surprise! There is such a skill in optimizing NC membranes and proteins.
Colloidal gold technology plays an immeasurable role in traditional diagnosis, and has been widely used in the detection of diseases, drugs, food, pets, and veterinary medicine. With the development of colloidal gold labeling technology and in-depth research by R&D personnel on colloidal gold rapid detection technology, the types of membrane-based rapid immunochromatographic detection products on the market have increased rapidly.
Although there are many types of packaging designs for immunochromatography products, in fact, the current mainstream commercial detection kits mainly come in the following two forms. The most commonly used testing formats are lateral flow or chromatographic quantitative testing, which are usually used in clinics or by direct sales customers. Another form of detection is vertical spot filtration, which requires strong operational skills and is limited to research use.
Regardless of the detection format used by the reagents, the production of sensitive and reproducible detection reagents requires reagent manufacturers to adopt effective methods to prepare detection line working solutions. Manufacturers of rapid diagnostic reagents are often interested in articles on how to optimize their assay lines. These articles may assist developers in the discussion of the fundamentals of protein attachment to nitrocellulose membranes and highlight common issues faced by developers in the development of immunochromatographic reagents. Since the problem of protein adsorption to NC membranes is common in lateral chromatography, this article focuses on this aspect.
Importance of protein adsorption:
In immunochromatographic detection, proteins are fixed to the NC membrane as a capture reagent for the sample to be tested. Since the detection results completely depend on the good adsorption effect of the capture reagent on the membrane, uniform and good adsorption of proteins on the membrane is very important for the detection results.
Since NC membranes were first used for protein adsorption, there have been considerable studies on protein adsorption to NC membranes, but the exact mechanism of protein adsorption to NC membranes remains unclear. Although multiple forces play a role in bonding, especially hydrophobic forces, hydrogen bonds, and electrostatic interactions, the importance and clear effects of each force remain elusive. There are currently two reasonable modes of action. The first model believes that the protein is initially adsorbed to the surface of the NC membrane through electrostatic interactions, and the long-term binding is accomplished through hydrogen bonds and hydrophobic interactions. Although this principle is difficult to prove, it is consistent with the experimental conclusions of published literature and is also the most accepted mechanism of action.
The second model believes that the protein first binds to the NC membrane through hydrophobic interactions and then firmly binds to the NC membrane through electrostatic forces. This binding model is consistent with a large number of published literature results. However, the electrostatic interaction mechanism cannot provide a reasonable explanation for the long-term stable adsorption of proteins on NC membranes using drying or ethanol adsorption methods.
Regardless of how protein binding forces are balanced, researchers must consider all forces affecting protein adsorption when optimizing protein adsorption to a specific NC membrane. This point of view inevitably affects the selection of test strip materials and their processing technology. For example, if product developers use buffers that extremely reduce electrostatic or hydrophobic interactions, the adsorption capacity of the protein will be drastically reduced. In the same way, sufficient drying after protein spotting is very important to ensure that the protein is stably fixed to the NC membrane for a long time.
The material selected by the manufacturer can effectively adsorb proteins to the NC membrane. There are usually three types of materials that affect the adsorption of proteins to 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 hydrogen bond formation (such as formamide, urea), and 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. Their mechanism of action may be the result 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 will be very obvious on the detection line of the test results (. If the amount of protein bound to the membrane is too low, then the detection in the results The color development 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 development instead of bright and clear, making the detection The results are difficult to interpret. Under extreme conditions, if the physical adsorption between the protein and the NC membrane is too weak, the flowing protein detector and surfactant solution may wash away the fixed protein from the NC membrane, resulting in a wider display or not at all. Unclear test lines make it difficult to interpret test results.
In vitro diagnostic reagent developers often encounter the above problems, and these problems significantly extend the development cycle of immunochromatographic detection reagents. In order to understand how to solve the above problems, researchers should first have a firm grasp of the various factors that affect the binding of proteins to NC membranes, including the inherent properties of the material and its pre-detection processing.
Factors affecting protein adsorption:
When studying the binding of protein capture agents to NC membranes, developers should consider each of the following five key factors that influence the protein binding mechanism.
Ø Working buffer to dissolve captured proteins;
Ø NC membrane used to fix and capture proteins;
Ø Capture the protein itself;
Ø A system for spotting captured proteins on NC membrane;
ØEnvironmental humidity when capturing protein spots.
Optimization:
Although many R&D laboratories have thoroughly studied the properties of 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 overlooked steps are often well thought out before development and therefore have little opportunity for further adjustments during development. By not optimizing those factors, developers often focus on optimizing what they think must be optimized. ,
1. Capture agent: With different detection items, the proteins used as capture agents vary. Even if the capture agents differ slightly, no one capture agent is exactly the same as another. Different protein capture agents have different adsorption capabilities to different membranes, and perhaps this factor is crucial. As a more uniform protein capture agent, monoclonal antibodies are relatively simple to optimize the binding process to the NC membrane, 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 and membrane binding conditions is relatively complex. For example, IgA and IgM have structural or steric hindrance and other factors, making it difficult to optimize their binding conditions to membranes. For example, BSA, protein A and protein G are easily adsorbed to solid-phase carriers due to their chemical properties or too large molecules, making it very difficult to adsorb them to the NC membrane. ,
2. Instruments and equipment: There are still some problems with the capture agent spraying system. Most of the commercially available instruments and equipment (capture agent spraying film, marking) have their own advantages and disadvantages. Variable parameters include the ability to spray a given volume, access to strips, pads or membranes, spray speed and post-spray treatments. The best solution that in vitro diagnostic reagent manufacturers 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 specific capture line spray equipment.
3. Environmental humidity: The ambient humidity during film dispensing seriously affects the quality of the capture line, especially the film spraying system. If the air humidity is too low, static charges will accumulate on the NC film, causing spots to easily appear when proteins are sprayed on the surface of the NC film, and hydrophobic spots will easily appear on the surface of the NC film. If the air humidity is too high, the capillary effect of the NC membrane on the captured protein will be strengthened, which will easily cause the capture line to broaden or spread. Under normal circumstances, the optimal relative humidity of the spot film environment should be maintained at 45-65%. In order to ensure the uniformity of raw materials, the NC film should be balanced in the working environment according to the optimal balancing time determined by the corresponding test before dispensing the film.
4. Optimization of working buffer: Since protein capture agents vary widely, the working buffer systems with the maximum binding capacity of different proteins vary. There are two important factors that affect the spot film working buffer. ,
Ø Protein solubility (i.e., the amount of protein used to adsorb to the NC membrane);
Ø Stability of protein molecules (i.e. tendency to aggregate or dissolve in water)
In order to ensure sufficient protein spraying on the capture line, the capture protein must first be dissolved in the spotting buffer, and maintaining a certain ion concentration in the spotting buffer can ensure the solubility of the protein. Although the dot film working solution has a certain ionic strength that helps control the pH value of the capture agent, it can also interfere with the electrostatic interactions of protein binding. Therefore, it is critical to determine the lowest possible ionic strength that maintains sufficient concentration of the captured protein.
If a specific concentration of protein molecules is stable in a solution, it will dissolve in the solution. But if its energy state is conducive to the formation of a solid, then the amount of protein adsorbed to the NC membrane is more than the amount of protein stably dissolved in the solution. This energy state can be induced using destabilizers or precipitants, but excessive induction can cause other problems. If the protein precipitates before spotting the membrane, the entire reagent system will be highly unstable and almost completely non-reproducible, resulting in a drastic reduction in the amount of remaining dissolved protein adsorbed to the NC membrane, and the precipitate can also cause problems such as clogging the spray equipment pipeline or NC membrane micropores and other issues. In some cases, the protein must be in an unstable state in order to achieve the appropriate amount of protein adsorption during the spotting process. There are exceptions in some cases.
The above analysis shows that the binding effect of protein and NC membrane can be changed by adjusting the properties of the buffer system for protein spraying. The core properties involve the ionic strength, acidity and concentration of the 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 the solution, the ionic strength of the solution should be as low as possible, which can increase the speed of protein binding to the NC membrane. At the same time, researchers should also note that high concentrations of salt can cause protein precipitation, and a large amount of salt during the drying process after spraying can interfere with the stability and sensitivity of the detection reagent.
