Preparation method of polymerase-antibody complex with bead structureTechnical Field
The invention belongs to the technical field of medical detection, and particularly relates to a preparation method of a polymerase-antibody complex with a bead structure.
Background
Histochemistry (immunochemistry) is a technology for locating, qualitatively and relatively quantitatively detecting specific antigens in tissue cells by using the specific binding principle of antigens and antibodies and a special labeling technology, and making secondary antibodies labeled with fluorescein, enzyme, metal ions and isotopes develop color under the action of a display agent through chemical reaction. Immunohistochemical (IHC) staining has extremely important significance for judging tumors and guiding medicines, the analysis of the immunohistochemistry of the tumors in the hospital pathology department is extremely important for parting tumor molecules, and accurate tumor parting detection is beneficial to guiding patients to accurately take medicines, improves treatment effect and reduces toxic and side effects.
The development of the immunohistochemical secondary antibody technology is carried out by a direct method, an indirect method, an enzyme-labeled streptavidin-biotin staining method and an enzyme-labeled polymer staining method. Among them, the direct method and indirect method have low sensitivity due to weak signal amplification efficiency, and are rarely applied at present; in the enzyme-labeled streptavidin-biotin staining method, the ABC complex can be combined with biotin commonly existing in cell tissues, so that severe nonspecific staining is generated, interpretation of results is greatly interfered, and therefore, the method is phased out in clinical immunoassay at present; enzyme-labeled multimeric staining is the mainstream technology in clinical application at present, and the method is to label enzymes on a main chain taking inert glucan, polylysine, polypeptide, dendrimer and the like as a framework to form multimeric enzymes, and then connect antibodies to the multimeric enzymes to obtain multimeric enzyme-antibody complexes. Each main chain can be connected with 4-70 enzyme molecules and 1-10 antibody molecules, so that the method has extremely strong signal amplification effect, and simultaneously, the non-biotin detection system is used, so that non-specific staining caused by endogenous biotin is avoided, and the method is widely applied clinically. However, this method is not perfect, and its main technical drawbacks are: the method adopts a glucan polymer skeleton with the molecular weight of more than or equal to 50 ten thousand, and is connected with 4-70 enzyme molecules and 1-10 antibody molecules, so that the structure is loose, the hydrodynamic volume is huge, and the great steric hindrance is caused, so that the penetration of the polymer to a cell nuclear membrane is low. Thus, the polymer exhibits poor performance in the detection of various different types of antigens, especially in the detection of low-expression antigens in some nuclei.
In addition, since most secondary antibodies are prepared by serially connecting a large amount of horseradish peroxidase and antibodies by adopting an amino-bearing skeleton, the molecular weight of the obtained secondary antibodies is basically more than 1000kd, and the large secondary antibody molecules have strong charges and strong hydrophobicity and are easy to generate nonspecific binding with tissue sections through static electricity and hydrophobic interaction, so that nonspecific background staining is caused, and the secondary antibodies are a great difficulty which is most difficult to solve in the development of the secondary antibodies.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a preparation method of a polymerase-antibody complex with a bead structure, which does not adopt a skeleton to prepare the polymerase, adopts small-molecule diammine polyethylene glycol as a cross-linking agent to synthesize the polymerase with the bead structure, so that the polymerase molecule is as compact as possible, the space occupation is smaller, and the prepared polymerase-antibody complex has better tissue penetration capability and higher detection sensitivity to smaller tissue cell structures when being used for tissue section staining; meanwhile, as a framework is not adopted and a small-molecule hydrophilic cross-linking agent is adopted, the chargeability of the secondary antibody is reduced, the hydrophilicity of the secondary antibody is improved, the electrostatic and hydrophobic combination between the secondary antibody and a tissue slice is greatly weakened, and the background dyeing caused by the nonspecific effect between the secondary antibody and the tissue slice is effectively reduced.
The invention adopts the technical scheme that:
a method for preparing a polymerase-antibody complex of a bead structure, comprising the steps of:
step one: horseradish peroxidase was aldehyde-formed using sodium periodate:
a. weighing a certain amount of horseradish peroxidase, and dissolving the horseradish peroxidase in the buffer solution A to obtain a horseradish peroxidase solution with a certain concentration;
b. weighing a certain amount of sodium periodate to be dissolved in purified water;
c. adding a sodium periodate aqueous solution into a horseradish peroxidase solution, and reacting for 5-60 minutes at 15-40 ℃;
d. finishing the reaction, and passing through a desalting column to obtain aldehyde activated horseradish peroxidase;
step two: synthesis of bead structured multimeric horseradish peroxidase using a diamine cross-linking reagent:
e. preparing aldehyde-activated horseradish peroxidase into a certain concentration;
f. adding a diamine crosslinking reagent, and adjusting the pH of a reaction system to be 7.5-10.0,20-30 ℃ for reaction for 1-16 hours;
g. adding a reducing agent, and reacting for 1-4 hours;
h. adding a blocking reagent, and reacting for 2-6 hours;
i. finishing the reaction, and passing through a desalting column to obtain the poly horseradish peroxidase with a bead structure;
step three: introducing a hydrophilic arm chain on the horse radish peroxidase with a bead structure:
j. preparing the polymeric horseradish peroxidase with a bead structure into a concentration of 1-20 mg/mL;
k. dissolving a hydrophilic arm chain reagent in a proper buffer solution to prepare 5-20 mg/mL;
mixing hydrophilic arm chain reagent and horse radish peroxidase in the molar ratio of 10 to 1-80 to 1, and reacting at room temperature for 0.5-4 hr;
finishing the reaction, and passing through a desalting column to obtain the poly-horseradish peroxidase containing the hydrophilic arm chain;
step four: coupling the antibody to the polyhorse radish peroxidase with a hydrophilic arm chain to finally obtain the polyenzyme-antibody complex with a bead structure:
dissolving the antibody in a proper solution to prepare 1-10 mg/mL;
dissolving sulfhydrylation reagent in proper solution to prepare 0.5-10 mg/mL;
p, mixing the sulfhydrylation reagent and the antibody together according to the mol ratio of 1:1-20:1, and reacting for 0.5-3 hours at room temperature;
finishing the reaction, and passing through a desalting column to obtain the sulfhydryl modified antibody;
and r, the mole ratio of the poly horseradish peroxidase to the sulfhydryl modified antibody is 8-20: 1, mixing the two materials together, and reacting for 12-72 hours at the temperature of 2-8 ℃;
and s, passing through a molecular sieve purification column, and collecting target peaks to obtain the polymerase-antibody complex with the bead structure.
In the first step, the buffer solution A is a citric acid buffer solution, the pH value is 4.0-7.0, and the preferable pH value is 5.0;
in the first step, the concentration of the sodium periodate aqueous solution is 5-50 mg/mL, preferably 20-30 mg/mL, and the concentration of the horseradish peroxidase solution is 1-50 mg/mL, preferably 20-40 mg/mL.
In the first step, the molar ratio of the sodium periodate to the horseradish peroxidase is 10-400, and the preferable molar ratio is 40-100; the reaction temperature in the first step is 15-40 ℃ and the reaction time is 5-60 minutes;
the desalting column in the first step is a PD-10 desalting column.
In the second step, the chemical structure of the diamine cross-linking reagent is shown in the following formula 1,
the diamine cross-linking agent of formula 1 has a chemical structure wherein n represents the degree of polymerization of polyethylene glycol (PEG) and ranges from 2 to 100, preferably from 10 to 50.
In the second step, the concentration of aldehyde-activated horseradish peroxidase is 1-40 mg/mL, preferably 10-30 mg/mL. In the step f, the pH of the reaction system is 8-10.5, preferably 9-10, the reaction temperature is 15-40 ℃, preferably 25-35 ℃ and the reaction time is 4 hours
In the second step, the reducing agent is sodium borohydride.
The blocking reagent in the second step comprises any one of ethylenediamine, 1, 3-propylenediamine, 3' -diaminodipropylamine, lysine and diammine polyethylene glycol with the polymerization degree of 2-5 shown in the formula 1.
In the second step, the serial number of the horseradish peroxidase in the polymer horseradish peroxidase with the bead structure is 6-32, preferably 10-20.
In the third step, the chemical structure of the hydrophilic arm chain reagent is shown in the following formula 2,
the chemical structural formula of the hydrophilic arm chain reagent of formula 2, wherein n represents the polymerization degree of polyethylene glycol (PEG), is in the range of 6 to 50, preferably in the range of 10 to 30.
The concentration of the polymeric horseradish peroxidase with the bead structure in the third step is 1-40 mg/mL, preferably 10-20 mg/mL; the concentration of the hydrophilic arm chain reagent is 10-50 mg/mL, preferably 20-40 mg/mL, the buffer solution is 10mM PBS buffer solution, and the pH is 5.0-8.0, preferably 7.0-8.0
The molar ratio of the horseradish peroxidase polymer to the hydrophilic arm chain reagent is 1:10-1:60, preferably 1:15-1:30.
The reaction time is 1.5 hours, and the reaction temperature is 15-25 ℃.
In the fourth step, the concentration of the antibody is 1-10 mg/mL, the concentration of the solution for dissolving the antibody is 10mM PBS,10mM EDTA,pH8.0, the concentration of the sulfhydrylation reagent is 1mg/mL, the proper solution is 10mM PBS,10mM EDTA,pH8.0, and the molar ratio of the horseradish peroxidase to the antibody in the obtained polymerase-antibody complex with the bead structure is 6:1-20:1, preferably 6:1-12:1.
In step four, the antibodies referred to include monoclonal antibodies, polyclonal antibodies, fab fragments, fab'2 fragments, wherein monoclonal antibodies include, but are not limited to, (CK PAN, CK19, MUC1 CK5/6, p63, calponin CK19, TPO, CD56 MUC-1TPO MC CK34. Beta.E12 Galectin-3CT CEA SYN CgA TTF-1Napsin A Ki-67 PMS2 P16 CD34 S100 DESMIN P53); polyclonal antibodies include, but are not limited to, goat anti-rabbit and goat anti-mouse; fab fragments and Fab '2 fragments include, but are not limited to, fab fragments and Fab'2 fragments of the monoclonal and polyclonal antibodies listed above.
The serial number of the polymerase-antibody complex with the bead structure prepared by the method is 6-32, the structural characteristics are shown in the following formula 3, and the HRP is firstly connected in series to form the multimeric HRP enzyme by taking small-molecule diammine polyethylene glycol as a cross-linking agent; then connecting the antibody or antibody fragment to the polymeric HRP through a hydrophilic cross-linking agent with a long arm chain, so that the polymeric enzyme molecule is as compact as possible, the space occupation is smaller, and the prepared polymeric enzyme-antibody compound has better tissue penetration capability when being used for tissue section dyeing and higher detection sensitivity to smaller tissue cell structures; meanwhile, as a framework is not adopted and a small-molecule hydrophilic cross-linking agent is adopted, the chargeability of the secondary antibody is reduced, the hydrophilicity of the secondary antibody is improved, the electrostatic and hydrophobic combination between the secondary antibody and a tissue slice is greatly weakened, and the background dyeing caused by the nonspecific effect between the secondary antibody and the tissue slice is effectively reduced.
The polyase-antibody complex with the bead structure has very wide application in the aspects of intraoperative frozen section immunohistochemistry, conventional paraffin section immunohistochemistry, chemiluminescence in-vitro diagnosis and the like.
The structure of the polymerase-antibody complex with the bead structure is shown in the formula 3, wherein circles represent horseradish peroxidase and Y represents an antibody.
The polyase-antibody complex with the bead structure has wide application in chemiluminescent in-vitro diagnostic reagents, elisa detection reagents, conventional paraffin section immunohistochemical detection reagents and intraoperative frozen section immunohistochemical detection reagents, and can greatly improve the detection sensitivity.
The beneficial effects are that:
compared with the related art, the preparation and application of the polymerase-antibody complex with the bead structure provided by the invention have the following advantages:
(1) The macromolecular glucans, dendritic polymers and other skeletons are not adopted, and small-molecule diammine polyethylene glycol is adopted as a cross-linking agent to synthesize the polymerase with a bead structure, so that the polymerase molecules are as compact as possible, and the space occupation is smaller;
(2) Meanwhile, hydrophilic diammine polyethylene glycol is used as a cross-linking agent, so that the cross-linking effect is achieved, the hydrophilic modification effect on the polymerase is achieved, the prepared polymerase is better in water solubility, and the aggregation of the polymerase and the dyeing background caused by the hydrophobic effect during tissue slice dyeing are reduced;
(3) A long hydrophilic arm chain is introduced into the polymerase, so that the antibody connected to the polymerase has higher flexibility, and is favorable for free combination of the antibody and antigen; meanwhile, the hydrophilic arm chain is introduced to greatly improve the hydrophilicity of the polymerase-antibody complex, and reduce the aggregation of the polymerase-antibody complex and the dyeing background caused by the hydrophobic effect during tissue section dyeing;
the 3 points are combined, so that the polymerase-antibody complex has better tissue penetration capability when being used for tissue section staining, has higher detection sensitivity on smaller tissue cell structures, and can effectively reduce the staining background.
Drawings
FIG. 1AR shows the secondary antibody prepared in example 5 to the left of the imported secondary antibody and to the right of the imported secondary antibody, with the advantage that the nuclear staining intensity is superior to the imported secondary antibody;
FIG. 2BerEP4 lung adenocarcinoma is followed by the imported secondary antibody and the secondary antibody prepared by the method of example 5, with the advantage that the cytoplasmic color intensity is superior to the imported secondary antibody;
FIG. 3 shows the right side of the CD10 lymph node with the imported secondary antibody and the right side with the secondary antibody prepared by the method of example 6, wherein the cell membrane staining intensity is superior to that of the imported secondary antibody;
FIG. 4KI67 shows the secondary antibody on the left of the tonsil and the secondary antibody on the right of the secondary antibody prepared by the method of example 6, with the advantage that the nuclear staining intensity is superior to that of the secondary antibody;
FIG. 5CK5/6 shows the secondary antibody of the intra-operative frozen section prepared by the method of example 2 on the left and the commercial quick freezing reagent on the right, and the staining intensity is superior to that of the secondary antibody of the commercial intra-operative frozen section.
Detailed Description
EXAMPLE 1 preparation of multimeric horseradish peroxidase-CK 5/6 antibody Complex with 16-24 peroxidase tandem
a. 300mg of horseradish peroxidase is weighed and dissolved in 100mL of citric acid buffer solution with pH of 6.0, so as to obtain 10mg/mL of horseradish peroxidase solution;
b. 80mg of sodium periodate is weighed and dissolved in purified water to obtain a sodium periodate aqueous solution with the concentration of 20mg/mL;
c. adding a sodium periodate aqueous solution into a horseradish peroxidase solution, and reacting for 15 minutes at 37 ℃;
d. finishing the reaction, and passing through a PD-10 desalting column to obtain aldehyde activated horseradish peroxidase;
e. preparing aldehyde-activated horseradish peroxidase into 20mg/mL;
f. adding 10mg of diamino polyethylene glycol with the polymerization degree of 24, adjusting the pH value of a reaction system to 10.0, and reacting for 4 hours at room temperature;
g. 5mg of sodium borohydride is added and the mixture is reacted for 2 hours at room temperature;
adding 0.5g of blocking reagent ethylenediamine, and reacting for 2 hours at room temperature;
i. finishing the reaction, and passing through a desalting column to obtain the poly horseradish peroxidase with a bead structure;
j. preparing the polymeric horseradish peroxidase with a bead structure into 15mg/mL;
k. dissolving a hydrophilic arm chain reagent with the polymerization degree of 8 in 10mM PBS buffer solution, and preparing 20mg/mL;
mixing the two materials together, and reacting for 1.5 hours at room temperature;
finishing the reaction, and passing through a PD-10 desalting column to obtain the horseradish peroxidase polymer containing hydrophilic arm chains; dissolving cytokeratin CK5/6 antibody in 10mM PBS,10mM EDTA,pH8.0 to prepare 6mg/mL; dissolving a sulfhydrylation reagent in 10mM PBS,10mM EDTA,pH8.0 solution to prepare 1mg/mL; p, mixing the two materials together, and reacting for 1 hour at room temperature;
finishing the reaction, and passing through a desalting column to obtain the sulfhydryl modified antibody;
mixing the thiol-modified cytokeratin CK5/6 antibody with the horseradish peroxidase with a hydrophilic arm chain according to the mol ratio of 1:30, and reacting for 24 hours at the temperature of 2-8 ℃;
s, passing through a molecular sieve purification column, and collecting a first out target peak to obtain the poly horseradish peroxidase-CK 5/6 antibody complex with a bead structure.
EXAMPLE 2 preparation of multimeric horseradish peroxidase-Ki-67 antibody Complex with 16-24 horseradish peroxidase tandem numbers
The procedure is as in example 1, except that the cytokeratin CK5/6 antibody is replaced with Ki-67.
(FIG. 5)
Example 3 preparation of a multimeric horseradish peroxidase-P63 antibody complex with 16-24 horseradish peroxidase tandem numbers.
The procedure is as in example 1, except that the cytokeratin CK5/6 antibody is replaced with P63.
EXAMPLE 4 preparation of multimeric horseradish peroxidase-Calponin antibody Complex with 16-24 horseradish peroxidase tandem numbers
The procedure is as in example 1, except that the cytokeratin CK5/6 antibody is replaced with Calponin. Example 5 preparation of multimeric horseradish peroxidase-goat anti-rabbit polyclonal antibody complex with number of horseradish peroxidase tandem of 8-12
a. 300mg of horseradish peroxidase is weighed and dissolved in 100mL of citric acid buffer solution with pH of 6.0, so as to obtain 10mg/mL of horseradish peroxidase solution;
b. 80mg of sodium periodate is weighed and dissolved in purified water to obtain 20mg/mL solution;
c. adding a sodium periodate aqueous solution into a horseradish peroxidase solution, and reacting for 10 minutes at 37 ℃;
d. and (3) finishing the reaction, and passing through a desalting column to obtain the aldehyde-activated horseradish peroxidase.
e. Preparing aldehyde-activated horseradish peroxidase into 20mg/mL;
f. adding 10mg of diamino polyethylene glycol with the polymerization degree of 24, adjusting the pH value of a reaction system to 10.0, and reacting for 4 hours at room temperature;
g. 5mg of sodium borohydride is added and the mixture is reacted for 2 hours at room temperature;
h. adding 0.5g of blocking reagent ethylenediamine, and reacting for 2 hours at room temperature;
i. finishing the reaction, and passing through a desalting column to obtain the poly horseradish peroxidase with a bead structure;
j. preparing the polymeric horseradish peroxidase with a bead structure into 15mg/mL;
k. dissolving a hydrophilic arm chain reagent with the polymerization degree of 8 in 10mM PBS buffer solution, and preparing 20mg/mL;
mixing the two materials together, and reacting for 1.5 hours at room temperature;
finishing the reaction, and passing through a PD-10 desalting column to obtain the horseradish peroxidase polymer containing hydrophilic arm chains;
dissolving goat anti-rabbit polyclonal antibody in 10mM PBS,10mM EDTA,pH8.0 to prepare 6mg/mL;
dissolving a sulfhydrylation reagent in 10mM PBS,10mM EDTA,pH8.0 solution to prepare 1mg/mL;
p, mixing the two materials together, and reacting for 1 hour at room temperature;
finishing the reaction, and passing through a PD-10 desalting column to obtain a sulfhydryl modified antibody;
mixing the thiol-modified goat anti-rabbit polyclonal antibody with the poly horseradish peroxidase with the hydrophilic arm chain according to the mol ratio of 1:20, and reacting for 24 hours at the temperature of 2-8 ℃;
s, passing through a molecular sieve purification column, and collecting a first separation peak to obtain the poly horseradish peroxidase-goat anti-rabbit polyclonal antibody complex with a bead structure. (FIG. 1 and FIG. 2)
Example 6 preparation of multimeric horseradish peroxidase-goat anti-mouse polyclonal antibody Complex with number of horseradish peroxidase tandem of 8-12
The procedure was as in example 5, except that goat anti-rabbit polyclonal antibody was replaced with goat anti-mouse polyclonal antibody (fig. 3, fig. 4).
Application example
Conventional immunohistochemical on-machine flow dewaxing: dewaxing liquid 3min x 2 dewaxing liquid 1min alcohol rinse 3 times TBS rinse 3 times;
repairing: repairing liquid 3 times repairing liquid 10min x 2 repairing liquid 18min TBS washes 4 times;
dyeing: blocking agent 8min TBS rinse 3 primary antibody 18min TBS rinse 3 primary antibody enhancement 10min TBS rinse 3 secondary antibody 20min TBS rinse 3 pure water rinse 3 DAB5min pure water rinse 5 hematoxylin 5min pure water rinse 2 TBS rinse two pure water rinse one pass;
sealing piece: sealing with xylene and gradient alcohol resin;
conventional immunohistochemical Manual flow
Dewaxing: xylene 5min 3;
antigen retrieval: EDTA9.0, cold water in 2200W, air valve air injection time 3min;
peroxidase blocking: 3% hydrogen peroxide methanol solution and ovalbumin for 8min;
an antibody selection: 32 DEG for 60min;
resistance enhancement: the method is free;
secondary antibody selection: HRP-labeled goat anti-mouse or goat anti-rabbit secondary antibody for 20min;
DAB：1:19 5min；
DAB enhancement: the method is free;
hematoxylin: 30 DEG for 5min;
returning blue: TBS for 1min;
sealing piece: sealing with xylene and gradient alcohol resin;
conventional immunohistochemical demonstration panels compare: the secondary antibody VS was imported and self-developed to immunize the secondary antibodies.
Quick freezing process
Quick freezing and dyeing: enzyme-labeled primary antibody 5min TBS washing 3 times mixing DAB5min pure water washing 4 times hematoxylin 2min pure water washing 2 times TBS washing 2 times pure water washing 1 time
Sealing piece: sealing with xylene and gradient alcohol resin;
quick freezing manual flow
Quick freezing and dyeing: enzyme-labeled primary antibody for 5min DAB:1:1:5min hematoxylin 3min blue-returning TBS1min sealing piece: sealing with xylene and gradient alcohol resin.