interaction is a critical 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 human serum albumin antibodies (anti-HSA) interaction with human serum albumin (HSA) immobilized on a microchip. As hypothesized, commercial anti-HSA binds strongly to HSA and dissociates extremely slowly from HAS. The interactions' steady-state affinity and kinetic binding parameters are calculated from the interactions.

Two. 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.

Three.  Roles in cancer treatment

Using biosensors, the viability of biological agents and antibody titers in patient serum can be detected and quantified. 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.

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

Human serum albumin (HSA) is an essential protein in the plasma due to its abundance. Its primary function is to transport fatty acids and maintain the colloidal osmotic pressure of blood, and it is an essential carrier for many hormones and drugs, especially hydrophobic ones. The binding of drug molecules to HSA increases the drug's half-life and decreases the concentration of free drug molecules in the blood, making it extremely important for clinical care. Early in the drug discovery process, it is vital to determine the binding of plasma proteins, as it evaluates the dose required and the clearance of the drug from the body.

Human serum albumin (HSA) was immobilized on the chip surface by amino coupling, and the SPR technique studied the interaction between small molecule drugs and HSA. Steady-state affinity values and kinetic data of binding were determined by measurement.

Five. Study the binding kinetics of single-chain antibodies with particular specificity

PKCε (protein kinase ε) plays an essential role in various signaling systems. Dysregulation of PKCε is associated with several fatal diseases, such as cancer, type II diabetes, and Alzheimer's. 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 monoclonal antibody class that precisely activates or inhibits human PKCε (protein kinase ε). A recent publication reports the analysis of the dynamics of this 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 sensors can detect a variety of biochemical indicators rapidly, sensitively, accurately, and efficiently and monitor the interaction between biomolecules and the active link of influencing factors in real-time, which can be used as an effective tool for disease diagnosis and mechanism research.

Six. Biacore in the field of angling research

In the research of natural products such as Chinese medicine, the core identifies its single practical components. The discovery of these unknown factors is referred to as ligand fishing, 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 flows across the microarray surface, the unknown interaction factors can be combined with the "decoy molecules" to capture 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.

4. Aquatic bio-optimization breeding and disease resistance research.

5. Isolation and identification of natural herbal active ingredients, etc.

Recently, several vital proteins based on Biacore pendant technology have been discovered and reported. The group of Prof. Junping Zhang at the Second Military Medical University used the tumor necrosis factor receptor (TNFR) as the "bait protein." It 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. Finally, the ligand of TNFR was isolated and recovered from rhubarb.

Structural identification using mass spectrometry and chromatography indicated that the recovered ligand was rhodopsin methyl ether-8-o-β-D-glucoside (PMG) (image from web)

The authors used Biacore to determine the affinity of the pure PMG molecule to TNFR at 376 nM, further confirming that PMG is the ligand of TNFR (image from the web)

The results of subsequent cellular experiments were highly consistent with the Biacore results, verifying the accuracy of the Biacore draping method (images from the web)

This is the first time humans discovered 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., the SPR-MS method has high sensitivity, high resolution, high specificity, simple operation, and high automation, which provides excellent accuracy and convenience for finding unknown molecules.

Seven. 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 (medroxyprogesterone 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. In contrast, the stability and specificity of the antibody were analyzed. Due to the short detection time and easy portability, it is suitable for rapid detection in the field and has a broad application prospect. The SPR bioanalyzer was used to detect sulfadimethoxine, the milk's main component of veterinary drug residues. The method was simple, rapid, sensitive and reproducible and suitable for detecting 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 screen the source of infection and rapidly minimizes 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 biomolecules' more profound biological properties. Its application areas involve scientific research, drug development and production, quality control and biotechnology, 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.