The biological analysis described in this article is to determine the concentration of biological drugs in animal and human body fluids or tissues (this article specifically refers to protein biological drugs, including monoclonal antibodies, cytokines, auxins, fusion proteins, etc.). Most bioanalytical methods are based on immunoassays (Immunoassays), or more broadly called, ligand binding assays (LBA). These methods involve the use of a series of reagents, such as anti-drug antibodies, other antibodies, biological drug target proteins, and so on. Another major bioanalysis platform for macromolecules is liquid chromatography-mass spectrometry (LC-MS/MS), and a combination of LBA and LC-MS/MS to a certain extent (LBA-LC-MS/MS).
In short, in the analysis of macromolecules, the target analyte (analyte of interest) is a protein in a very complex biological matrix background; therefore, its quantitative analysis has a series of unique challenges. This series begins with an introduction to the basic knowledge of LBA.
1. Overview of LBA test methods in bioanalysis
Ligand binding test method (LBA, also called immunoassays) is a commonly used analytical tool for quantitative determination of biomolecules (target analytes, Analyte) based on binding interactions with other biomolecules Concentration in biological fluids. LBA requires the use of at least one biomolecule to quantify the target analyte; this biomolecule is usually called the key reagent, which specifically binds the target analyte. LBA can be divided into two categories: (1) liquid phase binding test, in which the binding reaction between the target analyte and the labeled detection reagent occurs in the solution phase (Figure 1A); (2) solid phase binding test, one of which Key reagents are immobilized on a solid surface, such as a microplate or resin (Figure 1B), to capture the target analyte in the sample. Liquid phase testing usually requires separation steps such as chromatography or electrophoresis and/or centrifugation to separate the target analyte-labeled detection reagent from unbound molecules for quantification. These separation processes are lengthy and/or cumbersome. The current application of magnetic beads alleviates these challenges; however, separation steps such as precipitation of complexes are still required. Most modern LBAs use stationary phase testing, such as microtubes, microplates, or coated chips.
The LBA test method is the main quantitative analysis method when evaluating the PK/TK of biological drugs; the specificity and selectivity of this method depends on the target analyte and other biological molecules, such as receptors, antibodies against candidate biological drugs, and nucleic acid compatibility. Ligand (aptamer), the interaction. The instrument response observed in this method is indirectly related to the concentration of the biological drug, that is, the source of the detection signal is an enzymatic chemical reaction or a radiochemical reaction, and these reactions are part of various binding interactions. In general, the physical and chemical properties of macromolecules cannot directly generate detection signals for such quantitative analysis methods. Due to the inherent nature of these binding interactions, the dynamic (quantitative) range of the LBA standard (calibration) curve is relatively narrow, and it is also a non-linear, sigmoidal curve.
Figure 1. Schematic diagram of the liquid phase (A) and solid phase (B) binding test using radiolabeled detection reagents. In the liquid phase test, the binding reaction occurs in solution; then, the bound and unbound detection reagents are separated. In solid phase testing, a key reagent is passively immobilized on a solid surface, such as a microplate or resin. This reagent binds the target analyte, and the target analyte binds to the labeled detection reagent.
2. The birth of radioimmunoassay
The method for measuring human insulin developed by Yalow and Benson in 1960 is a landmark example of radioimmunoassay (RIA); Yalow won the Nobel Prize in 1977 for this reason. Briefly, guinea pigs were immunized with protamine zinc bovine insulin or commercial conventional bovine insulin to obtain insulin-binding antibodies as the key reagent of the RIA method. In this method, due to the lack of crystals of human insulin, I131-labeled crystalline bovine insulin (Insulin-I131) is used as a tracer. The principle of this RIA is based on the strong binding of human insulin in the sample to the insulin-binding antibody in the guinea pig serum (step 1 in Figure 2), and competition with the binding of insulin-I131 of known concentration to the antibody (step in Figure 2) 2-3). The binding of insulin-I131 and insulin-binding antibody is carried out in solution. The separation of free insulin-I131 and antibody-bound insulin-I131 is achieved by paper chromatography electrophoresis technology, which makes it possible to quantitatively determine insulin-I131; The concentration of -I131 represents the concentration of insulin in the sample. In this method, the lowest quantifiable insulin concentration is 1.4 μU.
After the publication of this landmark study, RIA was widely used in the late 20th century. When using appropriate key reagents, RIA has extremely high sensitivity and selectivity; and its main challenges include the licensing requirements for the use and disposal of radioactive materials, the labeling efficiency of radioisotopes, cost and its limited half-life. Within ten years of the application of RIA, that is, in the early 1970s, non-isotopic immunoassays, such as enzyme immunoassays (EIA), appeared. EIA is often called ELISA, or enzyme linked immunosorbent assay (enzyme linked immunosorbent assay).
Engvall and Perlmann first described an ELISA method to quantify rabbit immunoglobulin G (rabbit IgG) in 1971. Simply put, anti-rabbit IgG extracted from goat serum is used to bind rabbit IgG. Then, rabbit IgG combined with an alkaline phosphatase (ALP) is used to compete with rabbit IgG and bind with anti-rabbit IgG serum, thereby establishing the relationship between concentration and instrument response (signal). The instrument signal generated is inversely proportional to the amount of IgG in the sample. After that, different enzymes such as horseradish peroxidase (HRP), β-galactosidase (GAL) and luciferase (luciferase) are used to make EIA have the same sensitivity as RIA.
Figure 2. The schematic diagram of the insulin radioimmunoassay originally invented by Yalow. The insulin in the sample is combined with the anti-insulin serum in solution (step 1). With a known concentration, I131-labeled bovine insulin is added to the reaction solution (step 2); then, the insulin in the sample and I131-labeled bovine insulin (step 3) compete with each other for binding to the antibody. Using a standard curve prepared from human insulin, the concentration of insulin replaced in the sample can be calculated from the concentration of I131-labeled bovine insulin bound to the insulin antibody.
3. Common ELISA format
The competitive ELISA format is based on the binding competition in the initial insulin RIA and IgG EIA methods. The competition between the target analyte from the sample and the binding of the reference analyte to the analyte-specific antibody is the key to this format. Either antibody or target analyte can be labeled with enzyme and used in the binding reaction. When using labeled antibodies (Figure 3Ai), the reference analyte is first passively adsorbed into the wells of the microplate. Then add a sample containing the target analyte, such as cell lysate, serum, etc., and incubate with a fixed concentration of labeled antibody. The target analyte in the sample competes with the immobilized analyte to bind a limited number of labeled antibodies, and the signal generated by the subsequent enzymatic reaction is inversely proportional to the concentration of the target analyte in the sample. Alternatively, solid-phase adsorbed antibodies and labeled analytes can be used (Figure 3Aii). The target analyte in the sample is incubated with a fixed amount of labeled analyte and competes with each other for binding to the immobilized antibody. As in the previous example, the signal generated is inversely proportional to the concentration of the target analyte in the sample.
Direct ELISA is the simplest ELISA format. The target analyte in the sample is adsorbed into the wells of the microplate. The detection of the target analyte can be directly achieved through enzyme coupling reagents or labeled detection antibodies, as shown in Figure 3B.
Figure 3. Schematic diagram of competitive (Ai & Aii), direct (B) and indirect (C) ELISA test formats. Analyte represents the target protein to be determined. In the competitive ELISA, the target analyte or antibody can be labeled; in the direct or indirect ELISA format, only the detection antibody is labeled.
Indirect ELISA is similar to direct ELISA, but the primary antibody is not labeled. The detection of the target analyte is achieved by adding an enzyme coupling reagent or labeling the detection antibody again to bind it to the main antibody, as shown in Figure 3C.
Direct ELISA is faster because it has one less step than indirect ELISA. It is usually used for routine tests that need to be completed quickly; a commercial home pregnancy test is an example of this format. Although indirect ELISA involves another step, signal amplification is usually better than direct ELISA. Therefore, indirect ELISA is generally more sensitive than direct ELISA and can measure lower abundance proteins. There are some disadvantages to allowing the target analyte from the sample to be directly adsorbed to the microplate. Because in the plate washing step, the target analyte may be washed away, which may increase the variability of the test. In addition, other proteins that bind non-specifically to the microplate may cause false positive results. To overcome these challenges, other more reliable formats have evolved; among them are commonly referred to as sandwich, bridge, or competitive ELISA (in the sandwich or bridge ELISA format). In these formats, a biological reagent that specifically binds to the target analyte is first adsorbed into the microplate.
Sandwich ELISA requires two different reagents or antibodies, one for capture and the other for detection, target analyte. These reagents bind to different epitopes (binding sites) of the target analyte to form a sandwich format (Figure 4A). The resulting interaction has a particularly high specificity, because both the capture and detection steps require specific epitope recognition to generate a detection signal. Bridge-linked ELISA is often used to determine the concentration of antibodies or other target analytes with two identical antigen binding sites (divalent). The same reagent as the capture reagent can be labeled to detect a divalent target analyte; therefore, the target analyte can be regarded as a bridge between the capture and detection reagents (Figure 4B). Sandwich or bridge ELISA formats can be designed to be competitive.
Figure 4. Schematic diagram of sandwich ELISA (A) and bridge (B) ELISA.
The above format is the basic design of ELISA. All formats can be adjusted using competition or inhibition conditions to determine antigen or antibody (Figure 5). All methods require pre-reaction/incubation with reagents to achieve the best state. These optimal states can then be challenged by adding antigens (Figure 5a) or antibodies (Figure 5b). As the free antigen (antibody) in the solution increases, the amount of antibody (antigen) that can bind to the immobilized substrate decreases. After the washing step, a chromophore substrate is added to generate a signal (color change or light emission). The signal changes caused by the antibody/antigen challenge reveal information about competing antigens/antibodies. Competitive ELISA is particularly useful for determining the concentration of antigen in complex mixtures, especially when comparing unknown samples that may contain antigens with similar samples that contain known amounts of purified antigen.
4. Key analytical reagents in LBA method
Key term Immune epitope (Epitope)
Immune epitope (epitope) is a part of antigen molecules generally recognized by the host immune system. When the epitope of the antigen interacts with the complementarity determining region of the antibody induced by it in the form of a non-covalent bond, specific recognition occurs.
Antibody affinity (affinity) represents the intensity or strength of binding of an antibody to its single target analyte/antigen, and is represented by the dissociation constant (Kd). Low-affinity antibodies bind weakly to the antigen and are easily dissociated; while high-affinity antibodies bind very strongly to the antigen and are not easily dissociated during multiple washing steps. From an ELISA point of view, the latter is the first choice and is usually used to capture the target analyte.
Avidity is a measure of the overall strength of binding of an antibody to
multiple antigenic determinants. The affinity of an antibody to a certain
binding site does not always reflect the strength of the antibody-antigen
interaction. For example, immunoglobulin G (IgG) has two antigen binding sites
(2-valent), while IgM has 10 antigen-binding sites (10 valent). Affinity refers
to the overall strength of IgG or IgM for 2 or 10 antigen molecules,
Figure 5. The detailed principle and flow chart of competitive ELISA. Antigen competition ELISA (a); antibody competition ELISA (b).
The most important part of an ELISA method is the test reagent, which determines the sensitivity, specificity and quality of the ELISA method. ELISAs are often used in drug development: in PK evaluation, quantitative determination of the concentration of protein biopharmaceuticals; determination of endogenous proteins and biomarkers; detection of the presence of anti-drug antibodies for immunogenicity evaluation, etc. The key reagents of choice for the LBA method are monoclonal antibodies (MAbs) or polyclonal antibodies (PAbs), rather than recombinant target proteins. In order to produce PAbs or MAbs, it is necessary to immunize biological drugs, or biomarker proteins, and their adjuvants or carriers to appropriate host animal species according to the relevant application. For PAbs, rabbits, goats and sheep are the most commonly used host species; because of their large size (the amount of antibodies produced), blood vessels and strong immune responses are easy to find.
Each antigen used for immunization is highly complex, so it can present a large number of epitopes that can be recognized by different lymphocytes; these lymphocytes are then activated. The activated lymphocytes proliferate, differentiate into plasma cells, and secrete a mixture of polyclonal antibodies. The PAb library (mixed body) represents a collective population of different antibodies that can recognize multiple epitopes of the antigen (for ELISA analysis). In order to produce MAbs, individual lymphocytes need to be further separated and fused with myeloma cells to produce immortal hybridoma cells, thereby continuously producing specific MAbs. Therefore, antibodies of the same clone only recognize a single epitope of an antigen. MAbs or PAbs are frequently used in ELISA, and their choice (for each ELISA format) depends on many considerations: such as availability, affinity, specificity and cross-reactivity (Cross- reactivity).
Specificity and cross-reactivity (Cross-Reactivity)
The specificity of an antibody is the ability to specifically bind to the relevant antigen. It is not the same as the selectivity of the ELISA method, but is related. Selectivity is a quantitative analysis method. In the presence of other potentially interfering components, that is, the ability to identify and quantify the concentration of the target analyte from many other unrelated proteins. Cross-reactivity refers to the ability of an antibody to bind to multiple antigens in addition to the target antigen. Cross-reaction occurs when the epitope of the antigen (target analyte) bound to the antibody reagent is similar to other proteins. Generally speaking, specificity and cross-reactivity have a great influence on the selectivity and matrix effect of the ELISA method. If a high-affinity antibody is used as a capture reagent, the antibody/target analyte complex can be effectively formed in a “mixed” sample containing multiple proteins; the matrix effect will be reduced and the selectivity of the method will eventually be improved .
5. 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.