② The antigen or antibody is linked to an enzyme to form an enzyme labeled antigen or antibody, which retains both its immune activity and enzyme activity.During the determination, the tested samples (antibodies or antigens in them) and enzyme labeled antigens or antibodies are reacted with the antigens or antibodies on the surface of the solid carrier in different steps.Use washing method to makeAntigen antibody complexThe amount of enzyme separated from other substances and finally combined on the solid carrier and tested in the sampleAmount of substanceIn a certain proportion.joinEnzyme reactionThe substrate isEnzyme catalysisIt becomes a colored product, and the amount of the product is directly related to the amount of the tested substance in the sample, so it can be determined according toColor responseFor qualitative or quantitative analysis.Because of the high catalytic frequency of the enzyme, the reaction effect can be greatly amplified, so that the determination method can reach a highSensitivity。
ELISA can be used to detect antigen and antibody.There are three necessary reagents in this determination method:
③ Enzymatic substrate（Chromogenic agent）。Different types of detection methods can be designed according to the source of reagents, the characteristics of samples and the conditions for detection.
2. OnELISAIt is very important to select various experimental conditions, including:
⑴ Selection of solid carrier: many substances can be used as solid carrier, such aspolyvinyl chloride、polystyrene, Polypropylamide andcelluloseEtc.It can be in the form of concave plate, test tube, bead, etc.Currently, 40 hole polystyrene concave is commonly usedOrifice plate。No matter what kind of carrier, it can be screened before use: use the same amount of antigen to coat, react under the same experimental conditions, and observe itsChromogenic reactionwhetherHomogeneityAnd judge whether its adsorption performance is good.
(2) Selection of coated antibody (or antigen): when the antibody (or antigen) is adsorbed on the surface of the solid carrier, the purity is required to be good, and the general requirements for adsorptionPHBetween 9.0 and 9.6.The adsorption temperature, time and protein content also have a certain impact, generally 4 ℃, 18-24 hours.proteinThe optimum concentration of coating needs to be titrated: different protein concentrations (0.1, 1.0 and 10μg/Ml, etc.) after coating, when other test conditions are the same, observe theOD（optical density-optical density）Value.choiceOD valueThe highest concentration with the lowest protein content.It is usually 1-10 μ g/ml for most proteins.
⑶ Enzyme labelled antibodySelection of working concentration: first, use direct ELISA method for preliminarytiter Titration of (see the section of enzyme labeled antibody).Then fix other conditions or adopt the "square matrix method" (the coating, the reference substance of the sample to be tested and the enzyme labeled antibody are differentDilution）Titrate the working concentration accurately in the formal experimental system.
⑷ Enzyme substrate andHydrogen donorSelection of hydrogen donor: the selection requirements are cheap, safe and obviousChromogenic reaction, but it is colorless.Some hydrogen donors (such as OPD) have potentialCarcinogenesisPay attention to protection.If possible, non carcinogenic and highly sensitive hydrogen donors should be used, such asTMBandABTSIt is currently a relatively satisfactory hydrogen donor.After the substrate acts for a period of time, addstrong acidOr strong base to stop the reaction.Generally, the substrate action time should be 10-30 minutes.Substrate solution must be freshly prepared, especially H2O2 added before use.
Test steps
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ELISA uses serum for detection. First, the blood must be agglutinated for at least half an hour, and then the serum is taken.Use the enzyme complexDiluentAfter dilution, add serum, negative and positive controls, and quality control products (this is a strict requirement, and its scope must be within the quality control range).After an hour of incubation, the plate is washed, the substrate is added, and after half an hour of reaction in the dark, the reaction part is completed by adding the termination solution, and then the reading is made.The negative or positive result is judged by the numerical value.
Double antibody sandwich methodIt is the most commonly used method for antigen detection, and the operation steps are as follows:
1、 SetSpecificityThe antibody is connected with the solid carrier to form the solid antibody: the unconjugated antibody and impurities are washed and removed.
2、 Add the tested sample: make it contact with the solid antibody for reaction for a period of time, let the antigen in the sample combine with the antibody on the solid carrier to form a solid antigen complex.Wash to remove other unconsolidated substances.
3、 Add enzyme labeled antibody: make solid phaseImmune complexThe antigen on is bound to the enzyme labeled antibody.Wash the unbound enzyme labeled antibody thoroughly.At this time, the amount of enzyme carried on the solid carrier and the amount of tested substance in the samplepositive correlation。
4、 Substrate addition: enzyme catalyzed substrate in sandwich complex becomes colored product.The qualitative or quantitative determination of the antigen is based on the degree of color reaction.
According to the same principlemacromoleculeThe solid phase antigen and enzyme labeled antigen conjugates were prepared by the antibody, that is, the antibody in the sample can be determined by the double antigen sandwich method.
stayClinical testThis method is applicable to the detection of various proteins and other macromolecular antigens, such asHBsAg、HBeAg、AFP、hCGEtc.As long as it is obtained thatAntigenic anisotropyAntibodies can be used to coat solid carrier and prepare enzyme conjugate to establish this method.For example, the source of antibody isantiserumAntibodies for coating and enzyme labeling should preferably be taken from animals of different species and genera.As appliedmonoclonal antibodyGenerally, two different antigens are selectedDeterminative clusterOfMonoclonal antibody, respectively for coating solid phase carrier and preparing enzyme conjugate.This double locusSandwich methodIt has a high specificity, and can be used for one-step detection by holding the sample and enzyme labeled antibody together.
In the one-step method, when the content of the tested antigen in the sample is very high, the excess antigen will bind to the solid phase antibody and enzyme labeled antibody respectively, instead of forming a "sandwich complex".Similar toPrecipitation reactioninSuperantigenOfBackband phenomenon, at this time, the absorbance value of the color after reaction (located on the antigen excess band) andstandard curve (onAntibody excess band(I) The absorbance value of a certain antigen concentration is the same, if measured by normal method, the result will be lower than the actual content, which is calledHook effect(hook effect), because the standard curve bends like a hook after reaching the peak.When the hook effect is serious, the reaction may not even be colored and falseNegative results。Therefore, when using one-step reagents to determine substances with abnormally high content in samples (such as HBsAg, AFP in serum, and hCG in urine, etc.), attention should be paid to the highest value of the measurable range.The preparation of such reagents with high affinity monoclonal antibodies can weaken the hook effect.
If there are multiple identical determinants at different sites of the tested molecule, such as the a-determinant of HBsAg, the solid phase and enzyme conjugate can also be coated with the same monoclonal antibody for this determination.However, attention should be paid to subtypes in the detection of HBsAg. HBsAg has four subtypes: adr, adw, ayr and ayw. Obviously, each subtype has the same a-determinantReactivityThis is also a problem that should be paid attention to when using monoclonal antibody as sandwich method.
Another point of attention for antigen detection by double antibody sandwich method isRheumatoid factor(RF).RF is aautoantibody, most of which are IgM type, and can combine with Fc segment of IgG of many animals.Used for double antibody sandwich detectionSerum specimenIf RF is contained in, it can act as an antigen component and combine with solid phase antibody and enzyme labeled antibody to showfalse positiveReaction.Use F (ab ') orFab clipThe reagent used as enzyme conjugate can eliminate RF interference due to the removal of Fc segment.Whether the double antibody sandwich ELISA reagent is affected by RF has been listed as an assessment indicator of this kind of reagent (see 6.2).
The double antibody sandwich method is applicable to the determination of bivalent or above macromolecular antigens, but not applicable to the determination ofHaptenandSmall moleculeMonovalent antigen, because it cannot form a two site sandwich.
Double antigen sandwich method for antibody detection
The reaction mode is similar to that of the double antibody sandwich method.useSpecific antigenCoating and preparing enzyme conjugates to detect corresponding antibodies.The difference between indirect method and ELISA is that ELISA antigen replaces ELISA antibody.In this method, the tested sample does not need to be diluted and can be directly used for determination, so its sensitivity is relatively higher than that of the indirect method.hepatitis BMarkerThis method is often used to detect intermediate anti HBs.The key of this method lies in the preparation of enzyme labeled antigen, and appropriate labeling methods should be found according to different antigen structures.
When using the double antibody sandwich method to determine the antigen, if the application is aimed at two different antigen moleculesAntigenic determinantOfmonoclonal antibodyAs solid phase antibody and enzyme labeled antibody respectively, the addition of sample and enzyme labeled antibody can be made in two steps and one step.This double site one-step not only simplifies the operation, but also shortens thereaction timeIf high affinity monoclonal antibodies are used, the sensitivity and specificity of the assay will also be significantly improved.The application of monoclonal antibodies has improved the ELISA for antigen detection to a new level.
In one-step determination, attention should be paid to hook effect, which is similar to the phenomenon of the back band of excess antigen in precipitation reaction.When the concentration of the antigen to be measured in the sample is quite high, the excess antigen will bind to the solid phase antibody and enzyme labeled antibody respectively, instead of forming a sandwich complex, and the result will be lower than the actual content.When the hook effect is serious, false negative results may even occur.
Indirect detection of antibodies
indirect method
Indirect method is the most commonly used method to detect antibodies. Its principle is to use enzyme labeled antibodies to detect the tested antibodies that have been combined with solid phase, so it is called indirect method.The operation steps are as follows:
⑴ Connect specific antigen with solid carrier to form solid antigen: wash and remove unconjugated antigen and impurities.
⑵ Add diluted tested serum: the specific antibody in it combines with the antigen to form a solid phase antigen antibody complex.After washing, onlySpecificityAntibodies.Other antibodies and impurities in the serum are washed away in the washing process because they cannot bind to the solid phase antigen.
⑶ Enzyme labeled anti antibody: it binds with the antibody in the solid phase complex, so that the antibody is indirectly labeled with the enzyme.After washing, the amount of enzyme on the solid carrier represents the amount of specific antibody.For example, if you want to test human antibodies to certain diseases, you can use enzyme labeled sheep anti humanIgG antibody。
⑷ Color rendering with substrate:color depthRepresents the amount of antibody tested in the sample.
This law is mainly used forpathogenAntibody detection is used to diagnose infectious diseases.The advantage of indirect method is thatCoating antigenThe same enzyme labeled antibody can be used to establish a method for detecting the corresponding antibody.
The key to the success of indirect method is the purity of antigen.Although sometimes the actual and effective results can be obtained by coating with crude extracted antigen, it should be purified as far as possible to improve the specificity of the test.Special attention should be paid to removing impurities that can react with the serum of normal healthy people, such as EColi is the recombinant antigen of engineering enzyme, such as EColi composition, probably similar to EThe anti EColi antibody reacts.The antigen shall not contain substances that react with the enzyme labeled anti human Ig, such as antigens from human plasma or human tissues. If the Ig is not removed, false positive reactions will also occur in the test.In addition, if the antigen contains unrelated proteinsCompetitive adsorptionAnd affect the coating effect.
Another interference factor in the indirect method is the high concentration of non-specific antibodies contained in normal serum.The specific IgG detected in the patient's serum only accounts for a small part of the total IgG.IgGAdsorbabilityVery strong, non-specific IgG can be directly adsorbed onto the solid carrier, and sometimes onto the surface of the coated antigen.Therefore, in the indirect method, the antigen is usually coated with unrelated proteins (such asBovine serum albumin）Wrap it again to block the empty gap on the solid phase.In addition, the sample must be diluted first (1:40~1:200) during the test to avoid too high negative background affecting the judgment of the results.
competition law
The competitive method can be used to determine antigen and antibody.Take the determination of antigen as an example, the tested antigen and enzyme labelAntigenic competitionAndsolid phaseThe antibody binds, so the amount of enzyme labeled antigen bound to the solid phase is similar to the amount of tested antigeninverse ratio。The operation steps are as follows:
⑴ Connect the specific antibody with the solid phase carrier to form the solid phase antibody.wash.
⑵ Add the mixture of the tested sample and a certain amount of enzyme labeled antigen in the tube to be testedsolutionAnd make it react with solid antibody.If there is no antigen in the tested sample, the enzyme labeled antigen can be successfully combined with the solid phase antibody.If the tested sample contains antigen, it will bind to the solid antibody with the same opportunity as the enzyme labeled antigen, occupying the opportunity to bind the enzyme labeled antigen to the solid carrier competitively, reducing the amount of the enzyme labeled antigen bound to the solid carrier.Only enzyme labeled antigen is added to the reference tube. After heat preservation, the combination of enzyme labeled antigen and solid antibody can reach the maximum amount.wash.
⑶ Color development with substrate: the reference tube has the darkest color because it has the most enzyme labeled antigens.The difference between the color depth of the reference tube and the color depth of the tube to be measured represents the amount of antigen in the tested sample.The lighter the color of the tube to be tested, the more antigen content in the sample.Generally, the culture system is detected by square wave voltammetrypeak current, through peak current andAntigen antibodyOflinear relationshipTo finally determine the concentration of the final detection target of the system.
When theInterfering substanceThis method can be used when it is difficult to remove or obtain enough purified antigenSpecificityAntibodies.The principle is that the antibody in the sample competes with a certain amount of enzyme labeled antibody to bind to the solid phase antigen.The more antibodies in the sample, the less enzyme labeled antibodies bound to the solid phase, so the positive reaction is lighter than the negative reaction.If the antigen is of high purity, it can be directly coated with solid phase.Such as antigenChina CouncilIf there are interfering substances, it is not easy to directly coat them. The capture coating method can be used, that is, first coat the antibody corresponding to the solid phase antigen, then add the antigen to form the solid phase antigen.Wash to remove impurities in antigen, and then add samples and enzyme labeled antibodies for competitive binding reaction.There are many modes for competitive antibody detection, which can combine the sample and enzyme labeled antibody with solid phaseAntigenic competitionThis method is generally used for anti HBc ELISA.The other mode is to add the sample and antigen together into the solid phase antibody for competitive binding, and then add enzyme labeled antibody after washing to react with the antigen bound on the solid phase.This method is generally used to detect anti HBe.
In serum, certainAntigen specificityIgMIt often coexists with specific IgG, and the latter will interfereMP-IgM Determination of.Therefore, capture method is often used to determine IgM antibody. First, fix all serum IgM (including specific IgM and non-specific IgM) on solid phase, and then determine specific IgM after removing IgG.The operation steps are as follows:
⑴ Connect anti human IgM antibody to solid phase carrier to form solid phase anti human IgM.wash.
⑵ Add diluted serum sample: IgM antibody in serum after heat preservation reaction is captured by solid antibody.Wash to remove othersimmunoglobulinAnd impurities in serum.
⑶ Add specific antigen reagent: it only binds to specific IgM on solid phase.wash.
⑷ Add the targetSpecificityEnzyme labeled antibody: make it react and bind to the antigen on the solid phase.wash.
(5) Adding substrate color: if there is color display, it means that there is specific IgM antibody in the serum sample, which is a positive reaction.
Applied to ELISA
AvidinIs a kind ofglycoprotein, can be extracted from egg white.Molecular weight 60kD, each molecule consists of 4SubunitComposition, can be combined with 4BiotinMolecular intimate combination.Vitamin H, molecular weight 244.31, found in egg yolk.Derivative made by chemical method, biotin hydroxyamberImineBiotin hydroxysuccinide (BNHS) can be formed with proteins, sugars, enzymes and other types of large and small moleculesBiotinylationThe product of.The combination of avidin and biotin, although notimmune reaction, but the specificity is strong and the affinity is large. Once they are combined, they are extremely stable.Since one avidin molecule has four binding positions of biotin molecules, more biotinylated molecules can be connected to form a lattice likecomplex。Therefore, the sensitivity of ELISA can be greatly improved by coupling avidin and biotin with ELISA.
The avidin biotin system can be used in ELISA in many forms, including indirect coating and final reaction amplification.The avidin can be pre coated on the solid phaseAdsorption methodAntibodies or antigens coated with solid phase bind to biotin, and biotinylated antibodies or antibodies are in phase through avidin biotin reaction.This coating method can not only increase the amount of adsorbed antibodies or antigens, but also fully expose their binding points.In addition, the enzyme labeled antibody in conventional ELISA can also be replaced by biotinylated antibody, and then the avidin enzyme conjugate is connected to amplify the reaction signal.
Reagents for ELISA
Commercial kits are generally used in clinical tests.There are three necessary reagents in ELISA:Immunosorbent、ConjugatesAnd enzyme substrates.The complete ELISA kit contains the following components:
ELISA reagent
⑴ Solid phase carrier (immunoadsorbent) coated with antigen or antibody; ⑵Enzyme labeled antigen or antibody (conjugate);
The purity of the enzyme is expressed in RZ: RZ=OD403/OD275
The RZ of pure enzyme is more than 3.0, the highest is 3.4.Enzymatic products with RZ below 0.6 are crude enzymes, and non enzymatic proteins account for about 75%, which cannot be used for labeling.RZ above 2.5 can be used for marking.The acting substrate of HRP ishydrogen peroxide, CatalyticReaction timeThere are several kinds of hydrogen donors for: (1)O-phenylenediamine(OPD), the product is orange, soluble, high sensitivity, maximumAbsorption valueAt 490nm, it can be judged by naked eye, and is easy to be concentratedH2SO4Stop reaction, the color can remain unchanged for several hours, which is the most commonly used ELISA in China;(2) General AssemblyfennelAmine (OD), the product is orange, the maximum absorption value is 400nm, and the color is relatively stable;(3) 5-aminosalicylic acid (5-AS): the product is dark brown, the maximum absorption value is 449nm, partially dissolved, and has poor sensitivity;（4）O-toluidine（OT）The product is blue, the maximum absorption value is 630nm, partially dissolved, unstable, and acid resistant, but the reaction is fast and the color is obvious.
alkaline phosphatase
The intestinal mucosa andEscherichia coliExtracted from multipleIsoenzymeform.There are many kinds of substrates, commonly used asnitrobenzenephosphate, cheap and non-toxic.EnzymolysisThe product is yellow, soluble, and the maximum absorption value is 400nm.Enzymatic activity: hydrolyze 1 μ g at 37 ℃ for 1 minute in pH10 reaction systemDisodium phenyl phosphateIs a unit.
Enzyme labeling
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Enzyme and antibody cross-linking, commonly usedglutaraldehydeMethod and periodate oxidation method.The modified sodium periodate method of HRP labeled antibody established by Guo Chunxiang is simple and easy to use, with good labeling effect, especially suitable for small batch preparation in the laboratory.The labeling procedure is: dissolve 5 μ g HRP in 0.5ml distilled water, add 0.06 freshly preparedmol/LSodium periodate（NaIO4）0.5ml water solution,Evenly mixPlace in 4 ℃ refrigerator for 30 minutes, take out and add 0.16mol/Lglycol0.5ml of water solution, add 5g after 30 minutes at room temperaturePurified antibody1ml of water solution, mix and loaddialysisBag, use 0.05mol/L, pH9.5 carbonate buffer solution in a 4 ℃ refrigerator to slowly stir for dialysis for 6 hours (or overnight) to combine, and thenAspirate, plusSodium borohydride(NaBH4) solution 5 (g/ml) 0.2ml, put it in a 4 ℃ refrigerator for 2 hours, and transfer the above combinationMixed liquidAdd an equal volume of saturated ammonium sulfate solution, place it in a 4 ℃ refrigerator for 30 minutes, and centrifuge the obtainedprecipitateDissolve it in a little 0.02mol/L, pH7.4PBS, and dialysis it overnight (4 ℃). Centrifuge it the next day to remove the insoluble matter to obtain enzyme labeled antibody. Dilute it to 5ml with 0.02mol/L, pH7.4PBS, and measure it,freeze dryingorCryopreservation。
Effect measurement
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Enzyme labeled antibody labeling effect test: the test content includes enzyme andAntibody activity、ConjugatesMedium enzyme content andIgGContent, enzyme and IgGmoleRatio and binding rate.
