ELISA reagents

l Immunosorbent

The solid phase carrier coated with antigen or antibody can generally be stored for more than 6 months under the condition of low temperature (2-8 ℃) drying. Some incomplete test kits only supply coating antigens or antibodies, and the testing personnel need to coat themselves. The following briefly describes the solid phase carrier and coating process.

1 Solid-phase carrier The solid-phase carrier is used as an adsorbent and a container during the ELISA measurement process and does not participate in chemical reactions. There are many materials that can be used as carriers in ELISA, the most commonly used is polystyrene. Polystyrene has a strong ability to adsorb proteins, and antibodies or protein antigens retain the original immunological activity after being adsorbed on it, and its low price makes it widely used. Polystyrene is plastic and can be made into various forms.

There are three main shapes of ELISA carriers: microtiter plates, beads and small test tubes. The microtiter plate is the most commonly used. The product dedicated to EILSA is called the ELISA plate. The international standard microtiter plate is an 8 × 12 96-well format. In order to facilitate the detection of a small number of specimens, there are 8-well strips or 12-well strips, which are the same size as standard ELISA plates after being placed in the holder. The characteristic of the ELISA plate is that a large number of specimens can be tested at the same time, and the results can be quickly read on a special colorimeter. There are a variety of automated instruments for ELISA detection of microtiter plates, including the steps of sample addition, washing, heat preservation, and colorimetry, which are extremely beneficial to the standardization of operations. After polystyrene is irradiated with radiation, its adsorption performance, especially for immunoglobulin, is increased. The application of the double antibody sandwich method can increase the amount of antibody on the solid phase, but the blank value is larger when it is used for indirect method for antibody measurement.

A good ELISA plate should have good adsorption performance, low blank value, and high transparency at the bottom of the well. The performance is similar between the plates, between the wells of the same plate, and between the wells of the same plate. Due to the difference in raw materials and the manufacturing process of polystyrene ELISA plates, the quality of various products varies greatly. Therefore, the performance of each batch of ELISA plates must be checked before use. The commonly used inspection method is: coating each well of the ELISA plate with a certain concentration of human IgG (generally 10ng / ml), adding an appropriate dilution of enzyme-labeled anti-human IgG antibody to each well after washing, washing after incubation, and adding substrate After developing the color and stopping the enzyme reaction, the absorbance of each well solution was measured. Control the reaction conditions so that the reading of each well is around 0.8 absorbance. Calculate the average of all readings. The difference between all single readings and the average of all readings should be less than 10%.

The plastic similar to polystyrene is polyvinyl chloride. As an ELISA solid phase carrier, polyvinyl chloride is characterized by a thin, flexible board that can be cut and is inexpensive, but its finish is not as good as that of polystyrene board, and the bottom of the well is not as smooth as polystyrene. The adsorption performance of polyvinyl chloride on protein is higher than that of polystyrene, but the blank value is also slightly higher.

In order to compare the advantages and disadvantages of different solid phases in an ELISA assay, the following test can be applied: a typical positive specimen and a negative specimen are selected by other immunological measurement methods, and they are serially diluted to different solid phases. The phase carrier is measured according to predetermined ELISA procedures, and then the results are compared. On which carrier the positive result differs most from the negative result, this carrier is the most suitable solid phase carrier for this ELISA test item.

In ELISA, the beads used as a solid phase carrier are generally round beads with a diameter of 0.6 cm, and the surface area is greatly increased after the matte treatment. The adsorption area of ​​the ELISA plate well is about 200mm2, and the beads are all 1000mm2, which is nearly 5 times of the ELISA plate well. An increase in the adsorption area means an increase in the amount of solid-phase antigen or antibody. Furthermore, the surface curvature of the spherical beads is more conducive to the optimal response state of the adsorbed antigenic determinant or antibody binding site, so the bead ELISA reaction is often more sensitive. Another feature of the beads is that it is easier to make the washing thoroughly. The use of a special washing machine makes the beads roll and wash during the washing process. The washing effect is much better than the soaking type of the plate hole. However, due to the difficulty of the frosting process, the uniformity of the beads is poor.

As a solid phase carrier, the small test tube also has a large adsorption surface, and the reaction volume of the specimen also increases accordingly. The sample volume of plate and bead ELISA is generally 00-200ul, and the small test tube can increase the reaction volume according to the need. The increase of the sample reaction volume helps to improve the test sensitivity. The small test tube can also be used as a cuvette, and finally placed directly into the spectrophotometer for colorimetry.
There are also particles made of polystyrene latex or other materials used as ELISA solid phase carriers. The advantage is that the surface area is extremely large, the reaction is carried out in suspension, and its rate is similar to the liquid phase reaction. Iron-containing magnetic particles are used as ELISA solid-phase carriers. After the reaction, they are separated by the attraction of magnets. It is easy to wash. The kits are generally equipped with special instruments.

2 The coating method to fix the antigen or antibody in the process is called coating. In other words, coating is the process of binding an antigen or antibody to the surface of a solid support. The solid phase carrier of protein and polystyrene is combined by physical adsorption, relying on the interaction between the hydrophobic groups on the molecular structure of the protein and the hydrophobic groups on the surface of the solid phase carrier. This physical adsorption is non-specific and is affected by the molecular weight, isoelectric point, concentration, etc. of the protein. The adsorption capacity of the carrier to different proteins is different. Larger proteins and smaller molecules usually contain more hydrophobic groups, so they are more easily adsorbed on the surface of the solid phase carrier. IgG has a strong adsorption force on solid phases such as polystyrene, and its coupling occurs mostly on the Fc segment, and the antibody binding point is exposed to the outside. Therefore, the direct coating method is generally used for antibody coating. Most protein antigens can also be coated by methods similar to antibodies. When the antigenic determinant is in or adjacent to the hydrophobic region, the direct adsorption of the antigen and the solid phase carrier can make the antigenic determinant not fully exposed. In this case, the direct coating effect is not good, and indirect capture coating can be used The method is to pre-coat a specific antibody against the antigen, and then solidify the antigen through the antigen-antibody reaction. The antigen indirectly bound to the solid phase is far away from the surface of the carrier, and its antigenic determinants are also fully exposed. The indirect coated antigen is subjected to affinity chromatography with solid-phase antibodies, and the purity of the antigen coated on the solid phase is greatly improved. Therefore, the antigen with more impurities can also be captured and coated. The specificity and sensitivity of the test Both have been improved and repeatability is improved. Another advantage of indirect coating is that the amount of antigen is small, only 1/10 or even / 100 of direct coating. Non-protein antigens that are not easily adsorbed on polystyrene carriers can be coated in a special way. For example, when detecting anti-DNA antibodies, DNA is used as the coating antigen, and the general solid-phase carrier generally cannot directly bind to nucleic acids. The polystyrene board can be irradiated with ultraviolet rays (for example, 30W ultraviolet lamp, 75cm irradiation for 12 hours) to increase its adsorption performance. The solid-phase carrier is first pre-coated with alkaline proteins, such as polylysine, protamine, etc., which can also improve the binding capacity of nucleic acids. The avidin biotin system can also be used for indirect coating, that is, the carrier is coated with avidin first, and then biotinylated DNA is added. This coating method is uniform and firm, and has been widely applied to the quantification of various antigenic substances. Determination.

The lipid cannot be combined with the solid phase carrier. It can be dissolved in an organic solvent (such as ethanol) and added to the well of the ELISA plate. The lid is opened and placed in the refrigerator overnight or dried with cold air. After the alcohol volatilizes, the lipid is allowed to dry naturally On the solid surface. Anti-cardiolipin antibody ELISA reagents generally adopt this coating method.

3 coated antigen

The antigens used to coat the solid phase carrier can be divided into three categories: natural antigens, recombinant antigens and synthetic polypeptide antigens according to their sources. Natural antigens can be taken from animal tissues, microbial cultures, etc., and must be extracted and purified before being used for coating. For example, HBsAg can be extracted from the carrier's serum, general bacterial and viral antigens can be extracted from its culture, protein component antigens can be extracted from materials rich in this antigen, etc. (for example, AFP is extracted from cord blood or fetal liver ). Recombinant antigens are protein antigens expressed by antigen genes in plasmids, and E. coli or yeast are mostly used as plasmids. The advantage of recombinant antigens is that apart from the components of engineering bacteria, other impurities are few and non-infectious, but the purification technology is more difficult. Recombinant antigens using E. coli as a plasmid can not sufficiently remove E. coli components and used in ELISA. False positives may appear in the reaction. Anti-E. Coli antibodies are present in the serum because many subjects are infected with E. coli. Another characteristic of recombinant antigens is that they can use genetic engineering to prepare certain antigenic substances that cannot be separated from natural materials. For example, the hepatitis C virus (HCV) has not been successfully cultured, and the serum HCV antigen content in patients with hepatitis C is extremely small. At present, most of the coating antigens used in the detection of anti-HCV ELISA are recombinant antigens prepared based on the HCV gene clone expression. In the diagnosis of infectious diseases, many recombinant antigens such as HBsAg, HBeAg and HIV antigens have been used in ELISA. Synthetic polypeptide antigen is a polypeptide fragment artificially synthesized based on the amino acid sequence of an antigenic determinant of a protein antigen molecule. Peptide antigens generally contain only one antigenic determinant, which has high purity and high specificity. However, because the molecular weight is too small, it is often difficult to directly adsorb on the solid phase. The coating of polypeptide antigen generally needs to be coupled with unrelated proteins such as bovine serum albumin (BSA), etc., and indirectly bound to the surface of the solid phase carrier by means of adsorption of the conjugate and the solid phase carrier. Another point of attention when using peptide antigens is that he can only detect the corresponding antibodies. A protein antigen often contains many different determinants that can cause antibody production, so other antibodies in the tested serum cannot react with the polypeptide antigen. In addition, some microorganisms often undergo antigen structure changes when they are mutated. In this case, coating with individual polypeptide antigens can cause missed detection of other antibodies.

4 Coating antibody

The antibody coated with the solid phase carrier should have high affinity and high specificity, and can be obtained from antisera or ascites or culture fluid containing monoclonal antibodies. If the antigen used for immunization contains impurities (even trace amounts), hybrid antibodies will appear in the antiserum and must be removed (available by absorption method) before it can be used in ELISA to ensure the specificity of the test. Antiserum cannot be directly used for coating, IgG should be extracted first, usually using ammonium sulfate salting out and Sephadex gel filtration method. Generally, the crude IgG extracted by ammonium sulfate salting out can be used for coating, and the highly purified IgG is unstable. If high-affinity antibody coating is needed to improve the sensitivity of the test, affinity chromatography can be used to remove the non-specific IgG that is more in the antiserum. The concentration of monoclonal antibody in ascites is higher and the specificity is stronger. Therefore, absorption and affinity chromatography treatments are not necessary. Generally, the ascites can be directly diluted after appropriate coating, and purified IgG can also be used if necessary. When applying monoclonal antibody coating, it should be noted that one monoclonal antibody is only directed against one epitope. In some cases, a mixed coating with multiple monoclonal antibodies can achieve better results.

5 Coating conditions The concentration of coating antigen or antibody, the temperature and time of coating, the pH of the coating solution, etc. should be selected according to the characteristics of the test and the nature of the material. Antibodies and protein antigens generally use carbonate buffer of pH 9.6 as the diluent, as well as phosphate buffer of pH 7.2 and Tris-HCL buffer of pH 7-8 as the diluent. Generally, after adding the coating solution to the well of the ELISA plate, it is placed in a refrigerator at 4-8 ° C overnight, and it is considered to have the same coating effect by incubating at 37 ° C for 2 hours. The most suitable concentration of coating can vary greatly with the properties of the carrier and the coating. Each batch of material needs to be selected in harmony with the concentration of the enzyme conjugate through experiments. The general protein coating concentration is 100ng / ml-20ug / ml.

6 Blocking Blocking is the process of recoating with a high concentration of irrelevant protein solution after coating. The antigen or antibody is coated at a lower concentration. After absorption, there are still unoccupied voids on the surface of the solid phase carrier. Closing is to allow a large number of unrelated proteins to fill these voids, thereby rejecting interfering substances in the subsequent steps of ELISA Resorption. The procedures for closure are similar to coating. The most commonly used blocking agent is 0.05% -0.5% bovine serum albumin, and 10% calf serum or 1% gelatin is also used as the blocking agent. Skimmed milk powder is also a good sealant. Its biggest feature is its low price, which can be used in high concentration (5%). High-quality instant edible low-fat milk powder can be used directly as a blocking agent, but due to the complex composition of the milk powder and the carrier after sealing is not easy to store for a long time, it is rarely used in the preparation of kits.
Whether blocking is necessary depends on the ELISA mode and the specific experimental conditions. Not all ELISA solid phases need to be blocked. Improper blocking will increase the negative background. Generally speaking, the double antibody sandwich method, as long as the enzyme label is highly active, is washed thoroughly during operation, and satisfactory results can be obtained without blocking. Especially when directly coated with monoclonal antibody ascites, because a large amount of non-antibody protein is also adsorbed on the surface of the solid phase when coated, it has already played a similar role as a blocking agent. But in the indirect method of determination, closure is generally indispensable. The coated ELISA plate is dried and placed in a sealed bag or tin bag, which can be stored at low temperature for several months.

l enzyme conjugate (conjugate)

Conjugates are enzyme-labeled antibodies (or antigens) and are the most critical reagents in ELISA. A good conjugate should retain both the catalytic activity of the enzyme and the immunological activity of the antibody (or antigen). There is an appropriate molecular ratio between the enzyme and antibody (or antigen) in the conjugate. The binding reagent should contain as little or as little free (unbound) enzyme or free antibody (or antigen) as possible. In addition, the conjugate must have good stability.

1 Enzyme The enzyme used in ELISA should meet the following requirements: high purity, high conversion rate of catalytic reaction, strong specificity, stable nature, rich source, inexpensive price, and it will continue to retain its active part after being prepared as an enzyme conjugate And catalytic capacity. It is best that the same enzyme is not present in the specimen being tested. In addition, its corresponding substrate is easy to prepare and store, inexpensive, and colored products are easy to measure.

In ELISA, the commonly used enzymes are horseradish peroxidase (horseradish peroxidase, HRP) and alkaline phosphatase (alkaline phosohatase, AP). In a few commercial ELISA reagents, the enzymes used are glucose oxidase, β-D-galactosidase and urease. Domestic ELISA reagents generally use HRP to prepare conjugates. HRP is a glycoprotein with a sugar content of about 18% and a molecular weight of 44,000. It is a complex enzyme composed of a main enzyme (enzyme protein) and a prosthetic group (heme) and is a porphyrin protein. The main enzyme colorless glycoprotein has the highest absorption peak at a wavelength of 275 nm, the prosthetic group is a dark brown iron-containing porphyrin ring, and the highest absorption peak at a wavelength of 403 nm. The purity of HRP is represented by RZ (Reinheit Zahl, German, meaning purity number), which is the ratio of the absorbance at 403 nm to the absorbance at 280 nm. The RZ of high-purity HRP ≥? .0.

In addition to meeting the requirements of the above-mentioned ELISA labeling enzyme, HRP has the characteristics of lower price and more stable properties. It is worth noting that when selecting enzyme preparations, in addition to its purity RZ, attention should be paid to enzyme activity. If the high-purity enzyme is improperly stored, its activity will be reduced. The activity of the enzyme preparation is expressed in units of enzyme activity contained in it, which can be tested by measuring the amount of product produced after the action of the substrate.

Many foreign ELISA reagents use alkaline phosphatase (AP) as the labeling enzyme. There are two common sources of AP, which are extracted from E. coli and calf intestinal membranes. The biochemical characteristics of enzymes from different sources are slightly different. The molecular weight of AP extracted from E. coli is 80,000 and the most suitable pH for enzyme action is 8.0; the molecular weight of AP extracted from calf intestinal membrane is 100,000 and the optimal pH is 9.6. In ELISA, the sensitivity of the AP system is generally higher than that of the HRP system, and the blank value is also low, but the AP is expensive and the yield of conjugate preparation is also lower than that of HRP.

2 Antigens and antibodies used in the preparation of conjugates are generally high-purity IgG, so as to avoid interference with other contaminants when coupled with enzymes. It is best to use antibodies purified by affinity chromatography, so that all enzyme conjugates have specific immunological activities and can be reacted at high dilutions. The experimental results have a light background. If F (ab) 2 is used for labeling, RF interference in the specimen can be avoided. There are not many modes of using enzyme-labeled antigen in ELISA. The general requirement is that the antigen must be of high purity.

3 Preparation of conjugates There are two main methods for preparing enzyme-labeled antibodies, namely glutaraldehyde cross-linking method and periodate oxidation method.

(1) Glutaraldehyde cross-linking method: Glutaraldehyde is a kind of bifunctional reagent, it can make the amino group of enzyme and protein link through it. Basic phosphoric acid is generally labeled by this method. There are two types of cross-linking methods: one-step method and two-step method. In the one-step method, glutaraldehyde is directly added to the enzyme and antibody mixture, and the enzyme-labeled antibody is obtained after the reaction.

Enzymes commonly used in ELISA are generally cross-linked by this method. It has the advantages of easy operation, effective (combination rate of 60% -70%) and good repeatability. The disadvantage is that the cross-linking reaction is random, the ratio between the molecules when the enzyme and antibody are cross-linked is not strict, the size of the conjugate is not uniform, and the enzyme and enzyme, antibody and antibody may also be cross-linked, affecting the effect.謹 讲 椒 ㄖ 校? Br> First, the enzyme is reacted with glutaraldehyde, and after dialysis to remove excess glutaraldehyde, then the antibody is reacted to form an enzyme-labeled antibody. The antibody can also be first reacted with glutaraldehyde and then linked to the enzyme. Most of the enzyme and protein in the product of the two-step method are combined in a 1: 1 ratio, which is more conducive to the improvement of the background than the one-step enzyme conjugate to improve the sensitivity, but the efficiency of the coupling Lower than the one-step method.

(2) Periodate oxidation method: This method is only applicable to enzymes with higher sugar content. Horseradish peroxidase labeling is commonly used in this method. During the reaction, the sodium periodate oxidizes the polysaccharide on the surface of the HRP molecule to aldehyde groups, which can form Schiff's base with the amino group on the protein. The enzyme markers are linked in molar ratio, and the optimal ratio is: enzyme / antibody = 1-2 / 1. This method is simple and effective, and is generally considered to be the most desirable labeling method for HRP, but some people think that all reagents are relatively strong, and the results of each batch of experiments are not easy to repeat.

The enzyme conjugate prepared according to the above method is generally mixed with unconjugated enzyme and antibody. Theoretically, the free enzyme mixed in the conjugate generally does not affect the final enzyme activity measurement in the ELISA. The free enzyme can be removed after thorough washing and does not affect the final color development. But the free antibody is different, it will compete with the enzyme-labeled antibody for the corresponding solid-phase antigen, thereby reducing the amount of enzyme-labeled antibody bound to the solid phase. Therefore, the prepared enzyme conjugate should be purified, and the free enzyme and antibody should be removed for detection, and the effect is better. There are many purification methods, and methods for separating macromolecular compounds can be applied. The ammonium sulfate salting-out method is the simplest, but the effect is not ideal, because this method can only remove the free enzyme remaining in the supernatant, but a considerable amount of free antibody is still precipitated together with the enzyme conjugate and cannot be separated. Ion exchange chromatography or molecular sieve separation is more preferable. High performance liquid chromatography can clearly divide the prepared conjugate into three parts: free enzyme, free antibody and pure conjugate to obtain the best separation effect, but the cost More expensive.