NC membrane adsorption antigen antibody common problem processing!
Developers of membrane-based assays should critically consider the many factors that affect protein binding to nitrocellulose membranes, including the inherent properties and handling of raw materials. With the development of colloidal gold labeling technology and the in-depth research of colloidal gold rapid detection technology by R&D personnel, the types of membrane-based rapid immunochromatographic detection products on the market have rapidly increased.
Although there are many types of packaging designs for immunochromatography products, in fact, the current mainstream commercial detection kits are mainly in the following two forms. The most commonly used assay format is lateral flow or quantitative chromatography, which is usually performed in the clinic or by direct marketing customers. Another form of detection is vertical dot percolation, which requires strong operator skills and is limited to research use.
Regardless of the assay format used, the production of sensitive, reproducible assays requires reagent manufacturers to employ efficient methods for preparing assay line working solutions. Manufacturers of rapid diagnostics are often very interested in articles on how to optimize their test lines. These articles can help researchers relate the discussion of the fundamentals of protein immobilization to nitrocellulose membranes and highlight common issues that researchers face when developing immunochromatographic reagents. Since the problems related to protein adsorption to NC membranes are common in lateral flow chromatography, this article focuses on this aspect.
In immunochromatographic detection, proteins are immobilized on NC membranes as capture reagents for samples to be tested. Since the detection result depends entirely on the good adsorption effect of the capture reagent on the membrane, the uniform and good adsorption of protein on the membrane is very important for the detection result.
Since NC membranes were first used for protein adsorption, there have been quite a few studies on protein adsorption on NC membranes, but the exact mechanism of protein adsorption on NC membranes is still uncertain. Although multiple forces play a role in binding, such as hydrophobic forces, hydrogen bonds, and electrostatic interactions among others, the importance and precise effects of each force remain elusive. There are currently two plausible modes of action. The first model holds that proteins are initially adsorbed to the NC membrane surface through electrostatic interactions, while the long-term binding is accomplished through hydrogen bonding and hydrophobic interactions. Although this principle is difficult to prove, it is consistent with the experimental conclusions of published literature and is the most accepted mechanism of action.
The second model holds that proteins first bind to the NC membrane through hydrophobic interactions, and then firmly bind to the NC membrane through electrostatic forces. This binding mode is consistent with the results of a large number of published literatures. However, the electrostatic interaction mechanism cannot provide a reasonable explanation for the long-term stable adsorption of proteins on NC membranes by drying or ethanol adsorption methods.
Regardless of how the protein binding forces are balanced, researchers must comprehensively consider all forces that affect protein adsorption when optimizing protein adsorption to a specific NC membrane. This point of view inevitably affects the choice of test strip sheet and its processing technology. For example, if a product developer chooses a buffer that greatly reduces electrostatic or hydrophobic interactions, the adsorption capacity of the protein is drastically reduced. In the same way, sufficient drying after protein spotting is very important to ensure long-term stable fixation of proteins on NC membranes.
The material selected by the manufacturer is capable of efficiently adsorbing proteins to the NC membrane. There are generally three types of materials that affect protein adsorption on NC membranes: non-specific proteins, substances that affect electrostatic interactions, and hydrophobic interactions. Common substances that can reduce protein binding include other proteins that compete for protein binding sites, such as BSA, animal serum, substances that interfere with the formation of hydrogen bonds (such as formamide, urea), substances that affect the formation of hydrophobic interactions (such as Tween, Triton, Brij). Synthetic conjugates such as polyvinyl alcohol, polyethylene glycol, and polyvinylpyrrolidone can also affect protein binding, and their mechanism of action may be the result of the combined effect of inhibiting the binding of one or more proteins to the NC membrane.
If the amount of protein bound to the NC membrane is insufficient or the protein binding force is not strong enough, there will be quite a few problems, which are very obvious on the detection line of the test result (. If the amount of protein bound to the membrane is too low, then it will be detected in the result The color of the line is weak and the detection sensitivity is reduced. If the protein cannot be firmly adsorbed on the NC membrane, diffusion will occur before the protein is adsorbed on the NC membrane, resulting in a wider detection line and weaker color instead of bright and clear, making the detection The results are difficult to interpret. Under extreme conditions, if the physical adsorption of the protein to the NC membrane is too weak, the flowing protein assay and surfactant solution may wash the immobilized protein from the NC membrane, resulting in a wider or no display. Unclear test line, difficult to interpret test results.
Developers of in vitro diagnostic reagents often encounter the above problems, and these problems significantly prolong the development cycle of immunochromatographic detection reagents. In order to understand how to solve the above problems, researchers should first firmly grasp the various factors that affect the binding of proteins to NC membranes, including the inherent properties of materials and their pre-detection processing.
Factors affecting protein adsorption:
When studying the binding of protein capture agents to NC membranes, researchers should consider each of the following five key factors that affect the mechanism of protein binding.
Ø Working buffer to dissolve captured protein;
Ø NC membrane used to immobilize the capture protein;
Ø Capture the protein itself;
Ø A system for spotting captured proteins on NC membranes;
Ø Capture the ambient humidity when protein spotting. While many R&D laboratories have thoroughly studied the properties of the working buffers and membranes used in immunochromatographic assays, it is not possible for them to fully study or optimize the systems and capture reagents they use. These neglected steps are usually considered well in advance of development, so there is little opportunity for further adjustment during development. By neglecting to optimize those factors, developers often focus on optimizing what they think must be optimized.
Capture agent: With the different detection items, the proteins used as capture agents are different. Even if the capture agents differ slightly, no one capture agent is exactly the same as another capture agent. Perhaps this factor is critical because different protein capture agents have different adsorption capacities to different membranes. As a relatively uniform protein capture agent, monoclonal antibodies are relatively simple to optimize the binding process to NC membranes, while polyclonal antibodies contain antibodies against a large number of different antigenic determinants, and the optimal binding conditions of different antibodies may be slightly different, resulting in The optimization process of protein-membrane binding conditions is complex. For example, there are factors such as structure or steric hindrance of IgA and IgM, and it is difficult to optimize the conditions for their membrane binding. For example, BSA, protein A, and protein G are easily adsorbed on solid phase carriers due to chemical properties or too large molecules, so it is very difficult to adsorb them on NC membranes.
Instrumentation: There are still some issues with capture agent spray systems, and most of the commercially available (capture agent coating, scribing) instrumentation has pros and cons. Variable parameters include the ability to spray a given volume, access to strips, pads or membranes, spray speed, and post-spray handling. The best solution that manufacturers of in vitro diagnostic reagents need is to find the most effective solution to the problems faced in the actual production process, such as raw material problems and production capacity problems. Optimizing other factors can also optimize a particular capture line spray equipment.
Ambient Humidity: The ambient humidity when dispensing the film seriously affects the quality of the capture line, especially for the spray film system. If the air humidity is too low, static charges will accumulate on the NC membrane, which will cause spots when the protein is sprayed on the surface of the NC membrane, and hydrophobic spots will easily occur on the surface of the NC membrane. If the air humidity is too high, the capillary action of the NC membrane on the captured protein will be strengthened, which will easily cause the capture line to widen or spread. Under normal circumstances, the relative humidity of the best spot film environment should be kept at 45-65%. In order to ensure the uniformity of the raw material, the NC film should be balanced in the working environment according to the optimal balance time determined by the corresponding test before filming.
Optimization of working buffer: Due to the wide variety of protein capture agents, the maximum binding capacity of different proteins is different in the working buffer system. There are two important factors that affect the working buffer of the dot membrane.
Ø Protein solubility (that is, the amount of protein used to adsorb on the NC membrane);
Ø Stability of protein molecules (i.e. tendency to aggregate or dissolve in water)
In order to ensure that enough protein sprays the capture line, the capture protein must first be dissolved in the spotting buffer, and the spotting buffer maintains a certain ion concentration to ensure protein solubility. Although the ionic strength of the working solution helps to control the pH of the capture reagent, it can also interfere with the electrostatic interactions that bind proteins. Therefore, it is critical to determine the lowest possible ionic strength that maintains a sufficient concentration of captured protein.
If a protein molecule at a particular concentration is stable in solution, it will dissolve in solution. But if its energy state favors the formation of a solid, then more protein is adsorbed to the NC membrane than is stably dissolved in solution. This energy state can be induced with destabilizers or precipitants, but can cause other problems if induced too much. If the protein precipitates before spotting the membrane, the entire reagent system is highly unstable and almost completely irreproducible, resulting in a drastic reduction in the amount of remaining dissolved protein adsorbed to the NC membrane, and the precipitate can also cause, for example, clogging of spray equipment pipes or NC membrane microporosity and other issues. In some cases, the protein must be destabilized during spotting to achieve adequate protein adsorption, although there are some exceptions.
The above analysis shows that the binding effect of protein and NC membrane can be changed by adjusting the properties of protein spraying working buffer system, in which the core properties involve the ionic strength, acidity and concentration of precipitant used in the buffer.
Ionic Strength: Within a given ionic strength range, protein solubility increases proportionally with increasing salt concentration in the working buffer. In order to reduce the stability of captured protein molecules in solution, the ionic strength of the solution should be as low as possible, which can increase the speed of protein binding to NC membrane. At the same time, developers should also be aware that high concentrations of salt can cause protein precipitation, and a large amount of salt in the drying process after spraying the film can interfere with the stability and sensitivity of the detection reagent.
