Four main aspects that affect the binding of proteins to nitrocellulose membranes
1. Buffer for dissolving the capture reagent
2, the film itself
3, the capture reagent itself
4, the surrounding environment when the protein is combined (mainly refers to humidity)
2, the film itself
3, the capture reagent itself
4, the surrounding environment when the protein is combined (mainly refers to humidity)
Buffer use
A suitable buffer must be capable of solubilizing the capture protein to achieve the desired concentration for the application and to remain stable (in other words, to solubilize the protein and maintain its binding capacity). Getting the best combination means creating the best conditions for protein to be dispersed in the solid phase. The optimal conditions for protein to membrane binding vary with capture proteins. The use of a capture line in a buffer must require that the protein dissolved therein be at a suitable concentration. If the concentration is too low, you will not be able to capture enough protein at the capture line. If the protein forms a precipitate, it will impede the pores on the membrane and cause irregular flow when the sample passes through the test area of ​​the test strip. Precipitation can also block instrumentation tubes and holes and damage the instrumentation. Although destabilizing agents and coprecipitants can facilitate the distribution of proteins on the surface of the solid phase, it is necessary to find a suitable protein solvent at the outset. pH and ion concentration are two important parameters that need to be optimized.
A suitable buffer must be capable of solubilizing the capture protein to achieve the desired concentration for the application and to remain stable (in other words, to solubilize the protein and maintain its binding capacity). Getting the best combination means creating the best conditions for protein to be dispersed in the solid phase. The optimal conditions for protein to membrane binding vary with capture proteins. The use of a capture line in a buffer must require that the protein dissolved therein be at a suitable concentration. If the concentration is too low, you will not be able to capture enough protein at the capture line. If the protein forms a precipitate, it will impede the pores on the membrane and cause irregular flow when the sample passes through the test area of ​​the test strip. Precipitation can also block instrumentation tubes and holes and damage the instrumentation. Although destabilizing agents and coprecipitants can facilitate the distribution of proteins on the surface of the solid phase, it is necessary to find a suitable protein solvent at the outset. pH and ion concentration are two important parameters that need to be optimized.
pH level
The pH level in the buffer largely affects the binding of the protein to the membrane. Since the NC membrane has no acid protons, it is the nature of the protein that can only be changed when pH is adjusted. The solubility of a protein is minimal at its isoelectric point (pI). Too large or too small a pH can denature the protein, causing a dramatic change in its binding properties, and even aggregation and precipitation. To sum up, when exploring the conditions for making the protein solution ideally distributed in the solid phase, the optimal buffer conditions for the detection line are when the pH is equal to or close to pI.
Common buffer systems are phosphate, borate, carbonate and Tris salt buffers. It is worth noting that in the lateral flow detection application, the capture molecules need to be dried on the membrane, which means that all components of the buffer, except the volatile components, need to be dried on the membrane. This makes volatile buffers such as ammonium acetate or ammonium carbonate particularly preferred. However, high concentrations of primary ammonium (eg, from Tris buffer) can cause a salt bridge between the acidic amino acid residues (glutamic acid, aspartic acid) and the buffered ionic amino group in the capture protein to cover the binding site.
Ion concentration
The dissociation of ions in the electrolyte solution reduces the interaction between the protein and the protein, so the solubility of the protein in the solution is significantly enhanced (salt solution effect) in a specific ion concentration range. The too high ionic strength is the opposite (salting out effect). This is because a large amount of dissolved ions occupy more and more water molecules at this time, causing the dissolution layer around the protein molecules to collapse, and the protein molecules interact to form aggregation and precipitation. According to this principle, the protein should be dissolved in the application buffer and kept in an environment conducive to the solid phase distribution of the membrane. The salting out effect can reasonably reduce the stability of the molecules in the solution. However, certain salts such as ammonium sulfate can stabilize the protein solution and increase the difficulty of control. Small changes in salt concentration (as caused by evaporation) can severely affect the extent of precipitation. In addition, the use of these types of buffers to dry a large amount of salt into the membrane system can seriously interfere with the entire test. The most popular opinion is to make the ionic strength in the buffer as low as possible to reduce the salt dissolution effect. Buffer salts (PBS, TBS) are generally not recommended.
Membrane effect
The size of the membrane pore size determines the internal surface area of ​​the membrane, while the membrane surface area affects protein binding as well as summary and ability. The smaller the membrane pore size, the higher the protein knot and capacity. In addition, since the capture reagent diffuses after the start of the test, the size of the aperture can affect the appearance of the test line. In membranes with high lateral flow rates, the protein begins to diffuse rapidly from the application area, forming a wider and more diffuse line. For all membranes, the capture reagent bound in the deep part of the membrane failed to produce a signal, the only exception being the dye binder, which was still visible at 10 μm under the nitrocellulose membrane. The larger the membrane pore size, the greater the percentage of diffusion of the capture protein in the membrane. If the detection reagent is a magnetic bead, then the above situation need not be considered. Because the particulate matter at the detection line, regardless of its position in the membrane, produces a signal. For membranes with a constant surface area, the level of protein binding is only related to the type of polymer and the processing reagents that affect the surface energy of the membrane. The base polymers used for film production, although available from different suppliers, have slightly different material properties from each source. Nitrocellulose is produced by esterification of cellulose and is therefore a natural based product. In addition, different membrane manufacturers have different treatment methods for the membrane. For product developers, it is preferable to arrange some experiments to evaluate the protein binding properties of various membranes that may be used for testing. The range of protein binding capabilities should also be included in the membrane instructions used to test strips.
The size of the membrane pore size determines the internal surface area of ​​the membrane, while the membrane surface area affects protein binding as well as summary and ability. The smaller the membrane pore size, the higher the protein knot and capacity. In addition, since the capture reagent diffuses after the start of the test, the size of the aperture can affect the appearance of the test line. In membranes with high lateral flow rates, the protein begins to diffuse rapidly from the application area, forming a wider and more diffuse line. For all membranes, the capture reagent bound in the deep part of the membrane failed to produce a signal, the only exception being the dye binder, which was still visible at 10 μm under the nitrocellulose membrane. The larger the membrane pore size, the greater the percentage of diffusion of the capture protein in the membrane. If the detection reagent is a magnetic bead, then the above situation need not be considered. Because the particulate matter at the detection line, regardless of its position in the membrane, produces a signal. For membranes with a constant surface area, the level of protein binding is only related to the type of polymer and the processing reagents that affect the surface energy of the membrane. The base polymers used for film production, although available from different suppliers, have slightly different material properties from each source. Nitrocellulose is produced by esterification of cellulose and is therefore a natural based product. In addition, different membrane manufacturers have different treatment methods for the membrane. For product developers, it is preferable to arrange some experiments to evaluate the protein binding properties of various membranes that may be used for testing. The range of protein binding capabilities should also be included in the membrane instructions used to test strips.
Capture reagent
Classic capture reagents used in immunoassays are antibodies, mostly IgG. Despite this, there are still no two identical capture reagents present. The most straightforward way to optimize binding is to use monoclonal antibodies as capture reagents. Polyclonal antibodies make the entire optimization more difficult to control due to impure protein components. Each subgroup can be slightly different in terms of optimization. IgA or IgM may be more difficult to optimize due to potential structural or spatial problems. The capture protein other than the antibody has an increased difficulty in optimization due to its chemical nature or molecular weight. In general, the stronger the molecular weight, the stronger the binding of the protein to the surface. Other protein impurities in the preparation of the product as well as the carrier protein will compete with the capture antibody for binding to the surface. Generally, the protein binding capacity of the NC membrane exceeds the amount of capture protein that needs to be fixed. However, in the preparation, when the ratio of the capture molecules of the test line is too low, the experiment will not be possible.
Environmental humidity
Nitrocellulose membranes are highly susceptible to environmental influences, and dry environments can enhance membrane hydrophobicity. Low ambient humidity has a significant effect on the membrane, causing the membrane to generate considerable static electricity. Especially for the film without backing, not only the handling is difficult, but also the dust particles are easily adsorbed, and the dust can hardly be removed without damaging the film. Low humidity will have a major impact on test line results when adding capture reagents, especially when using contactless applications. At low humidity, water droplets splash on the membrane, which is repelled due to its own negative charge, resulting in a "satellite point." In extreme cases, continuous lines cannot be formed because most of the water droplets cannot travel in a straight line. Protein samples flow rapidly at very high ambient humidity, creating a broad or diffuse capture line. Although most experienced test developers have their own set of optimal humidity ranges, the best known humidity is around 50% RH (40-60% at 18-22 °C normal room temperature). It is recommended that the membrane be equilibrated in air prior to squeezing the capture zone, and the time required for equilibration should be determined experimentally.
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