1. The principle of combining protein with membrane.
According to the bonding principle of protein and membrane, the known bonding forces are hydrophobic force \H bond \ electrostatic force, etc. The exact binding principle is not clear, and it is mainly supported by hypothesis. There are two main assumptions:
1) firstly combines through electrostatic force, and then combines for a long time through H bond and hydrophobic interaction.
2) First combine by hydrophobic action, and then combine by electrostatic action for a long time.
Both hypotheses show that the combination process is divided into two steps, first combination and then long combination. Because of the fuzziness of combination principle, the work in this field depends on practical experience to a great extent.
2. Effect of film on adhesion
1) membrane pore size
Some technicians tend to use membrane pore size to distinguish different membranes, but please note that this is 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 chromatographic speed has been described above.
With the decrease of membrane pore size, the actual available surface area of membrane increases, and the amount of membrane-bound protein also increases. The parameter used to estimate the surface area is the surface area ratio (the ratio of the actual available surface area to the flat area of the film used).
In addition, the smaller the pore size of the membrane, the lower the chromatographic speed, and the longer the time for the gold-labeled complex to pass through the T-line, the more complete the reaction.
Based on the above two points, it is concluded that the smaller the pore size of the membrane, the higher the sensitivity. But it also slows down the running speed and increases the chance of nonspecific binding, that is, the higher the false positive. Therefore, it is necessary to select the membrane suitable for practical engineering according to the test results and find a suitable balance point.
2) membrane differences of different manufacturers
This difference mainly comes from two points:
1 & gt； When producing membranes, the sources, types and quantities of polymers and surfactants used are different. Similarly, in membrane treatment, these two substances generally have a great influence on the performance.
2> The treatment process is different.
3. Formulation of biological raw materials, reagents and buffer solutions
1) Biological materials are used in different situations as biological materials for CT lines, and only a brief introduction is made here.
Firstly, the binding of monoclonal antibody to membrane is better than polyclonal antibody, mainly because polyclonal antibody has many different surface sites, and the optimal binding conditions of each site to membrane are slightly different, which undoubtedly increases the difficulty of optimization.
Secondly, the greater the molecular weight, the more difficult it is for protein to combine with solid substances.
2) Buffer solution
People may be most concerned about obtaining a formula with excellent performance, including buffer solution, sealing solution and so on.
In fact, I can't give you a list of general formulas. Because different reaction systems need different formulas to support them, and the reaction systems of different institutions are different. If you want to get "fish", you must learn to fish first. In order not to mislead everyone, I only provide ideas on the following questions related to the formula. Please explore the specific formula yourself.
Generally, the composition of buffer solution is PBS (or other buffer system)+active substance (for a specific problem) +PH adjustment. Referring to the previous information, my personal opinion is that the formula principle should be simple and uncomplicated, and active substances should be added according to your own needs. Many active substances that need to be added are no longer needed because of the improvement of membrane technology.
The recommended buffer system is 0.0 1M PBS PH7-7.2, which has good adaptability to various antigens and antibodies.
The situation of active substances is roughly listed as follows:
A small amount of NACL can reduce the signal intensity and eliminate false positives.
Organic alcohols (methanol, isopropanol, etc.). ) wetting the film, reducing the static electricity carried by the film, which is beneficial to combining the coating. Personally, I don't recommend it because of the improvement of film production technology.
Surfactant (TW20, TX 100) can increase hydrophilicity, avoid hollow line phenomenon and increase color.
Sugar, a protective agent, can slow down the aging speed and increase hydrophilicity.
Adjusting the pH to a certain position can eliminate false yang.
4. Sample environment
Environmental humidity is very important to the film deposition process. The optimum humidity is generally 45-65%.
When the humidity is too low, electrostatic charge is easy to accumulate on the film, and speckle is easy to appear on the film, which leads to hydrophobic spots in the test.
When the humidity is too high, the capillary action on the film is strengthened, and the dotted film is easy to cause the CT line to widen or even spread.
In order to ensure the uniformity of membrane humidity during sampling, the membrane is usually placed in this humidity condition for a period of time before sampling.
5. Relationship between sampling instrument and membrane surface condition.
At present, there are two sampling methods, namely, scraping film type and non-contact point film type. Non-contact point film type is better than scraping film type, and imported scraping film type is better than domestic scraping film type.
Because the membrane scraping method is to scrape the antibody on the surface of the membrane with a hose, and the physical properties of the membrane itself are soft and brittle, the membrane scraping tube will leave traces on its surface. Because the imported film scraper adopts better materials and control system, the scratches left are lighter, while the domestic instruments are worse and the scratches left are more serious. Scratches are easy to form resistance to chromatographic gold-labeled complexes, leading to false positives. At the same time, it is easy to appear if there is a thin line (ghost line) at the position of T line during running.
6. Film width and spot position
The width of the membrane is generally 18mm (or 20mm) and 25mm, which are used for test paper and test board respectively.
However, different T-line sampling positions will bring different sensitivities. When the sampling position moves up, the speed of gold labeled compounds passing through the T-line position will slow down, the reaction time will increase and the sensitivity will improve. On the contrary, the sensitivity will decrease. This method can be used to change sensitivity and eliminate false positives.
7. Diffusion of solution on membrane
Generally speaking, the sample size of the solution on the membrane is 1ul/cm.
The diffusion of the solution on the membrane tends to both ends, and the spraying point is a uniform antibody solution. But when drying, the drying speed of the edge of the line is higher than that in the middle, and the antibody in the middle will continue to spread to both sides, so the antibody will gather at both ends of the line after drying. Generally, it will not affect your experiment. If you find that the two ends of the line are red and the middle is bright, you should consider this problem. It can be solved by adding the above-mentioned functional substances.
8. Sealing effect before and after film pasting
The films purchased from suppliers are basically optimized and can be used directly. However, we often encounter discussions about closure.
In fact, closure is not what everyone thinks. Once closed, everything will be solved. The old problem has gone, and the new problem has come again, which is even more troublesome. What I want to say is to use closures with caution.
I am extremely opposed to the method of sealing first and then ordering the film. The reason is that when manufacturers produce films, all kinds of formulas are mixed with the original pulp. When they seal the film before ordering it, they must soak the film in the sealing liquid, which will inevitably disturb the normal distribution of substances in the film. This has caused many problems and reduced work efficiency. Some manufacturers encounter the problem that they can't cover the film without sealing the film. In fact, they can solve it in other ways, and sealing the film is bound to be measured.
There are two kinds of sealing techniques that I often use.
The flow is turned off and the active substance is treated on the sample pad.
The membrane is sealed at a fixed point, and the active substances are mixed into a solution and sprayed at a specific position of the membrane. This method requires the use of BIODOT air jet nozzle, which can revive the unqualified semi-finished board.
As long as the film is sealed, it will inevitably affect the stability of the product. The specific degree of influence should be judged by stability test.
9. Storage of membrane
The newly produced film generally contains 5- 10% moisture. The aging mechanism of movies has theoretical support, but it is controversial. According to the theory, the aging of the film is due to the evaporation of water on the film, which makes the film hydrophobic, charged and fragile. The general requirement for storing films is light-proof sealing. Too dry or too wet is unfavorable. Under this storage condition, it can generally be preserved for two years. however
Due to the problems of production process, the sensitivity of some membranes will change within a period of time after use. In case of such problems, it is necessary to place the membrane for a period of time after ordering, and then put it into commissioning production after it is stable.