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The sandwich method is often used to detect macromolecular antigens. The general procedure is:
Adding the sample to be tested, if the sample contains the antigen to be tested, it will specifically bind to the antibody on the plastic well plate;
Wash away the specimen to be tested, add a secondary antibody labeled with an enzyme, and bind to the antigen;
Wash off the unconjugated secondary antibody, add the enzyme receptor to make the color, and read the color result with the naked eye or instrument.
The so-called ABC technology refers to Avidin: Biotinylated Enzyme Complex Technology, avidin-biotin-peroxidase complex technology, the so-called ABC system, which is widely regarded as the most sensitive, reliable and effective dyeing system at present. It is widely used in immunohistochemistry, immunoelectron microscopy, in situ hybridization and lectin chemistry. The system is still evolving to meet the diverse needs of researchers (such as multi-antigen labeling).
Fixing the known antigen on the plastic well plate and washing away the antigen of the polysaccharide after completion;
Adding the specimen to be tested, if the specimen contains the primary antibody to be tested, it will specifically bind to the antigen on the plastic orifice plate;
Wash away the specimen to be tested, add a secondary antibody with an enzyme, and bind to the primary antibody to be tested;
Wash away the unbound secondary antibody, add the enzyme receptor to color the enzyme, and read the color result with the naked eye or instrument.
The specific antibody is fixed on the plastic well plate, and the multi-antimony antibody is washed off after completion;
Adding the sample to be tested, so that the antigen to be tested in the specimen is specifically combined with the antibody on the plastic well plate;
Adding an antigen with an enzyme, this antigen can also specifically bind to the antibody on the plastic plate. Since the number of antibodies immobilized on the plastic plate is limited, the more the amount of antigen in the sample, the more the enzyme The fewer immobilized antibodies the antigen can bind, that is, both antigens compete with the antibodies on the plastic plate, the so-called competition law.
Wash off the specimen and the antigen with the enzyme, add the enzyme receptor to make the enzyme color. When the amount of antigen in the specimen is more, the less antigen with the enzyme left in the plastic plate, the lighter the color.
A competitive ELISA is generally considered when it is desired to detect antigens that are unable to obtain two or more single antibodies, or to obtain sufficient purified antibodies to immobilize on a plastic well plate.
(2) an enzyme-labeled antigen or antibody (conjugate);
(3) a substrate for the enzyme;
(4) Negative control and positive control (in qualitative determination), reference standard and control serum (quantitative determination);
(5) a dilute of the enzyme conjugate (conjugate) and the specimen;
(6) washing liquid;
(7) Enzyme reaction stop solution.
There are three main types of ELISA vectors: microtiter plates, beads, and small tubes. Microtiter plates are most commonly used. Products dedicated to EILSA are called ELISA plates. International standard microtiter plates are 8×12 96-well. In order to facilitate the detection of a small number of specimens, there are 8 joint strips or 12 joint strips, which are the same size as the standard ELISA plate after being placed in the mount. The ELISA plate is characterized by the ability to simultaneously perform a large number of specimens and quickly read the results on a special colorimeter. A variety of automated instruments are now available for ELISA assays for microtiter plate types, including loading, washing, incubation, colorimetric, etc., which are highly advantageous for standardization of operations. After the irradiation of polystyrene, the adsorption performance of the polystyrene increases especially for the immunoglobulin. The double antibody sandwich method can increase the amount of the antibody on the solid phase, but the blank value is larger when used for indirect method.
In the ELISA, the beads used as the solid phase carrier are generally 0.6 cm in diameter, and the adsorption area is greatly increased after the surface is frosted. The adsorption area of ​​the ELISA plate well is about 200 mm 2 , and the beads are 1000 mm 2 , which is 5 times that of the ELISA plate. 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 favorable for the optimal reaction state of the exposed antigenic determinant or the exposed surface of the antibody binding site, so the reaction of the bead ELISA is often more sensitive. Another feature of the beads is that it is easier to wash thoroughly, using a special scrubber to allow the beads to roll and rinse during the washing process, and the washing effect is much better than the immersion of the plate holes. However, due to the difficulty of the sanding process, the uniformity of the beads is poor.
The lipid substance cannot be combined with the solid phase carrier, and it can be dissolved in an organic solvent (such as ethanol), added to the well of the ELISA plate, and opened in a refrigerator or dried in a cold air. After the alcohol is evaporated, the lipid is naturally dried. On the solid surface. ELISA reagents for anticardiolipin antibodies generally employ this type of coating.
1.4 coated antibody <br> The antibody coated with the solid phase carrier should have high affinity and high specificity, and can be obtained from antiserum or monoclonal antibody-containing ascites or culture solution. If the antigen used in the immunization contains impurities (even in a very small amount), the hybrid antibody will appear in the antiserum and must be removed (absorbable method) before use in the ELISA to ensure the specificity of the test. Antiserum can not be used directly for coating, IgG should be extracted first, usually by ammonium sulfate salting and Sephadex gel filtration. Generally, crudely extracted IgG by ammonium sulfate salting has been used for coating, and highly purified IgG is unstable in nature. If high-affinity antibody coating is required to increase the sensitivity of the assay, affinity chromatography can be used to remove non-specific IgG from the antiserum. The concentration of monoclonal antibody in ascites is higher and the specificity is stronger. Therefore, absorption and affinity chromatography are not required. Generally, ascites can be directly diluted after appropriate dilution, 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 to one antigenic determinant, and in some cases, mixed with a plurality of monoclonal antibodies can achieve better results.
1.5 coating conditions <br> The concentration of antigen or antibody used for coating, 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. Antibody and protein antigens are generally used as a diluent in a carbonate buffer of pH 9.6, and a phosphate buffer of pH 7.2 and a Tris-HCL buffer of pH 7-8 are also used as the diluent. Usually, the coating solution was added to the wells of the ELISA plate, and it was left overnight in a refrigerator at 4-8 ° C, and the incubation at 37 ° C for 2 hours was considered to have an equivalent coating effect. The optimum concentration of coating can vary widely depending on the nature of the carrier and coating. Each batch of material is selected by experiment and concentration of the enzyme conjugate. Typical protein coating concentrations range from 100 ng/ml to 20 ug/ml.
1.6 Blocking <br> Blocking is the process of recoating with a high concentration of unrelated protein solution after coating. The concentration of the antigen or antibody is low, and there is an unoccupied void on the surface of the solid support after absorption. Blocking is to fill a large number of unrelated proteins to repel the interfering substances in the subsequent steps of the ELISA. Re-adsorption. The closed procedure is similar to the package. 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 a blocking agent. Skim milk powder is also a good sealer. Its biggest feature is its low cost and high concentration (5%). The high-quality instant edible low-fat milk powder can be directly used as a blocking agent, but since the composition of the milk powder is complicated and the sealed carrier is not easy to be stored for a long time, it is less used in the preparation of the kit.
Whether blocking is necessary depends on the mode of the ELISA and the specific experimental conditions. Not all ELISA solids need to be blocked, and improper closure will increase the negative background. In general, the double antibody sandwich method, as long as the enzyme label is highly active, is thoroughly washed during the operation, and satisfactory results can be obtained without blocking. Especially when directly coated with monoclonal antibody ascites, a large amount of non-antibody protein is also adsorbed on the surface of the solid phase during coating, and has played a similar role as a blocking agent. However, in the indirect method, closure is generally indispensable. The coated ELISA plate is dried and placed in a sealed bag or tin bag, which can be stored for several months at low temperatures.
The conjugate, the enzyme-labeled antibody (or antigen), is the most critical reagent in the ELISA. A good conjugate should be to retain both the catalytic activity of the enzyme and the immunological activity of the antibody (or antigen). There is a suitable molecular ratio between the enzyme and the antibody (or antigen) in the conjugate, and the binding reagent should contain as little or no free (unbound) enzyme or free antibody (or antigen) as possible. In addition, the combination still has good stability.
2.1 Enzymes Enzymes used in ELISA should meet the following requirements: high purity, high conversion rate of catalytic reaction, strong specificity, stable nature, abundant source, inexpensive price, and still retain its active part after preparation of enzyme conjugate. And catalytic ability. Preferably, the same enzyme is not present in the specimen being tested. In addition, its corresponding substrate is easy to prepare and store, and the price is low, and the colored product is easy to measure.
In ELISA, commonly used enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Among the 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). It is a porphyrin. protein. The main enzyme colorless glycoprotein has the highest absorption peak at 275 nm, and the prosthetic group is a dark brown iron-containing porphyrin ring with the highest absorption peak at 403 nm. The purity of HRP is represented by RZ (Reinheit Zahl, German, meaning purity), which is the ratio of the absorbance at 403 nm to the absorbance at 280 nm, and the RHP of high purity H? is ??.
In addition to the above-mentioned requirements for labeling enzymes in ELISA, HRP is more inexpensive and more stable. It is worth noting that in the selection of enzyme preparations, in addition to its purity RZ, more attention should be paid to the activity of the enzyme. High-purity enzymes, such as improper preservation, will also reduce their vitality. The viability of the enzyme preparation is expressed in units of enzyme activity contained, and can be tested by measuring the amount of product produced by the action of the substrate.
Many ELISA reagents abroad use alkaline phosphatase (AP) as a labeling enzyme. Commonly used APs come from two sources, 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 optimum pH for enzyme is 8.0. The molecular weight of AP extracted from calf intestinal membrane is 100,000, and the optimum pH is 9.6. In the 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 the prepared conjugate is also lower than that of the HRP.
2 .2 Antigens and antibodies The antibodies used in the preparation of conjugates are generally high purity IgG to avoid interference with other heteroproteins when linked to the enzyme. It is preferable to use an affinity-purified pure antibody, so that all the enzyme conjugates have specific immunological activities, and the reaction can be carried out at a high dilution, and the results of the experiment are light. If labeled with F(ab')2, RF interference in the specimen can be avoided. There are not many patterns of enzyme-labeled antigens in ELISA, and the general requirement is that the antigen must be of high purity.
2.3 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 bifunctional group reagent which allows an enzyme to bond with an amino group of a protein. Alkaline phosphoric acid is generally labeled by this method. The cross-linking method is one-step method and two-step method. In the one-step method, glutaraldehyde is directly added to the mixture of the enzyme and the antibody, and after the reaction, the enzyme-labeled antibody is obtained.
Enzymes commonly used in ELISA are generally cross-linked by this method. It has the advantages of simple and effective operation (combination rate of 60%-70%) and good repeatability. The disadvantage is that the cross-linking reaction is random. When the enzyme is cross-linked with the antibody, the ratio between the molecules is not strict, the size of the conjugate is not uniform, and the enzyme and the enzyme, and the antibody and the antibody may crosslink, which may affect the effect.诹Talk about ㄖ ㄖ school? br> First the enzyme and glutaraldehyde, dialysis to remove excess glutaraldehyde, and then react with antibodies to form enzyme-labeled antibodies. It is also possible to first react the antibody with glutaraldehyde and then with the enzyme. Most of the enzymes and proteins in the two-step process are combined in a 1:1 ratio, which is more helpful to the background improvement than the one-step enzyme conjugate, but the sensitivity is coupled. It is lower than one step.
(2) Periodate oxidation method: This method is only applicable to enzymes with high sugar content. This method is commonly used for the labeling of horseradish peroxidase. During the reaction, sodium periodate oxidizes the polysaccharide on the surface of the HRP molecule to an aldehyde group, which is active and forms a Schiff base with an amino group on the protein. The enzyme label is linked in a 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 are not easy to repeat.
The enzyme conjugates prepared as described above are generally mixed with unconjugated enzymes and antibodies. In theory, the free enzyme mixed in the conjugate generally does not affect the final enzyme activity assay in the ELISA, and after thorough washing, the free enzyme can be removed without affecting the final color development. However, the free antibody is different, and it competes with the enzyme-labeled antibody for the corresponding solid phase antigen, thereby reducing the amount of the enzyme-labeled antibody bound to the solid phase. Therefore, the prepared enzyme conjugate should be purified, and the free enzyme and antibody are removed for detection, and the effect is better. There are many methods for purification, and methods for separating macromolecular compounds can be applied. The ammonium sulfate salting out method is the easiest, but the effect is not satisfactory because this method can only remove the free enzyme remaining in the supernatant, but a considerable amount of free antibody still precipitates together with the enzyme conjugate and cannot be separated. It is more preferable to use ion exchange chromatography or molecular sieve separation. High performance liquid chromatography can clearly separate the prepared conjugate into three parts: free enzyme, free antibody and pure conjugate to obtain the best separation effect, but the cost More expensive.
After the conjugate is prepared, it is necessary to determine the appropriate working concentration before use as an ELISA reagent. The use of a concentrated conjugate is neither economical nor increases the background; if the concentration of the conjugate is too low, it affects the sensitivity of the detection. Therefore, the concentration of the conjugate must be selected. The optimum working concentration means that when the conjugate is diluted to this concentration, a low background can be maintained, and the optimum sensitivity of the measurement can be obtained, and the most suitable measurement conditions and measurement cost savings can be achieved. In the case of the enzyme-labeled antibody itself, its effective working concentration refers to the highest dilution at which a positive reaction can be obtained when tested against a carrier coated with its corresponding antigen. For example, a certain HRP: anti-human IgG preparation indicates a working concentration of 1:5000, indicating that the preparation will undergo a positive reaction when subjected to an ELISA test in a solid phase coated with human IgG after dilution of 1:5000. However, in the specific ELISA assay, the optimal working concentration of the enzyme-labeled antibody is affected by the nature of the solid phase carrier, the purity of the coated antigen or antibody, and the entire detection system such as the specimen, reaction temperature and time, etc. The "drip dispense" selection under actual measurement conditions can achieve the highest sensitivity maximum dilution as the working concentration in the kit.
2.4 Preservation of conjugates The enzymes and antibodies in the enzyme-labeled antibodies are biologically active substances, which are improperly stored and easily inactivated. The high concentration of the combination is relatively stable, and can be stored in an ordinary refrigerator for about one year after lyophilization, but the activity is reduced during the lyophilization process, and it needs to be reconstituted during use, which is quite inconvenient. The addition of an equal volume of glycerin to the conjugate solution can be stored for a longer period of time in the ice tray of a low temperature refrigerator or a conventional refrigerator. The conjugates in the early ELISA kits are generally supplied in the above two forms, with dilutions (see 3.2.5) diluted to the working solution at the indicated dilutions. Now more advanced ELISA kits have been formulated into working fluids with suitable buffers. No further dilution is required. The storage period is 4 months at 4-8 °C. Due to the low protein concentration, the conjugate is easily inactivated and a protein protectant is added. In addition, antibiotics (such as gentamicin) and preservatives (HRP conjugate plus sulfur pump, AP conjugate can be added with sodium azide) to prevent bacterial growth.
2.5 conjugate dilution
It is used to dilute a high concentration of the conjugate to prepare a working fluid. In order to avoid direct adsorption of the conjugate to the solid support in the reaction, a high concentration of unrelated protein (for example, 1% bovine serum albumin) is often added to the dilution buffer to compete for inhibition of conjugate adsorption. A nonionic surfactant having an inhibitory protein adsorbed on the surface of the plastic is also generally added, such as Tween 20, and a concentration of 0.05% is suitable. In the case of indirect determination of antibodies, serum samples should be diluted and assayed, and such dilutions can also be applied.
3.1HRP substrate
HRP catalyzes the oxidation of peroxides. The most representative peroxide is H2O2. The reaction formula is as follows:
DH2+ H2O2 D+ H2O
In the above formula, DH2 is an oxygen donor and H2O2 is a hydrogen acceptor. In ELISA, DH2 is generally a colorless compound which, upon enzymatic action, becomes a colored product for colorimetric determination. Commonly used hydrogen donors are O-phenylenediamine (OPD), tetramethylbenzidine (3,3',5,5'-tetramethylbenzidine, TMB) and ABTS [2,2'-azino-di- (3-ethylbenziazobine sulfonate-6)].
The product after oxidation of OPD is orange-red, and after stopping the enzyme reaction with acid, it has the highest absorption peak at 492 nm, high sensitivity, convenient colorimetry, and is the most commonly used substrate for HRP conjugate. OPD itself is hardly soluble in water, and OPD·2HCL is water soluble. It has been reported that OPD is anabolic and should be noted during operation. OPD is easy to deteriorate, and it is more unstable after being mixed with hydrogen peroxide to form a substrate application liquid. In the kit, OPD and H2O2 are generally divided into two components, and OPD can be made into a certain amount of powder or tablet form, and the tablet contains a foaming auxiliary solvent, which is more convenient to use. Hydrogen peroxide is added to the substrate buffer, and is made into a concentrate that is easy to store. It is diluted with distilled water when used. The advanced ELISA kit is directly formulated into a working solution containing a protective agent at a working concentration of 0.02% H2O2, and can be used as a substrate application liquid only after adding OPD.
The TMB was blue after the action of HRP, and the visual contrast was clear. TMB is relatively stable in nature and can be formulated into a solution reagent. It can be mixed with H2O2 solution to form an application solution, which can be directly used as a substrate. In addition, TMB has the advantages of no carcinogenicity, so it is widely used in ELISA. After the enzyme reaction is terminated with HCL or H2SO4, the TMB product is yellowish blue and can be quantified in a colorimeter with an optimum absorption wavelength of 405 nm.
Although ABTS is not as sensitive as OPD and TMB, the blank value is extremely low and is also used in some kits.
Another substrate for HRP is 3-(4-hydroxy)phenly propionic acid (HPPA). After HRP, the product is fluorescent and can be measured by a fluorometer. An advantage for ELISA is that it can broaden the linear range of quantitative assays.
HRP has a high specificity for hydrogen acceptors and acts only on H2O2, peroxides of small alcohols and urea peroxide. H2O2 is the most widely used, but urea peroxide is solid and is convenient and stable as a reagent compared to H2O2. The kit is supplied with a urea peroxide tablet, which is dissolved in distilled water and sealed in a substrate buffer at a low temperature (2 to 8 ° C) for one year.
3.2 AP substrate
AP is a phosphatase, and p-nitrophenyl phosphate (p-NPP) is generally used as a substrate to form a tablet, which is convenient to use. The product was yellow p-nitrophenol with an absorption peak at a wavelength of 405 nm. After the enzyme reaction was stopped with NaOH, the yellow color was stable for a time. AP also has a fluorogenic substrate (4-methylumbelliferone phosphate), which can be used for ELISA for fluorescence determination, and the sensitivity is higher than the colorimetric method using a chromogenic substrate.
In the plate ELISA, the usual dilution is 0.05% Tween 20 phosphate buffered saline.
5. Enzyme reaction stop solution
The commonly used HRP reaction stop solution is sulfuric acid, and its concentration varies depending on the amount of addition and the final volume of the colorimetric solution, and is generally 2 mol/L in a plate ELISA.
6. Positive control and negative control
The positive control and the negative control are the control products for verifying the validity of the test, and also serve as a control for the judgment result. Therefore, the basic composition of the reference substance, especially the positive control substance, should be as close as possible to the test sample. The composition is consistent. In the determination of human serum as a specimen, the control substance is preferably also human serum, since normal human serum can produce different degrees of background in various ELISA modes. Because a large number of normal human blood is more difficult to obtain, the reference materials in foreign kits mostly use recalcified human plasma as raw material, that is, calcium ions are added to the plasma to solidify the fiber protein, and the clot is removed. The resulting liquid has a composition similar to that of serum. The negative control must be tested first to determine that it does not contain the substance to be tested. For example, HBsAg should not contain HBsAg in the negative control, and anti-HBs are also negative. The positive control is mostly based on a buffer containing a protein protective agent, and a certain amount of the substance to be tested is added, and the amount is preferably indicated in the reagent specification. The amount to be added should be commensurate with the sensitivity of the reagent. The absorbance obtained in the assay is compared with the absorbance of the specimen to have a rough estimate of the amount of the test substance in the specimen. The sensitivity of the ELISA kit for detecting HBsAg abroad is about 0.5 ng/ml, and the content of the positive control is about 10 ng/ml. Antibiotics and preservatives are generally added to the control to facilitate storage.
7. Reference standard
AP can catalyze the substrate NADP+ dephosphorylation to produce NAD+ (coenzyme 1), using ethanol as reducing agent, dehydrogenation of alcohol under alcohol deoxygenase ratio, NAD+ reduction to NADH, and NADH dehydrogenation under catalysis of diaphorase. At the same time, the tetrazoliim is reduced to colored formazan. AP continuously catalyzes the formation of NAD+, and the conversion of NAD+ and NADH continuously generates wax. The entire reaction cycle consists of AP and alcohol dehydrogenase, diaphorase enzyme cascade, each AP molecule can catalyze the formation of 6X104 NAD+ molecules per minute, and each NAD+ can produce 60 waxy molecules per minute. This EIA amplification system, first reported by Selfl in 1984, can increase the sensitivity of EIA by about 250 times.
Review of ELISA technology
ELISA principle
The ELISA utilizes the specific binding characteristics of antigen-antibodies to detect the specimen; since the antigen or antibody on the solid support (usually a plastic well plate) can still have immunological activity, the binding mechanism is designed and matched. The color reaction of the enzyme can show the presence of a specific antigen or antibody, and can be quantitatively analyzed by the depth of color development. Depending on the sample to be tested and the binding mechanism, ELISA can design a variety of different types of detection methods, mainly in the "sandwich" (indirect method) (indirect), and "competitive" (Competitive) Mainly, the following is an introduction to various methods.
ELISA classification
See the double-anti-sandwich principle of ELISA;
The double-anti-sandwich method of ELISA is also known as the "sandwich method".
The specific antibody is coated on the plastic well plate, and the multi-purine antibody is washed away after completion;
The sandwich method uses two antibodies to identify the antigen in the specimen twice, so the specificity is quite high, but the antigen to be tested must be a multivalent antigen, so that more than two specific antibodies can be obtained. To separate the sandwiches; and this method requires sufficient epitope space for the sandwich of antigen and antibody, so it is not suitable for small molecular weight such as hapten or small molecule antigen.
Derivatives of the ELISA double-antibody sandwich method, such as the ABC method: the biotin-avidin system of ELISA, is an important revolution in detection methods. The biotin/avidin system is generally used in three forms, namely, bridging method (BAB), labeling method (BA) and ABC method. Many scholars have used different antigen-antibody systems to detect better. As a result, the BA method and the ABC method are more applied, and the sensitivity is generally 4 to 80 times higher than that of the conventional ELISA method.
Indirect method of ELISA:
Indirect methods are commonly used to detect antibodies. The general procedure is:
The principle of competition in ELISA;
The competition method is a less commonly used ELISA detection mechanism, which is generally used to detect small molecule antigens. The operation steps are as follows:
Commercialization kit components and classification of ELISA :
In clinical testing or scientific testing, ELISA experiments are usually available in commercial kits. There are three essential reagents in the ELISA kit: immunosorbent, conjugate and substrate for the enzyme.
The complete ELISA kit contains the following components: (1) a solid phase carrier (immunoadsorbent) that has been coated with an antigen or antibody;
1 , immunosorbent
The solid phase carrier coated with the antigen or antibody can be generally stored for more than 6 months under conditions of low temperature (2 to 8 ° C) drying. Some incomplete test kits only supply the antigen or antibody for the package, and the tester needs to coat it by himself. For example, the BioTNT website, the agent's products, some are complete kits, some are duoset that need to be coated, or other claims, are incomplete kits.
1.1 Solid phase carrier
The solid phase carrier acts as an adsorbent and container during the ELISA assay and does not participate in the chemical reaction. There are many materials that can be used as carriers in ELISA, the most common being polystyrene. Polystyrene has a strong ability to adsorb proteins, and the antibody or protein antigen retains its original immunological activity after being adsorbed thereon, and its price is low, so it is widely used. Polystyrene is plastic and can be made in various forms.
A good ELISA plate should have good adsorption performance, low blank value, high transparency at the bottom of the hole, and similar performance between the plates, between the holes of the same plate, and between the holes of the same plate. Polystyrene ELISA plates vary greatly in quality due to differences in raw materials and manufacturing processes. Therefore, each batch of ELISA plates must be inspected beforehand for performance. The commonly used test method is: coating the wells of the ELISA plate with a certain concentration of human IgG (generally 10 ng/ml), adding appropriate dilution of the enzyme-labeled anti-human IgG antibody to each well after washing, washing after heat preservation, adding substrate Color development, after terminating the enzyme reaction, the absorbance of each well solution was measured. The reaction conditions were controlled so that the readings of each well were at an absorbance of about 0.8. Calculate the average of all readings. The difference between the mean of all individual readings and all readings should be less than 10%.
A plastic similar to polystyrene is polyvinyl chloride. As an ELISA solid phase carrier, polyvinyl chloride is characterized by a thin, soft board that can be cut and is inexpensive, but the finish is not as good as that of polystyrene, and the bottom of the hole is not as flat as polystyrene. Polyvinyl chloride has higher adsorption properties for proteins than 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 and negative specimens are selected by other immunological methods, and they are subjected to a series of dilutions at different solids. The phase carrier was assayed according to a predetermined ELISA procedure and the results were compared. On which carrier the positive and the negative results differ the most, and this vector is the most suitable solid phase carrier for this ELISA assay.
The small test tube as a solid phase carrier also has a large adsorption surface, and the reaction amount of the specimen also increases accordingly. The sample size of the plate and bead ELISA is generally 00-200 ul, and the small test tube can increase the reaction volume as needed, and the increase of the reaction amount of the sample contributes to the improvement of the test sensitivity. The small test tube can also be used as a cuvette, and finally placed directly into the colorimetric meter in the spectrophotometer.
Microparticles made of polystyrene latex or other materials are also used as ELISA solid phase carriers. This has the advantage that the surface area is extremely large and the reaction proceeds in a suspension at a rate similar to that of the liquid phase. The magnetic particles containing iron are used as the ELISA solid phase carrier, and the reaction is separated by magnet attraction after the reaction, and the washing is convenient, and the kit is generally equipped with a special instrument.
The kits used in scientific research and quantitative testing have higher requirements for solid phase carriers, and generally use nunc or costar, or other well-known brands of enzyme-labeled plates for coating.
The solid phase carrier and coating process are briefly described below.
1.2 way of coating
Fixing an antigen or antibody in a process is called coating. In other words, the coating is the process by which the antigen or antibody binds to the surface of the solid support. The protein and polystyrene solid phase carrier are bound by physical adsorption, relying on the interaction between the hydrophobic group on the structure of the protein molecule and the hydrophobic group on the surface of the solid support. 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 for different proteins is different. Macromolecular proteins Smaller molecular proteins usually contain more hydrophobic groups, so they are more easily adsorbed onto the surface of the solid support. IgG has a strong adsorption force on solid phase such as polystyrene, and its linkage occurs mostly on the Fc segment, and the antibody binding site is exposed to the outside, so the coating of the antibody is generally carried out by direct adsorption. Most protein antigens can also be coated in a similar manner to antibodies. When the antigenic determinant is present in or adjacent to the hydrophobic region, the direct adsorption of the antigen and the solid phase carrier can prevent the antigenic determinant from being sufficiently exposed. In this case, the direct coating effect is not good, and the indirect capture coating can be used. In the method, a specific antibody against the antigen is pre-coated, and then the antigen is immobilized by an antigen-antibody reaction. The antigen bound indirectly on the solid phase is far from the surface of the carrier, and its antigenic determinant is also fully exposed. The affinity of the indirectly coated antigen by solid phase antibody, the purity of the antigen coated on the solid phase is greatly improved, so the antigen containing more impurities can also adopt the capture coating method, the specificity and sensitivity of the test. Both are improved and repeatability is good. Another advantage of indirect coating is that the amount of antigen is small, only 1/10 or even /100 of the direct coating. Non-protein antigens that are not readily adsorbed on a polystyrene carrier can be coated in a special manner. For example, when detecting an anti-DNA antibody, DNA is required as a coating antigen, and a general solid phase carrier generally cannot directly bind to a nucleic acid. The polystyrene plate can be first irradiated with ultraviolet rays (for example, a 30 W ultraviolet lamp, irradiated for 75 hours at 75 cm) to increase its adsorption performance. The solid phase carrier is pre-coated with a basic protein such as polylysine or protamine, and the binding force of the nucleic acid can also be improved. The avidin biotin system can also be used for indirect coating, that is, the carrier is first coated with avidin, and then the biotinylated DNA is added. The coating method is uniform and firm, and has been expanded to be used for quantification of various antigenic substances. Determination.
1.3 coated antigen
The antigens used for coating the solid phase carrier can be divided into three major categories: natural antigen, recombinant antigen and synthetic polypeptide antigen. Natural antigens may be obtained from animal tissues, microbial cultures, etc., and must be extracted and purified for coating. For example, HBsAg can be extracted from the serum of the carrier. General bacterial and viral antigens can be extracted from the culture. Protein antigens can be extracted from materials rich in this antigen (eg AFP is extracted from cord blood or fetal liver). ). The recombinant antigen is a protein antigen expressed by an antigen gene in a plasmid, and most of them are Escherichia coli or yeast. The advantage of the recombinant antigen is that in addition to the engineering bacteria components, other impurities are less, and no contagious, but the purification technology is more difficult. Recombinant antigens using Escherichia coli as a plasmid may not be sufficiently used for ELISA, and may be used for ELISA, and false positives may occur in the reaction, since many subjects are infected with E. coli and anti-E. coli antibodies are present in the serum. Another feature of recombinant antigens is the ability to genetically engineer certain antigenic materials that cannot be separated from natural materials. For example, hepatitis C virus (HCV) has not been successfully cultured, and the HCV antigen content in the serum of patients with hepatitis C is extremely small. At present, most of the coated antigens used in the detection of anti-HCV ELISA are recombinant antigens prepared according to the gene expression of HCV. In the diagnosis of infectious diseases, many recombinant antigens such as HBsAg, HBeAg and HIV antigens have been used in ELISA. A synthetic polypeptide antigen is a polypeptide fragment artificially synthesized based on the amino acid sequence of an antigenic determinant of a protein antigen molecule. Polypeptide 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 to the solid phase. The coating of the polypeptide antigen is generally first coupled to an unrelated protein such as bovine serum albumin (BSA) or the like, and indirectly bound to the surface of the solid support by adsorption of the conjugate to the solid support. Another point of attention in the application of the polypeptide antigen is that he can only detect antibodies corresponding thereto. A protein antigen often contains a number of different determinants that cause antibody production, so other antibodies in the serum to be tested cannot react with the polypeptide antigen. In addition, changes in antigen structure often occur when certain microorganisms are mutated. In this case, coating with individual polypeptide antigens may cause missed detection of other antibodies.
2. The enzyme conjugate (conjugate)
3. Enzyme substrate
4. Washing liquid
Quantitatively determined ELISA kits (eg, alpha-fetoprotein carcinoembryonic antigen assays, etc.) should contain reference standards for the preparation of standard curves, which should include 4-5 concentrations covering the detectable range, generally formulated with protein-containing protective agents and Preservative in the buffer.
Other content:
Multi-enzyme cascade amplification system for enzyme-linked immunosorbent assay:
First, AP substrate amplification system
Substrate ampiification system
However, Brooks et al. found that the acetaldehyde produced in the above substrate reaction cycle inhibited the entire enzyme cascade and affected the amplification effect, and when the semicarbazide hydrochloride was added, the reaction cycle was further extended, thereby increasing the sensitivity of the assay. It is because Yanjun semicarbazide can react with acetaldehyde to form semicarbatone and water, thus eliminating the inhibition of acetaldehyde on the reaction. This improved method was used to determine the protein A-containing Staphylococcus aureus in food, and the sensitivity was determined from the original method of (4-6) X103 colony forming units (c.{.u)/g or m1 to 20 colonies. The formation unit (c.f.u) / g or ml, the sensitivity of the measurement is increased by about 200-300 times.
Later, Self et al. replaced the tetrazolium salt in the original amplification mode with resazurin, which became a fluorescent resorufin under the action of diaphorase, and then obtained the result using a fluorescence colorimeter. The sensitivity of this method is also much higher than that of the original method, and only about 350 AP molecules per well can be measured.
Second, double enzyme cascade amplification system
Dual-enzyme cascade double enzyme cascade amplification system, also known as enzyme inhibition cascade amplification system, esterase inhibitor 4-(3-oxo-4,4,4-trifluorobutyl)phenylphosphonium salt due to its closure The -P04 group has no inhibitory activity on esterases, but when it is de-PO4 group under the action of AP, the inactivated inhibitor becomes an active inhibitor, which inactivates the added carboxylesterase. The color reaction determines the residual esterase activity to know the activity of the original AP, which is inversely related. The sensitivity of this amplification system is about 125 times higher than that of the conventional method.
Third, the enzyme activated cascade amplification system
Enzymeactivationcascade is also an amplification system that uses AP as a labeling enzyme. The flavin-adenine dinucleotide phosphate (FADP) is dephosphorylated to FAD under the catalysis of AP. FAD converts the apo-amino acid oxidase into a full-enzyme amino acid oxidase, which catalyzes the production of H2O2 by proline. Thus, in the presence of DCHBS, 4AAP and HRP, a colored product is obtained. The above amplification system can measure l{mol/LAP.
In addition, catalytic indicator deposition (CARD) is also a multi-enzyme cascade EIA amplification system. The free radical generated by HRP oxidized biotin or fluorescein-labeled tyramine can react with the tyrosine and tryptophan of the protein on the solid phase to deposit a large amount of biotin or fluorescein on the solid phase around the HRP. Inside, the deposited biotin can be determined by HRP and 6-galactosidase or AP-labeled streptavidin, and the precipitated fluorescein can be determined by the enzyme-labeled anti-fluorescein. This allows a single molecule of HRP to be converted to a large number of labels, thereby greatly improving (about 30 times) the sensitivity of the EIA assay. Diamandis et al. used the chelated Eu3+ streptavidin-thyroglobulin reagent instead of the above enzyme standard to determine AFP, which not only improved the sensitivity of the assay, but also improved the background "noise" by 6-8 times compared with the conventional method. We use the above CARD amplification system directly in the HBsAg commercial ELISA kit, which can increase the sensitivity of the kit by about 5 times without changing any components and procedures of the commercial kit.
Coagulation factor enzyme cascade amplification system
Lipopolysaccharide (LPS) activates the coagulation cascade of hemorrhagic cell lysates. First, LPS activates factor C to C, which in turn activates factor B to B, B converts precoagulase to coagulase, and coagulase catalyzes substrate (Boc-Le, u-Gly-Arg-p) to produce colored p-Nitroaniline (旷nitroa·niline, PNA), measured at A405nm. Therefore, in the immunoassay, if the antibody is labeled with LPS as a label, the sensitivity of the assay can be amplified by the above reaction, and IgG of 10-7 to 10-11 g/m1 and 10-7 to 10-11 g/ can be detected. The anti-IgG, Seki, etc. of ml used the above method for the detection of HBsAg by double sandwich method with sensitivity of 10-10~10-12g/ml.
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