The binding principle, influencing factors, formula, spotting environment, sealing, membrane storage of Nitrocellulose membrane and protein

The binding principle, influencing factors, formula, spotting environment, sealing, membrane storage of Nitrocellulose membrane and protein

1. The binding principle of protein and membrane

The binding principle of protein and membrane, the known binding force includes hydrophobic force\H bond\electrostatic force, etc. The exact binding principle is not clear, mainly supported by hypothesis. There are two main hypotheses:

(1) First, the two are combined by electrostatic force, and then they are maintained for a long time by H-bond and hydrophobic interaction.

(2) First, the two are combined by hydrophobic interaction, and then maintained by electrostatic interaction for a long time.

Both hypotheses indicate that the binding process is divided into two steps, the first combination and the long-term combination later. Due to the ambiguity of the binding principle, the work in this area is very dependent on practical experience.

2. Effect of membrane on binding

(1) Membrane pore size

Some technicians tend to use the membrane pore size to distinguish different membranes, but please note that this is only limited to the products of the same manufacturer. If the products are from different manufacturers, this comparison is meaningless. The relationship between membrane pore size and chromatography velocity has been described above.

As the membrane pore size decreases, the actual available surface area of the membrane increases, and so does the amount of membrane-bound proteins. A parameter for estimating surface area is the surface area ratio (the ratio of the actual usable surface area to the planar area of the membrane used).

In addition, the smaller the membrane pore size and the lower the chromatography speed, the longer the time for the gold-labeled complex to pass through the T-line, and the more fully the reaction will be.

Based on the above two points, the conclusion is that the smaller the membrane pore size, the higher the sensitivity. But at the same time, it also slows down the running speed and increases the chance of non-specific binding, that is, the higher the false positive. Therefore, it is necessary to select a membrane suitable for the actual project according to the test results and find a suitable balance point.

(2) Differences in membranes from different manufacturers

This difference mainly comes from two points:

A. When producing films, the sources, types and quantities of polymers and surfactants used are different. In the same way, these two types of substances generally have a greater impact on performance in membrane processing. B. The process is different.

3. Reagents and formulations of biological raw materials and buffer solutions

(1) The use of biological raw materials as biological raw materials for CT lines varies, so only an overview is given here. First of all, the binding of monoclonal antibody to membrane is better than that of polyclonal antibody, mainly because polyclonal antibody has many different surface sites, and the optimal binding conditions of each site and membrane are slightly different, which undoubtedly increases optimization difficulty. Second, the larger the molecular weight, the harder it is for the protein to bind to the solid phase material.

(2) Buffer

Perhaps what everyone is most concerned about is to obtain a formulation with excellent performance, including buffer solution, blocking solution and other processing solution formulations. In fact, I can't provide you with a list of universal recipes. Because different reaction systems require different formulas to support, and the reaction systems of different institutions are different. If you want to catch "fish", learn "fishing" first. In order not to mislead everyone, I only provide ideas on the following issues related to the formula. Please explore the specific formula yourself. The composition of the buffer is generally: PBS (or other buffer system) + action substance (for a specific problem) + pH adjustment. After referring to various previous materials, my personal opinion is that the principle of formula should be simple rather than complicated, and the active substances should be added according to my own needs. Many active active substances that need to be added in the past are no longer needed due to the improvement of membrane manufacturing technology. The recommended buffer system is 0.01M PBS PH7-7.2, which has good adaptability to various antigens and antibodies. The conditions of the active substances are roughly listed as follows: a small amount of NACL reduces the signal intensity and eliminates false positives. Organic alcohol (methanol, isopropanol, etc.) wets the membrane, reduces the static electricity on the membrane, and facilitates the binding and coating. I personally do not recommend it, due to the improvement of the film making process. Surfactants (TW20, TX100), increase hydrophilicity, avoid hollow lines, and enhance color.

Sugar protective agent, slow down the aging speed, and can also increase the hydrophilicity as above. Adjusting the pH to a certain position can eliminate false positives.

4. Sampling environment

Ambient humidity is very important to the dot film process. The optimum humidity is generally between 45-65%.

If the humidity is too low, electrostatic charge is easy to accumulate on the film, and scattered spots are easy to appear on the dotted film, resulting in hydrophobic spots in the test. If the humidity is too high, the capillary action on the film will be strengthened, and the film will easily cause the CT line to widen or even spread. In order to ensure the uniformity of the humidity of the membrane during sample application, the membrane is generally placed in the humidity condition for a period of time before sample application.

5. The relationship between the sampling instrument and the membrane surface

There are currently two spotting methods, film-scribing and non-contact point-membrane. The non-contact membrane type is better than the wiped membrane type, and the imported wiped membrane type is better than the domestic wiped membrane type. Because the scratching method uses a hose to scratch the antibody onto the surface of the membrane, and the physical properties of the membrane itself are soft and brittle, the scratching tube will leave marks on the surface. Due to the better materials and control system used in the imported film scribing machine, the scratches left are lighter, while the domestic instruments are poorer, and the scratches left are more serious. Scratching tends to form resistance to the chromatographic gold-labeled complex, resulting in false positives. At the same time, it is easy to appear a thin line (ghost line) at the position of the T line when running the board, and the ghost line disappears after the running board is over.

6. Film width and spotting position

The width of the film is generally 18mm (or 20mm) and 25mm, which are used for test strips and test boards respectively. However, different T-line sampling positions will bring different sensitivities. As the spotting position moves up, the speed of the gold-labeled compound passing through the position of the T line becomes slower, the reaction time increases, and the sensitivity increases. On the contrary, the sensitivity decreases. This method can be used to vary the sensitivity and eliminate false positives.

7. Diffusion of solution on the membrane

The sample volume of the solution on the membrane is generally 1ul/cm. The diffusion of the solution on the membrane tends to both ends, and what is sprayed is a uniform antibody solution, but when it is dry, the drying speed at the edge of the line is higher than that in the middle. The antibody in the middle will continue to diffuse to both sides, so after drying, the antibody gathers towards both ends of the line. Normally it does not affect your experiment. If you find that the lines are red at both ends and light in the middle, you should consider this problem. It can be solved by adding the active substances as mentioned above.

8. The sealing effect before and after dotting the film

The membranes purchased from suppliers are basically optimized and can be used directly. However, we still often encounter discussions about closures. In fact, the closure is not like everyone thinks, once it is closed, all problems are solved. The old problem is gone and the new problem is coming again, and it is more troublesome. What I want to say is to use closure with caution.

I am extremely opposed to the practice of closing before the dot film. The reason is that when the manufacturer produces the film, various formulas are mixed and added to the original pulp. However, before sealing the membrane, the membrane must be soaked in the sealing solution, which will inevitably disturb the normal material distribution in the membrane. Caused many problems thus, also reduced work efficiency. Some manufacturers also encounter the problem that they cannot be coated without sealing. In fact, other methods can be used to solve the problem. Sealing must be the next test. There are two types of closures that I often use. The flow is closed and the substance of action is treated on the sample pad. The membrane is sealed at a fixed point, and the active substance is made into a solution and sprayed on a specific position of the membrane. This method requires the use of BIODOT's AIRJET nozzle. Unqualified semi-finished slabs can be resurrected. As long as the seal is made on the membrane, it will inevitably affect the stability of the product. The specific degree of influence should be judged by the stability test.

9. Membrane storage

Freshly produced membranes generally contain 5-10% moisture. Regarding the aging mechanism of the membrane, there is a theoretical support, but the controversy is relatively large. The theory is that the aging of the membrane is due to the evaporation of water on the membrane, making the membrane hydrophobic, charged and brittle. The storage film is generally required to be protected from light and sealed. Too dry or too wet is not good. Under this storage condition, it can generally be placed for two years. However, if the sealing treatment is done on the membrane, it must be judged according to the specific test situation. Due to the problems of the production process, the sensitivity of some membranes will change within a period of time after use. If this problem is encountered, it needs to be placed for a period of time after the membrane is applied, and it can be debugged and produced after it stabilizes.