Influencing factors in the development of colloidal gold
1. Selection of consumables
1.1 Screening of different types of membranes
The type of nitrocellulose membrane is very important in the test, as the reaction carrier affects the success or failure of the whole test. The sources, types and quantities of polymers and surfactants used by different manufacturers in the production of nitrocellulose membranes are quite different, which have a great impact on the performance of the produced membranes—the pore size and distribution structure of the membranes are different. The membrane pore size decreases, the actual available surface area of the membrane increases, and the amount of membrane-bound proteins also increases; the smaller the membrane pore size, the slower the chromatography speed, the longer the time for the gold-labeled complex to pass through the T line, and the more fully the reaction ; Therefore, 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. The membranes used for gold standard immunoassays are mostly nitrocellulose membranes or nitrocellulose/cellulose acetate mixed membranes. Different coating proteins have specific requirements for the membranes. The tester should choose the appropriate pore size and distribution according to the properties of the protein. The structure of the membrane, find a suitable balance point, so that the flow rate of the colloidal gold marker on the membrane is the best.
1.2 Selection of binding pads
The binding pad is located in the middle of the chromatography system. It is generally required that the grid of the binding pad is uniform and the non-specific adsorption is low, so that it can well load the colloidal gold marker and the sample to be detected without being adsorbed; the glass cellulose membrane has the above advantages, At the same time, it has a certain hardness and is commonly used in experiments.
1.3 Selection of sample pad and absorbent paper
The sample pad and absorbent paper are located at the two ends of the colloidal gold immunochromatography system, which play a decisive role in the realization of the function of the colloidal gold chromatography system. During the test, the appropriate sample pad and absorbent paper should be selected according to the properties of the sample tested by the test strip to ensure that the sample flows quickly in the channel formed by the sample pad without being non-specifically adsorbed or changing the properties of the sample. If the test sample is serum, you can choose a glass cellulose membrane with a loose mesh; if the test sample is toxin, you can choose good quality absorbent paper and sample pads. Absorbent paper must have a good water storage capacity to ensure that the liquid used in the sample passes through the reaction zone of the membrane and is absorbed and accumulated by the sample pad.
2. About the preparation of colloidal gold
The preparation of colloidal gold with uniform particles and good dispersion is very critical in the gold standard immune rapid test. If the variation range of the gold particle diameter is too large, it will affect the stability and repeatability of the test. If the shape of the gold particles is irregular or The diameter is not uniform, so that the colloidal gold marker is easy to dissociate and precipitate, resulting in incomplete diffusion of the gold standard, too dark background color in the reaction area, and false positive phenomena; and the quality of the colloidal gold is not good, the colloidal gold conjugate cannot be quickly and completely Dissociate from the glass fiber, thereby affecting the test results.
The preparation of colloidal gold not only guarantees the quality of medicines, but the details in the preparation process are related to the success or failure of colloidal gold preparation. The first is the cleanliness of the operating environment and containers used. All dirt entering the sol will interfere with the generation of colloidal gold particles or cause the generated colloidal gold to pile up. It is best for the container to be pickled and siliconized. The operating environment should be kept clean and dust-free, and it is best to have a dedicated work area. Secondly, the preparation solution needs to be prepared with double distilled water or triple distilled deionized water, and it is best to use deionized water for firing colloidal gold. Another is the influence of different reducing agents on the quality of colloidal gold. The preparation of colloidal gold is based on the reduction method. By changing the nature and concentration of the reducing agent, colloidal gold suspensions with different particle sizes can be prepared. Select the particle size of colloidal gold according to the purpose of the test, and determine the reducing agent according to the selected particle size, such as preparing a colloidal gold solution with a diameter of 5 to 12 nm and reducing chloroauric acid with white phosphorus or ascorbic acid; Colloidal gold uses trisodium citrate to reduce chloroauric acid. In addition, different firing methods, the amount of colloidal gold prepared, the way of adding the reducing agent, the size of the heating container and the heating time also affect the size and uniformity of the colloidal gold particles. According to the particle size and quantity of the required colloidal gold, combined with its own test conditions, select the appropriate colloidal gold preparation method.
3. Preparation of labeled proteins and related antigens and antibodies
The purity and concentration of the antigen or antibody of the labeled protein, T-line and C-line directly affect the quality of the gold-labeled probe. The key to obtaining antigens and antibodies with high purity and moderate concentration lies in the selection of preparation and purification methods and the optimization of conditions. Before the test, high-speed centrifugation should be used to remove impurities, and a series of treatment methods such as saturated ammonium sulfate precipitation, affinity chromatography, and dialysis should be used to remove as much as possible the high-concentration impurities and excess in the monoclonal antibody, secondary antibody, and related protein solutions. ions, so as to avoid interfering with the adsorption and binding of the target protein and colloidal gold, or lead to the aggregation of colloidal gold particles; especially borate and phosphate are not conducive to the combination of colloidal gold and protein, and should be avoided as much as possible. At the same time, fully treat various macromolecular proteins to make them dispersed into monomers and have appropriate molecular weights, improve the binding ratio of colloidal gold and protein, and facilitate the full and stable combination with colloidal gold.
4. Marking of colloidal gold
The two key links in colloidal gold labeling are the pH of labeling and the determination of the optimal protein labeling amount. According to the principle of colloidal gold labeling, only when the pH is close to or slightly higher than the isoelectric point of the protein, the adsorption force of the colloidal gold protein is the strongest; too high or too low a pH is not conducive to the combination of the two, so choose a precise one when labeling. Use different methods to repeatedly calibrate pH measuring instruments or test strips, and conduct gradient tests to find the best pH for labeling. Whether the ratio of sol to protein to be labeled is appropriate is an important factor affecting the success of labeling. Too much protein labeling will cause waste and dragging of the test paper; too little protein labeling will lead to incomplete colloidal gold labeling, thereby reducing the sensitivity of the test paper and the appearance of false positives. It is also necessary to carry out a gradient test in the test, repeatedly compare the stability of markers with different labeling amounts in the destruction test, and determine the optimal labeling amount.
5. Optimization of membrane coating conditions
Membrane coating conditions, including membrane sealing, environmental humidity, concentration of antigen or antibody on T-line and C-line, spotting membrane conditions, temperature, coating time, etc., need to be adjusted repeatedly according to the actual situation of the experiment. During the coating process, special attention should be paid to the following two points: ① The coated amount of T-line and C-line antigens or antibodies on the membrane should be relatively saturated; ② The coated membrane must be thoroughly dried at a suitable temperature, otherwise it will cause dragging , The color rendering is not clear, and the sensitivity is also greatly affected.
5.1 Sealing and processing of the used membrane
In particular, sealing is a very controversial issue. Theoretically speaking, the purchased membranes are basically optimized and processed, and they can be used directly on the membrane. If the membrane is soaked in the sealing solution for sealing treatment before applying the membrane, it will inevitably disturb the normal material distribution in the membrane, which will cause many unnecessary troubles. However, if in the actual experimental operation, non-specific bands or dragging phenomena appear on the membrane that has not been blocked, or the problem that the protein or antibody cannot be coated on the membrane without the membrane being blocked, blocking should be considered. question. It is recommended to choose whether to block or not according to the properties of the respective markers and their color development on the membrane during the process of making test strips. If the marking effect is better and the color development on the membrane is clear without dragging and false positives, The sealing of the membrane is then completely unnecessary. On the contrary, the sealing of the membrane can be carried out to find out the reasons for the occurrence of undesired phenomena. There are many substances used to seal the membrane, and macromolecular proteins such as bovine serum albumin (BSA) and polyethylene glycol (PEGMW20000) are mostly used. Commonly used sealing methods include flow sealing and fixed-point sealing on the membrane. The former processes the sealing material on the sample pad, and the latter sprays the active substance into a solution on a specific position of the membrane.
5.2 Ambient Humidity
Ambient humidity is very important to the dot film process. The optimum humidity is generally 45% to 65%. If the humidity is too low, static charges are easy to accumulate on the film, and scattered spots are prone to appear on the film, resulting in hydrophobic spots during the test; if the humidity is too high, the capillary action on the film is strengthened, and the film is easy to cause T line, C line widened 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.
5.3 Concentration of antigen or antibody on T line and C line
The coating concentration of the antigen or antibody on the T-line and C-line directly affects the binding ratio of the antigen or antibody to the gold marker, and ultimately affects the color development of the test strip. If the coating concentration is too low, the color of the bands on the membrane will not be clear or appear hollow; if the coating concentration is too high, the C line will not develop color or appear dragging. In order to obtain a test strip with stable reaction and clear color development, it is necessary to adjust and compare the combined concentration of antigen or antibody on the two lines and the binding ratio of the two to the gold marker in order to obtain the best combination.
5.4 Spotting position
Different T-line and C-line sampling positions will bring different sensitivities. When the sampling position moves upward, the speed of the gold-labeled complex will slow down when passing the T-line position, the reaction time will increase, and the sensitivity will increase; otherwise, the sensitivity will decrease. This approach can be used to vary sensitivity and eliminate false positives.
5.5 Spotting instrument
There are currently two spotting methods, film-scribing and non-contact point-membrane. The non-contact membrane type is better than the scratched membrane type. The scratched membrane type needs to use a hose to scratch the antibody onto the surface of the membrane, but the physical properties of the membrane itself are relatively soft and brittle, and the scratched tube will leave marks on the surface. Scratches are easy 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 at the position of the T line when running the board, which affects the judgment of the result. The sample spotting instrument can not only control the spotting position of T line and C line, but also control the details such as the width of T line and C line and spray film speed through the adjustment of various parameters of the film spotting instrument to achieve the best color rendering effect. It is necessary to repeatedly adjust various parameters during the test process to do a comparison test and observe the color development of the test strip to determine.
6. Buffer
The buffer constitutes the liquid phase carrier of the colloidal gold immunochromatography technology, and provides the best combination reaction for them without affecting the combination of colloidal gold and protein, antigen and antibody, nitrocellulose membrane and T line and C line protein. Good acid-base environment. Buffers are not universal, and different reaction systems require different buffers to support, which requires experimenters to try different buffers in combination with their own experiments to determine the buffer formula that suits them. The commonly used buffer system is formulated by adding corresponding active substances to the selected buffer solution (phosphate, borate, etc.). The so-called active substances are corresponding substances added to solve a specific problem in the reaction system. For example, by adding an appropriate amount of surfactant in the buffer, it can increase the hydrophilicity, enhance the color and avoid the hollow phenomenon of the line; it can also add several substances at the same time, and solve related problems through their mutual synergy. For example, a small amount of NaCl can reduce the signal intensity and eliminate false positives; sugar and polyethylene glycol can be used as protective agents, which can slow down the aging speed and increase the hydrophilicity, but the addition of active substances should be simple rather than complicated. Many treatment solutions in the test strip preparation process are prepared by adding corresponding active substances on the basis of the selected buffer solution (such as blocking solution, binding pad treatment solution, etc.).
7. Sample processing and result judgment
7.1 Sample processing method and sample volume
For clinical specimens (such as whole blood, serum, and plasma), the processing methods adopted by different units and testing personnel are not uniform, which will also affect the judgment of results. For example, a large amount of fibrinogen in plasma affects the speed and uniformity of chromatography, directly affects the combination of antigen and antibody, and even a non-specific combination occurs if it is not handled properly. When testing, the amount of sample added should be relatively sufficient, so that the reaction on the membrane can fully proceed, so as to obtain clear results. If the amount of sample added is too low, it will lead to insufficient chromatography of the sample on the test strip and false negatives.
7.2 Judgment of results
Since the staff who use colloidal gold products are distributed in different levels and departments, there is a lot of randomness in judging the results, and the judging time is not uniform, especially under the influence of the working environment, temperature and humidity, according to the marking line The color development time can be judged arbitrarily, and the effective time set by the manufacturer is ignored. Therefore, during the test, the staff is required to be able to distinguish the false positives, false negatives and abnormal bands in the test results in a timely manner, and to retest after finding the cause or to retest for comparison and confirmation in combination with relevant detection methods to avoid missed detection and false positives.
8. Storage and verification of products
8.1 Storage of Products
The newly produced test strips or test cards generally contain 5% to 10% moisture, and the environment during storage is too dry and too humid to be conducive to product storage. If the environment is too dry, the water on the film evaporates, making the film hydrophobic, charged and brittle; the environment is too humid to affect the quality of gold markers and the coloring effect of T-line and C-line; therefore, the finished test strips and detection The card should be designed to be moisture-proof. If it is stored for a long time, it is generally required to keep it away from light and sealed. The temperature of the environment also affects the biological activity of markers, antigens, and antibodies, and the storage time of products can be effectively extended under low temperature conditions.
8.2 Finished product verification test
The verification test includes 10 tests including specificity test, linear sensitivity test, stability test, sample detection, interference test, inter-batch and intra-batch repeatability test, etc., to comprehensively evaluate the quality of the product. The finished product verification test takes a long time, especially the stability test of the product needs to be regularly sampled for a long period of time. Combining with your own tests and referring to the corresponding national or industry standards, you should formulate targeted tests to detect the specificity, stability, repeatability and sensitivity of the finished product, in order to further optimize and finally make high-quality test strips or The test card provides data support.
To sum up, each link of colloidal gold immunochromatography technology is affected by various factors, which also makes the detection method established by immunocolloidal gold technology also have some problems in the actual application process, such as poor stability of colloidal solution , The storage time is relatively short and the sensitivity is affected by various factors, which lead to certain limitations in the application of immune colloidal gold technology. With the advancement of science and technology, these problems will be gradually improved in future research, and immunocolloidal gold technology will further show its great advantages, realize semi-quantitative or quantitative detection and diversified detection, and broaden its scope in light microscopy, Electron microscopy, flow cytometry, western blotting, biochips and other fields.
