CN1164947C - Process for preparing cross-linking antibody by solid-phase immunoadsorption method - Google Patents
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Abstract
The present invention belongs to the technical field of label immunoassay with wide application in medical diagnosis and a biological chip thereof, and particularly relates to a method for preparing cross-linking antibodies by a solid-phase immunoadsorption method. Firstly, an antigen molecule is used for coupling an activated solid-phase carrier for preparing a solid-phase immunoadsorption agent; the solid-phase immunoadsorption agent is thoroughly mixed with a purified antibody so that the antibody is combined with the solid-phase immunoadsorption agent in an immune mode; an activating functional reagent is used for respectively activating a macromolecular probe and the antibody in immune combination with the solid-phase immunoadsorption agent; then, a mixed reaction is carried out for forming a cross-linking object; the labelled antibody is dissociated and desalted on the solid-phase immunoadsorption agent for obtaining the labelling object-a cross-linking antibody of the covalent combination of the macromolecular probe and the antibody. The solid-phase immunoadsorption agent can be circularly and repeatedly used for the solid-phase cross-linking preparation of the labelling object-the cross-linking antibody. The cross-linking antibody prepared by the present invention and the covalent combination of the macromolecular labelling object and the antibody have small influence on the antigen combining capability of the antibody; the cross-linking antibody has an integral antigen combining position.
Description
Technical field
The technical field of the invention is labelled immune analysis and a biochip most widely used in the medical diagnosis, particularly utilizes the solid-phase immunity absorption method to prepare the method for cross-linking antibody.
Background technology
In the preparation of detection kit that labelled immune is analyzed and biochip, need to select suitable probe to prepare label, label is a key reagents of setting up the labelled immune analytical approach, and its quality directly influences sensitivity, stability and the accuracy of check and analysis.
The molecular labeling of antibody promptly under the prerequisite of the immunological characteristic that does not change antibody, links to each other reporter molecules with antibody, obtain the antibody that has the antibody of spike effect or have therapeutic action.Antibody labeling is one of immunochemistry most common technique, can reach identify antigen, to purposes such as antigen position.Directly mark tense marker thing directly is marked on the specific antibody of purifying.
The method of mark is relevant with the probe of selecting for use.Probe commonly used comprises isotope radioactive nuclide, fluorescent dye and enzyme molecule etc.By the mark of these reporter molecules, can realize radiommunoassay, fluoroimmunoassay and enzyme-linked immuno assay etc. effectively.
At present, the crosslinking technological of big molecular probe and antibody is promptly with the labelling technique of fluorescin dye well enzyme etc.Because probe and antibody all are protein, so during mark, can use for reference the general flow of protein covalent cross-linking.Cross-linking reaction between the protein, the chemical functional group's type that is suitable for coupling that mainly is subjected to being had in the protein molecule and the restriction of quantity.Most of cross-linking reaction be by the α in the protein molecule-and the nucleophilic character of epsilon-amino, imino group and sulfydryl realize, can adopt suitable mobilizing function reagent.Optionally mobilizing function reagent comprises homobifunctional agent and heterobifunctional agent.Homobifunctional agent comprises glutaraldehyde etc., and heterobifunctional agent comprises that (brief note is N-succinimido 3-(2-pyridine radicals two sulphur) propionic ester: SPDP), (brief note is succinimido-4-(N-methyl maleimide) cyclohexane-1-carbonic ester: SMCC), succinimido-γ-(brief note is (right-phenyl maleimide) butyrate: SMPB) etc.When labelled protein and antibody protein carry out can adopting single step reaction or the reaction of two steps, but usually forming inhomogenous potpourri when crosslinked with the homotype bifunctional reagent, there is multiple polymers to exist simultaneously.The development of the heterobifunctional agent of synthetic and application have overcome this shortcoming of homotype bifunctional reagent, and the direct mark mode of covalency still is widely used so far.The SPDP more as present application, it is crosslinked to use SPDP label albumen and antibody protein can be carried out by both amino, and can not form the polymkeric substance of label albumen or antibody protein self, has higher joint efficiency.
Above-mentioned labeling method adopts the liquid phase cross-linking reaction to realize, when introducing label by the free amine group of antibody molecule, the covalent bond site may be positioned at or F (ab) section of contiguous antibody comparatively at random sometimes.During particularly with marks such as fluorescin dyestuff or enzymes, because marker molecules is bigger, this combination may cause the partly shielding effect of antigen binding site of cross-linking antibody or sterically hindered, hinders combining of antibody and antigen, influences the antigen-binding activity of labelled antibody.In the protein cross reaction, also have the phenomenon of certain antibody part or all of inactivation in coupling process once in a while, inactivation is because the sealing or the conformational change of antigen binding site cause sometimes.Need by selecting suitable locus specificity coupling method to solve.
Summary of the invention
The objective of the invention is at preparing in the cross-linking antibody in the liquid phase cross-linking reaction, big molecular probe causes the antigen binding capacity of cross-linking antibody impaired because of the randomness (binding site may be positioned at or contiguous antibody active site) in covalent bond site; A kind of method of utilizing the solid-phase immunity absorption method to prepare cross-linking antibody (covalent conjunct agent of big molecular probe and antibody) is provided.Based on Solid phase Immunoadsorbent, protected at the antigen-binding site of antibody, and under the prerequisite of the antigen binding capacity of preservation antibody, prepare the cross-linking agent of big molecular probe and antibody.Crosslinked mild condition, preparation technology is simple, is easy to grasp control, the cross-linking antibody of preparation is complete, and the antigen binding site of crosslinked back antibody be subjected to big molecular probe sterically hindered influence less.
Antibody has two important domains, and one is the antigen-antibody binding site, and another is a fixed area, and the maintenance of antibody activity depends on the complete preservation of antigenic determinant configuration in the antibody.Therefore should take into full account the antibody protein configuration during chemical crosslinking of antibody and other albumen, activating area is optimized, preferred plan is effectively to control cross-linking reaction, and makes activated group avoid the antigen-antibody binding site.
The present invention is directed to the crosslinked randomness of liquid phase to big molecular probe and the antibody linked defective of bringing, produced with antigen and antibody in advance immunity combine and carries out labeled molecule and antibodies again and prepare the orderly crosslinked new approaches of label.
The present invention is at label (as the R-phycoerythrin)---and in the preparation of cross-linking antibody, the distinct methods and the technology of preparation cross-linking antibody in proposition and the traditional liquid phase are the cross-linking antibodies for preparing big molecular probe and antibody on Solid phase Immunoadsorbent.At first antigen is immobilized onto solid phase carrier, then antibody mediated immunity is adsorbed on the solid phase antigen, it is crosslinked with big molecular probe and antibody linked to carry out solid phase again, at last crosslinked good antibody is disintegrated down from solid phase antigen, obtain the label of probe and antibodies, immobilization antigen can be recycled repeatedly.Antigen-binding site owing to antibody in cross-linking process combines with the antigen molecule immunity, when big molecular probe and antibody molecule covalent bond, the covalent bond site is away from the antigen-binding site of antibody, the influence of antagonist conjugated antigen ability is little, and the loss of activity of the cross-linking antibody label of acquisition is less.
The present invention at first with the solid phase carrier of antigen molecule coupling activation, prepares Solid phase Immunoadsorbent; Solid phase Immunoadsorbent is fully mixed with antibody purified, antibody mediated immunity is incorporated on the Solid phase Immunoadsorbent, antibody combines with the immobilized antigen immunity and forms comparatively stable immune complex; With the antibody that mobilizing function reagent is distinguished the big molecular probe of activation processing and combined with the Solid phase Immunoadsorbent immunity, hybrid reaction forms cross-linking agent then; The antibody that mark is good by dissociate on the Solid phase Immunoadsorbent, desalination, obtain the covalently bound label of big molecular probe and antibody---cross-linking antibody.
Applied Solid phase Immunoadsorbent can be reused for the crosslinked preparation of solid phase of cross-linking antibody with freshly prepd Solid phase Immunoadsorbent through reclaiming in the solid phase cross-linking method.
The present invention utilizes the solid-phase immunity absorption method to prepare the concrete grammar of cross-linking antibody:
(1) preparation Solid phase Immunoadsorbent
At first choose solid phase carrier, the activation by carrier surface group (as hydroxyl etc.) is immobilized onto surface of solid phase carriers with antigen molecule, make stable Solid phase Immunoadsorbent, remaining avtive spot through fully cleaning, obtains Solid phase Immunoadsorbent with the sealing of micromolecule closed reagent.
(2) immunoadsorption
At pH is to help under the environment of the neutrality of immune complex combination or meta-alkalescence and low ionic strength, the Solid phase Immunoadsorbent that step (1) is prepared is mixed with treating crosslinked antibody protein, the antibody addition is as the criterion with excessive saturated antigen, abundant incubation, with reaction mixture centrifugal after, remove excessive not binding antibody, get precipitation and fully clean, obtain the Solid phase Immunoadsorbent that combines with antibody mediated immunity.The antibody molecule of its solid phase carrier absorption combines with the immobilized antigen immunity with the Fab section, and its Fc section is away from surface of solid phase carriers.
(3) solid phase is crosslinked
It is crosslinked that the Solid phase Immunoadsorbent that combines with antibody mediated immunity that step (2) is obtained is used for solid phase; Adopt the crosslinked flow process of liquid phase method, with the mobilizing function reagent Solid phase Immunoadsorbent that combines with antibody mediated immunity that obtained of the big molecular probe of activation processing and step (2) respectively, and then hybrid reaction, wherein, antibody is 1: 1～1: 5 with the mol ratio of big molecular probe, adds stop buffer and ends cross-linking reaction; Centrifugal back collecting precipitation fully washs; Obtain the cross-linking antibody that combines with Solid phase Immunoadsorbent.
(4) label that dissociates
Adopt chaotropic dissociation solution to dissociate, dissociating is to carry out in the damping fluid that dissociates, and interrupts the immunity combination of antigen, antibody, and cross-linking antibody is disintegrated down from Solid phase Immunoadsorbent; The cross-linking antibody that disintegrates down is replaced rapidly in the neutral buffered liquid, remove the dissociation solution composition, obtain cross-linking antibody.
The present invention disintegrates down label one cross-linking antibody from Solid phase Immunoadsorbent, with reference to the dissociation solution prescription of affine separation: during affine separation, the damping fluid that dissociates commonly used, the damping fluid that dissociates is the acid dissociation solution of pH2～3, alkaline dissociation solution or the dense salt dissociation solution of pH 10～11.
Described acid dissociation solution is glycocoll-HCl distilled water solution, and alkaline dissociation solution is glycocoll-NaOH distilled water solution, and dense salt dissociation solution is the KCSN distilled water solution; Wherein, the concentration of described glycocoll-HCl is 0.1～0.5mol/L; The concentration of described glycocoll-NaOH is 0.1～0.5mol/L; The concentration of described KCSN is 0.5～3mol/L.
((carry out in the dissociation solution of glycocoll-NaOH) by glycocoll-HCl) or pH10～11 at pH 2～3 for the dissociation of most of antigen-antibody complexes; In addition, add the wash-out that some short molten reagent (as KCSN etc.) also can influence binding molecule.Select suitable dissociation solution can effectively interrupt the immunity combination of antigen, antibody, cross-linking antibody is disintegrated down from Solid phase Immunoadsorbent.The cross-linking antibody that disintegrates down is replaced rapidly in the neutral buffered liquid, remove the dissociation solution composition, obtain target product---the cross-linking antibody of solid phase cross-linking method preparation.
In addition, also can gather in the crops Solid phase Immunoadsorbent, through fully washing, be suspended in the activation damping fluid, the Solid phase Immunoadsorbent that this step reclaims can be mixed with the freshly prepd Solid phase Immunoadsorbent of step (1), be used to treat that the solid phase of cross-linking antibody is crosslinked, can prepare the cross-linking agent of antibody and big molecular probe.
Described solid phase carrier comprises Ago-Gel microballon, microcarrier or polystyrene microbeads etc.
Described micromolecule reagent comprises monoethanolamine or glycocoll etc.
Condition during described the sealing is to adopt shaking table room temperature reaction 1～8h, or spends the night smaller or equal to 10 ℃ greater than 0 ℃.
Described incubation conditions is 15～37 ℃, reaction 2～4h, or greater than 0 ℃ smaller or equal to 10 ℃, reaction 8～16h.
Described mobilizing function reagent comprises homobifunctional agent, as glutaraldehyde or sodium metaperiodate etc.; Or heterobifunctional agent, as SPDP, SMCC or SMPB etc.
Described activation processing and hybrid reaction condition are room temperature oscillating reactions 1～8h, or greater than 0 ℃ smaller or equal to 10 ℃, reaction 4～16h.
Described stop buffer comprises N-acetyl maleimide or sodium iodoacetate etc.
Described neutral buffered liquid comprises biochemical damping fluids commonly used such as phosphate buffer, carbonate buffer solution or the Tris/HCl damping fluid about pH6～8.
Described big molecular probe is a phycobniliprotein.Described phycobniliprotein is phycoerythrocyanin (pec), allophycocyanin, phycocyanin, B-phycoerythrin or R-phycoerythrin.
The present invention proposes a kind of new method of new preparation cross-linking antibody, be used to prepare covalent conjunct agent---the cross-linking antibody of big molecular probe and antibody, can keep the antigen binding capacity of antibody largely.
Similar on the methodological principle of this solid phase synthesis cross-linking antibody to liquid phase synthesizing method; because the chemical bond of aglucon (antigen) and solid phase carrier; solid phase antigen has to a certain degree tolerance to conditions such as pH, ionic strengths; solid phase antigen had both been protected the antigenic determinant of antibody effectively, and the means of comparatively easy separation marking thing are provided again.
For example: by the R-phycoerythrin fluorescent-labeled antibody HBsAb (seeing embodiment 1) of solid phase cross-linking method preparation, be used for double antibody sandwich method and detect serum soluble antigen HBsAg, the mark rate homogeneity of probe molecule is better, 1.18 R-phycoerythrin of per molecule antibody covalent bond molecule; The antibody titer height, be used for the detection (80 parts of HBsAg negative serums, 44 parts of HBsAg weak positive serums, 376 parts of HBsAg positive serums) of 500 parts of blood serum samples, has higher recall rate (accuracy of testing result), negative recall rate 100%, none official holiday positive, positive rate 99.5% is 95.5% to p+ recall rate, and total recall rate reaches 99.6%; Be used for the detection of the HBsAg serum dish purchased at the visiting center, recall rate reaches 100%.We in experimental study with the crosslinked R-phycoerythrin fluorescent-labeled antibody HBsAb that also prepared of liquid phase, its mark rate is also about 1.2 after measured, the immune detection that is used for above-mentioned 500 parts of blood serum samples, negative recall rate 100%, none official holiday positive, total recall rate 96.6%, more undesirable to the detection effect of weak positive serum, the recall rate of weak positive serum only is 61.4%.
Below in conjunction with drawings and Examples technical scheme of the present invention is further described.
Description of drawings
Fig. 1. utilize the solid-phase immunity absorption method to prepare the synoptic diagram of cross-linking antibody.
As shown in the figure, the cross-linking antibody that the method by this solid phase synthesis obtains has complete antigen-binding site, the ability of big its conjugated antigen of molecular probe little effect of introducing.
Earlier antigen is connected on the solid phase carrier with covalent as aglucon, makes Solid phase Immunoadsorbent (solid phase antigen); By the antigenic determinant of antibody, specifically with antigen generation immune response, form antigen one antibody mediated immunity compound, immune complex can effectively dissociate under certain condition; Introduce big molecular probe and antibody covalent bond; Select certain wash conditions, dissociate antibody and the big covalently bound label of molecular probe---cross-linking antibody make cross-linking antibody disintegrate down from immobilization aglucon (Solid phase Immunoadsorbent).
The antigenic determinant of the antibody in the solid-phase immunity compound is with after solid phase antigen combines, and the antigen-binding site of antibody has obtained protection to a certain extent.Therefore during activation processing solid-phase immunity compound, the probability that activated group is introduced the antigenic determinant of antibody can reduce significantly, when big molecular probe (as phycobniliprotein) by activated group and when antibody linked, cross-linking antibody still keeps complete antigen-binding site.The probe phycobniliprotein forms cross-linking agent by activated group and antibody.
Embodiment
Embodiment 1:
(macro-molecular protein, molecular weight 240KD) is example with fluorescence probe R-phycoerythrin, the cross-linking antibody of preparation R-phycoerythrin and hepatitis B virus surface antigen (HBsAg) monoclonal antibody.
1. preparation Solid phase Immunoadsorbent
(HBsAg) is immobilized onto solid phase carrier with hepatitis B virus surface antigen, makes stable immunosorbent-solid phase antigen.Adopting the Ago-Gel 4B of cyanogen bromide-activated is solid phase carrier, and the solid phase filler of using always when it is the affinity purification monoclonal antibody when being used to prepare solid phase antigen, has been set up the operating process of standard.The preparation method who adopts is as follows:
1. the freeze-dried powder of weighing 0.3g, swelling is in 10
-3N hydrochloric acid 30min, and clean with the hydrochloric acid solution of 60mL, more rapidly with 0.1mol/L, pH8.3 NaHCO
3Solution washing once obtains 1mL activation gel;
2. add immediately and treat coupled antigen HBsAg (5mg), HBsAg is in advance with 0.1mol/L, pH8.3 NaHCO
3Solution equilibria slowly stirs 6h under the room temperature;
3. centrifugal, draw supernatant, in gel, add 2mL 1.0mol/L ethanolamine solutions, oscillating reactions 2h under the room temperature is closed unnecessary reactive group;
4. with 50mM Tris, 1M NaCl, pH 8.0 and 50mM Glycine, 1M NaCl, pH3.5 alternately clean gel 8 times, to remove non-specific combinating substance;
5. with 50mM, pH 7.5, and PBS cleans gel, and is centrifugal, the results Solid phase Immunoadsorbent, i.e. and coupling has the gel 1mL of HBsAg antigen.
2. immunoadsorption
For making antibody and Solid phase Immunoadsorbent form comparatively stable immune complex, select to help the acidity-basicity ph 7.5 of immune complex combination, the buffered environment of low ionic strength (50mM PBS), with excessive antibody fully in and antigen.Operation steps is as follows:
1. (pH 7.5 for 3.2mg/mL, 50mM, PBS) mix with the 1mL Solid phase Immunoadsorbent, slowly stir, and 4 ℃ are spent the night with 1.5mL antibody;
2. centrifugal, draw supernatant (reservation), measure the absorbance value A of 280nm;
3. when A is not 0, adds an amount of PBS (50mM, pH 7.5) and clean gel, repeating step 2;
4. when A is 0, merge all supernatants, concentrate, record free antibodies 22mg, the amount of the antibody of immunoadsorption is 2.6mg altogether, needs the sulfhydrylation derivant 6.9mg of the R-phycoerythrin of adding.
3. solid phase is crosslinked
The solid phase cross-linking method is undertaken by the liquid phase cross-linking method substantially.It is crosslinked that heterobifunctional agent SMCC has been widely used in the liquid phase of protein.The NHS ester that its end has amine to reactivate, the other end are the maleimide end, and stabilizer pole, hydrolysis are slow, allow a kind of albumen its NHS ester end to be modified by amino, form stable amido link, produce terminal dimaleoyl imino, can be special crosslinked with the albumen that contains sulfydryl.Therefore contain amino carrier (Solid phase Immunoadsorbent that combines with antibody mediated immunity) and also can modify the terminal maleimide amino of generation, supply with aglucon (the big molecular probe of the sulfydryl activation) coupling that contains sulfydryl and use with SMCC.
Concrete steps are as follows:
1. preparation activates damping fluid (promote the pH of buffer 7.2 of NHS coupling, 0.1mol/L PBS contains 0.15mol/L NaCl), and 4 ℃ of precoolings are washed prepared gel with ice-cold activation damping fluid, and adjust gel strength 60% (v/v), and vibration mixes;
2. weighing SMCC 5mg is dissolved in the anhydrous DMSO of 20 μ L, is added dropwise in the gel suspension while vibrating;
3. continue hybrid reaction 4h at 4 ℃;
4. rapidly with cold activation damping fluid flushing gel,,, and the gel equal-volume is resuspended in the activation damping fluid with the cross-linking reagent of removing not coupling and the accessory substance of reaction again with after the 1mol/L NaCl flushing;
5. the R-phycoerythrin (protein concentration 5.5mg/ml) that adds 2-IT sulfydryl derivatization immediately, 4 ℃ are mixed oscillating reactions 4h;
6. gel is filled into post, washes with activation damping fluid stream, detect that no albumen flows out to the post, reach balance, get a little gel and observe in microscopically this moment, as seen the fluorescence microballon that becomes clear.
4. label dissociates
Correct selection elution requirement is related to the success of label and dissociates, the suitable elution buffer material of combination that not only will dissociate with the volume of minimum, and also the labelled antibody that dissociates still should be able to keep the ability that combines once again with antigen.With reference to the successful experience of dissociating antibody in the affinity chromatography separation, screened suitable dissociation solution prescription: the object of screening comprises 0.5mol/L～3mol/L KSCN, pH2.8,0.1～0.2mol/L glycocoll-HCl damping fluid and pH 10.6,0.1mol/L glycocoll-NaOH damping fluid etc.Therefrom obtain 0.2mol/L glycocoll-HCl damping fluid, pH 2.8, and it is best to contain 1mol/L NaCl dissociation effect.
Gel is mixed by 1: 1 volume ratio with above-mentioned dissociation solution, and vibration is centrifugal gently, collects supernatant, rapidly the cross-linking antibody that dissociates is crossed desalting column displacement damping fluid to 50mM, and pH 7.5, PBS.Obtain R-phycoerythrin fluorescence labeling HBsAg monoclonal antibody, can be used for the immune detection of serum HBsAg.
Meanwhile, the gel after dissociating is suspended in the activation damping fluid through fully washing.Can mix with the freshly prepd HBsAg of having coupling gel, the solid phase that is reused for antibody is crosslinked.
Embodiment 2:
Solid phase preparation with the cross-linking antibody of R-phycoerythrin and prostate specific antigen monoclonal antibody is an example, and the operating process of this invention is described below once more:
1. preparation Solid phase Immunoadsorbent
Adopting the Ago-Gel 4B of cyanogen bromide-activated is solid phase carrier, and (PSA) is immobilized onto solid phase carrier with prostate specific antigen, makes stable Solid phase Immunoadsorbent.The preparation method is as follows:
1. the freeze-dried powder of weighing 0.3g, swelling is in 10
-3N hydrochloric acid 30min, and clean with the hydrochloric acid solution of 60mL, more rapidly with 0.1mol/L, pH 8.3 NaHCO
3Solution washing once obtains 1mL activation gel;
2. add immediately and treat coupled antigen PSA (2mg), antigen is in advance with 0.1mol/L, pH 8.3NaHCO
3Solution equilibria slowly stirs 8h under the room temperature;
3. centrifugal, draw supernatant, in gel, add 2mL 1.0mol/L ethanolamine solutions, oscillating reactions 8h under the room temperature is closed unnecessary reactive group;
4. with 50mM Tris, 1M NaCl, pH 8.0 and 50mM Glycine, 1M NaCl, pH3.5 alternately clean gel 8 times, to remove non-specific combinating substance;
5. with 50mM, pH 7.5, and PBS cleans gel, and is centrifugal, the results Solid phase Immunoadsorbent, i.e. and coupling has the gel of PSA.
2. immunoadsorption
With excessive PSA monoclonal antibody fully in and the rapid prepared Solid phase Immunoadsorbent of previous step.Operation steps is as follows:
1. (pH 7.5 for 3.2mg/mL, 50mM, PBS) mix slowly stirring, 37 ℃, incubation 2h with the 1mLPSA Solid phase Immunoadsorbent with 1.5mL PSA antibody;
2. centrifugal, draw supernatant (reservation), thoroughly clean gel, 280nm does not have absorption to supernatant.
3. solid phase is crosslinked
1. preparation activates damping fluid (pH 7.2, and 0.1mol/L PBS contains 0.15mol/L NaCl), and 4 ℃ of precoolings are washed prepared gel with ice-cold activation damping fluid, and adjusts gel strength 60% (v/v), and vibration mixes;
2. weighing SMCC 5mg is dissolved in the anhydrous DMSO of 20 μ L, is added dropwise in the gel suspension while vibrating;
3. continue hybrid reaction 4h at 4 ℃;
4. rapidly with cold activation damping fluid flushing gel,,, and the gel equal-volume is resuspended in the activation damping fluid with the cross-linking reagent of removing not coupling and the accessory substance of reaction again with after the 1mol/LNaCl flushing;
5. R-phycoerythrin (protein concentration 2.5mg/mL) 1mL that adds 2-IT sulfydryl derivatization immediately, 10 ℃ are mixed oscillating reactions 16h;
6. thoroughly clean with the activation damping fluid, detect supernatant and do not have albumen.
4. label dissociates
Choosing dissociation solution is 0.1mol/L glycocoll-HCl damping fluid, and pH 2.8, contain 1mol/LNaCl.
The 3rd gel that obtained of step is mixed by 1: 1 volume ratio with above-mentioned dissociation solution, vibration gently, centrifugal after, collect supernatant, and the cross-linking antibody that dissociates is crossed desalting column replace damping fluid rapidly to 50mM, pH 7.5, PBS.Gel after fully washing is dissociated simultaneously, and be suspended in the activation damping fluid.
The prepared R-phycoerythrin and the cross-linking agent of PSA monoclonal antibody are used to detect the PSA purifying antigen of 5ng/mL, detect the result and are positive.
Embodiment 3:
(diameter 60～250 μ m) are solid phase carrier with polystyrene microbeads, the cross-linking antibody of solid phase preparation R-phycoerythrin and HBsAg monoclonal antibody:
1. preparation Solid phase Immunoadsorbent
1. the polystyrene microbeads of weighing 0.5g, room temperature, in fresh glutaraldehyde solution (percent by volume is 25%), soak 4h after, rapidly with 0.1mol/L, pH 8.3 NaHCO
3Solution washing once;
2. add immediately and treat coupled antigen HBsAg (5mg), antigen is in advance with 0.1mol/L, pH8.3 NaHCO
3Solution equilibria, 1 ℃ is slowly stirred 10h down;
3. centrifugal, draw supernatant, in microballon, add 2mL 0.1mol/L glycine solution, oscillating reactions 1h under the room temperature is closed unnecessary reactive group;
4. with 50mM Tris, 1M NaCl, pH 8.0 and 50mM Glycine, 1M NaCl, pH3.5 alternately clean gel 8 times, to remove non-specific combinating substance;
5. with 50mM, pH 7.5, and PBS cleans microballon, centrifugal after, results Solid phase Immunoadsorbent, i.e. coupling has the polystyrene microbeads of HBsAg.
2. immunoadsorption
With excessive HBsAg monoclonal antibody fully in and the rapid prepared Solid phase Immunoadsorbent of previous step.Operation steps is as follows:
1. (pH 7.5 for 3.2mg/mL, 50mM, PBS) mix with the Solid phase Immunoadsorbent of above-mentioned preparation, slowly stir, and 1 ℃ is spent the night with 1.5mL PSA antibody;
2. centrifugal, draw supernatant (reservation), thoroughly clean polystyrene microbeads, 280nm does not have absorption to supernatant.
3. solid phase is crosslinked
1. preparation activates damping fluid (pH 7.2, and 0.1mol/L PBS contains 0.15mol/L NaCl), and 4 ℃ of precoolings are with ice-cold activation damping fluid washing polystyrene microbeads;
2. weighing SMCC 5mg is dissolved in the anhydrous DMSO of 20 μ L, is added dropwise to while vibrating in the microballon suspending liquid;
3. continue hybrid reaction 4h at 4 ℃;
4. rapidly with cold activation damping fluid flushing microballon,,, and microballon is resuspended in the activation damping fluid with the cross-linking reagent of removing not coupling and the accessory substance of reaction again with after the 1mol/LNaCl flushing;
5. R-phycoerythrin (protein concentration 2.5mg/mL) 1mL that adds 2-IT sulfydryl derivatization immediately, 4 ℃ are mixed oscillating reactions 4h;
6. thoroughly clean with the activation damping fluid, detect supernatant and do not have albumen.
4. label dissociates
Choosing dissociation solution is 0.1mol/L glycocoll-HCl damping fluid, and pH 2.8, contain 1mol/LNaCl.
The 3rd microballon that obtained of step is mixed by 1: 1 volume ratio with above-mentioned dissociation solution, vibration gently, centrifugal after, collect supernatant, and the cross-linking antibody that dissociates is crossed desalting column replace damping fluid rapidly to 50mM, pH 7.5, PBS.Microballon after fully washing is dissociated simultaneously, and be suspended in the activation damping fluid.
The prepared R-phycoerythrin and the cross-linking agent of HBsAg monoclonal antibody are used to detect positive and negative serum, and it is correct to detect the result.
Embodiment 4:
(PEC, molecular weight 100KD) is fluorescence probe with phycoerythrocyanin (pec), with the crosslinked preparation fluorescent-labeled antibody of HBsAg monoclonal antibody solid phase.
1. preparation Solid phase Immunoadsorbent
1. the freeze-dried powder of weighing 0.3g, swelling be in 10-3N hydrochloric acid 30min, and clean with the hydrochloric acid solution of 60mL, more rapidly with 0.1mol/L, and pH8.3 NaHCO
3Solution washing once obtains 1mL activation gel;
2. add immediately and treat coupled antigen HBsAg (5mg), HBsAg is in advance with 0.1mol/L, pH8.3 NaHCO
3Solution equilibria slowly stirs 6h under the room temperature;
3. centrifugal, draw supernatant, in gel, add 2mL 1.0mol/L ethanolamine solutions, oscillating reactions 2h under the room temperature is closed unnecessary reactive group;
4. with 50mM Tris, 1M NaCl, pH 8.0 and 50mM Glycine, 1M NaCl, pH3.5 alternately clean gel 8 times, to remove non-specific combinating substance;
5. with 50mM, pH 7.5, and PBS cleans gel, and is centrifugal, the results Solid phase Immunoadsorbent, i.e. and coupling has the gel 1mL of HBsAg antigen.
2. immunoadsorption
1. (pH 7.5 for 5mg/mL, 50mM, PBS) mix slowly stirring, 15 ℃, incubation 4h with the 1mL Solid phase Immunoadsorbent with 1mL HBsAg antibody;
2. centrifugal, draw supernatant (reservation), measure the absorbance value A of 280nm;
3. when A is not 0, adds an amount of PBS (50mM, pH 7.5) and clean gel, repeating step 2;
4. when A is 0, merge all supernatants, concentrate, record free antibodies 1.3mg, the amount of the antibody of immunoadsorption is 3.7mg altogether, needs the sulfhydrylation derivant 4.6mg of the PEC of adding.
3. solid phase is crosslinked
1. preparation activates damping fluid (promote the pH of buffer 7.2 of NHS coupling, 0.1mol/L PBS contains 0.15mol/L NaCl), and 4 ℃ of precoolings are washed prepared gel with ice-cold activation damping fluid, and adjust gel strength 60% (v/v), and vibration mixes;
2. weighing SMCC 5mg is dissolved in the anhydrous DMSO of 20 μ L, is added dropwise in the gel suspension while vibrating;
3. continue hybrid reaction 4h at 4 ℃;
4. rapidly with cold activation damping fluid flushing gel,,, and the gel equal-volume is resuspended in the activation damping fluid with the cross-linking reagent of removing not coupling and the accessory substance of reaction again with after the 1mol/L NaCl flushing;
5. add immediately 2-IT sulfydryl derivatization PEC (2.5mg/mL, 1.85mL), 4 ℃ are mixed oscillating reactions 4h; Thoroughly wash with the activation damping fluid, centrifugal, get precipitation and be used to dissociate.
4. label dissociates
With above-mentioned precipitation and 0.2mol/L glycocoll-HCl damping fluid, pH 2.8, contain 1mol/LNaCl and mix by 1: 1 volume ratio, and vibration is centrifugal gently, collect supernatant, rapidly the cross-linking antibody that dissociates crossed desalting column displacement damping fluid to 50mM, and pH 7.5, PBS.Obtain phycoerythrocyanin (pec) mark HBsAg monoclonal antibody.
Meanwhile, the gel after dissociating is suspended in the activation damping fluid through fully washing.Can mix with the freshly prepd HBsAg of having coupling gel, the solid phase that is reused for antibody is crosslinked.
The cross-linking agent of prepared PEC and HBsAg monoclonal antibody is used to detect positive and negative serum, and it is correct to detect the result.
Claims (16)
1. a method of utilizing the solid-phase immunity absorption method to prepare cross-linking antibody at first with the solid phase carrier of antigen molecule coupling activation, prepares Solid phase Immunoadsorbent; Solid phase Immunoadsorbent is fully mixed with antibody purified, antibody mediated immunity is incorporated on the Solid phase Immunoadsorbent, antibody combines with the immobilized antigen immunity and forms comparatively stable immune complex; With the antibody that mobilizing function reagent is distinguished the big molecular probe of activation processing and combined with the Solid phase Immunoadsorbent immunity, hybrid reaction forms cross-linking agent then; The antibody that mark is good by dissociate on the Solid phase Immunoadsorbent, desalination, obtain the covalently bound cross-linking antibody of big molecular probe and antibody.
2. the method for claim 1, the concrete steps of described method are:
(1) preparation Solid phase Immunoadsorbent
At first choose solid phase carrier, the activation by the carrier surface group is immobilized onto surface of solid phase carriers with antigen molecule, make stable Solid phase Immunoadsorbent, remaining avtive spot through fully cleaning, obtains Solid phase Immunoadsorbent with the sealing of micromolecule closed reagent;
(2) immunoadsorption
At pH is to help under the environment of the neutrality of immune complex combination or meta-alkalescence and low ionic strength, the Solid phase Immunoadsorbent that step (1) is prepared is mixed with treating crosslinked antibody protein, the antibody addition is as the criterion with excessive saturated antigen, abundant incubation, with reaction mixture centrifugal after, remove excessive not binding antibody, get precipitation and fully clean, obtain the Solid phase Immunoadsorbent that combines with antibody mediated immunity;
(3) solid phase is crosslinked
It is crosslinked that the Solid phase Immunoadsorbent that combines with antibody mediated immunity that step (2) is obtained is used for solid phase; Adopt the crosslinked flow process of liquid phase method, with the mobilizing function reagent Solid phase Immunoadsorbent that combines with antibody mediated immunity that obtained of the big molecular probe of activation processing and step (2) respectively, wherein, antibody is 1: 1～1: 5 with the mol ratio of big molecular probe, and then hybrid reaction, add stop buffer and end cross-linking reaction; Centrifugal back collecting precipitation fully washs; Obtain the cross-linking antibody that combines with Solid phase Immunoadsorbent;
(4) label that dissociates
Adopt chaotropic dissociation solution to dissociate, dissociating is to carry out in the damping fluid that dissociates, and interrupts the immunity combination of antigen, antibody, and cross-linking antibody is disintegrated down from Solid phase Immunoadsorbent; The cross-linking antibody that disintegrates down is replaced rapidly in the neutral buffered liquid, remove the dissociation solution composition, obtain cross-linking antibody.
3. method as claimed in claim 1 or 2 is characterized in that: described solid phase carrier is Ago-Gel microballon, microcarrier or polystyrene microbeads.
4. method as claimed in claim 2 is characterized in that: described micromolecule reagent is monoethanolamine or glycocoll.
5. method as claimed in claim 2 is characterized in that: the condition during described the sealing is to adopt shaking table room temperature reaction 1～8h, or spends the night smaller or equal to 10 ℃ greater than 0 ℃.
6. method as claimed in claim 2 is characterized in that: described incubation conditions is 15～37 ℃, reaction 2～4h; Or greater than 0 ℃ smaller or equal to 10 ℃, reaction 8～16h.
7. method as claimed in claim 2 is characterized in that: described mobilizing function reagent comprises homobifunctional agent or heterobifunctional agent.
8. method as claimed in claim 7 is characterized in that: described bifunctional reagent is glutaraldehyde or sodium metaperiodate; Described heterobifunctional agent is SPDP, SMCC or SMPB.
9. method as claimed in claim 2 is characterized in that: described activation processing and hybrid reaction condition are room temperature oscillating reactions 1～8h; Or greater than 0 ℃ smaller or equal to 10 ℃, reaction 4～16h.
10. method as claimed in claim 2 is characterized in that: described stop buffer is N-acetyl maleimide or sodium iodoacetate.
11. method as claimed in claim 2 is characterized in that: the phosphate buffer that described neutral buffered liquid is pH6～8, carbonate buffer solution or Tris/HCl damping fluid.
12. method as claimed in claim 2 is characterized in that: the acid dissociation solution that the described damping fluid that dissociates is pH2～3, the alkaline dissociation solution of pH10～11 or dense salt dissociation solution.
13. method as claimed in claim 12 is characterized in that: described acid dissociation solution is glycocoll-HCl distilled water solution, and alkaline dissociation solution is glycocoll-NaOH distilled water solution, and dense salt dissociation solution is the KCSN distilled water solution; Wherein, the concentration of described glycocoll-HCl is 0.1～0.5mol/L; The concentration of described glycocoll-NaOH is 0.1～0.5mol/L; The concentration of described KCSN is 0.5～3mol/L.
14. method as claimed in claim 1 or 2 is characterized in that: described big molecular probe is a phycobniliprotein.
15. method as claimed in claim 14 is characterized in that: described phycobniliprotein is phycoerythrocyanin (pec), different algae prison albumen, phycocyanin, B-phycoerythrin or R-phycoerythrin.
16. method as claimed in claim 2, it is characterized in that: the Solid phase Immunoadsorbent after described the dissociating, through fully washing, be suspended in the activation damping fluid, mix with the freshly prepd Solid phase Immunoadsorbent of step (1), it is crosslinked to be reused for the solid phase for the treatment of cross-linking antibody, the cross-linking antibody of preparation antibody and big molecular probe.
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| CN101936987B (en) * | 2010-09-16 | 2013-07-24 | 厦门大学 | Protein A immobilized antibody-based micro-sphere immunoassay method |
| CN101980019B (en) * | 2010-09-17 | 2013-12-25 | 山东轻工业学院 | Method for preparing allophycocyanin-marked fluorescent antinuclear antibody |
| CN101980020B (en) * | 2010-09-17 | 2013-12-25 | 山东轻工业学院 | Preparation method of R-phycocyanin (RPC)-marked fluorescent anti-antibody |
| CN101957377B (en) * | 2010-09-17 | 2013-11-13 | 山东省农业科学院畜牧兽医研究所 | Method for preparing fluorescent antibody for detecting avian influenza virus and solid phase immunofluorescence detection kit |
| CN102062780A (en) * | 2010-11-23 | 2011-05-18 | 北京正旦国际科技有限责任公司 | Polypeptide immunoassay kit and detection method thereof |
| CN103776996A (en) * | 2014-01-23 | 2014-05-07 | 中国人民解放军第一一七医院 | CIC (circulating immune complex) antibody buffer solution dissociation agent for immune complex buffer dissociation agent |
| CN111701576B (en) * | 2020-05-26 | 2023-07-28 | 武汉瑞法医疗器械有限公司 | Preparation method of West Nile virus immunoadsorbent, immunoadsorbent and application thereof |
| CN113042016A (en) * | 2021-04-25 | 2021-06-29 | 上海捷门生物技术有限公司 | Solid phase affinity adsorbent for purifying antiserum and method for obtaining purified antibody |
| CN115350507B (en) * | 2022-05-25 | 2025-02-14 | 河北省食品检验研究院（国家果类及农副加工产品质量监督检验中心、河北省食品安全实验室） | Immunomyosin immunoaffinity column, preparation method and application thereof |
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