CN108593909A - A kind of method, labelled immune reagent and the application of fixed point labelled immune reagent - Google Patents
A kind of method, labelled immune reagent and the application of fixed point labelled immune reagent Download PDFInfo
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Abstract
The present invention relates to a kind of methods of fixed point labelled immune reagent,Labelled immune reagent and application,This method is first in antibody or antigen end connecting peptides segment Leu Pro X Thr Gly,The polypeptide fragment for containing at least three glycine in end is connected on signaling molecule again,Finally by engineered antibody or antigen,Signaling molecule and transpeptidase Sortase A hybrid reactions is transformed,Make signaling molecule quantify fixed point to be tagged on antigen or antibody,Polyol is additionally added in reaction as protein protective agent,The activity of can effectively protect antigen or antibody,Effectively avoid antigen caused by transpeptidase SortaseA or affinity of antibody impaired,Stability is impaired etc.,Greatly improve the thermal stability and specificity of labelled immune reagent,The labelled immune reagent differences between batches of preparation are small,Antigen or antibody can be also greatly saved when being applied to immunoassay.
Description
Technical field
The present invention relates to a kind of method, labelled immune reagent and the applications of fixed point labelled immune reagent, belong to labelled immune
Determination techniques field.
Background technology
Clinical immunoassay technology occurs to trace back at the end of the 19th century earliest, and it is classics to immunoprecipitate with immune agglutination technology
Immunoassay, the label immunoassay technology that developed on this basis can solve the immunoassays of many classics
The clinical assays problem that technology cann't be solved.
Since label immunoassay technology develops, finds and the new marker of application is to improve the sensitive of immunoassays
Degree, specificity, stability and simplicity are always one of the main direction of studying of label immunoassay technology, current marker
There are the signaling molecules such as isotope, enzyme, fluorescein, colloidal gold, light emitting molecule.
Labelled immune reagent is usually formed by signaling molecule labelled antibody/antigen, and traditional labelling technique is to utilize antigen
Or the lysine residue that antibody has is reacted with the signaling molecule of various activated forms, and since the lysine of antigen or antibody is residual
Radix amount is various (according to statistics, the antibody of 150kDa has more than 100 lysine residues), and label has randomness, marks species
The factors such as class, reaction temperature, rate of charge, reaction system size, reaction time, stirring can all influence to mark effect, signaling molecule
The quantity of labelled antibody/antigen and site cannot control well, and excessive signaling molecule, which is tagged on antigen/antibody, to lead
Its affinity is impaired, stability is impaired etc. is caused, signaling molecule is tagged to critical sites (such as antigenic determinant) meeting of antigen/antibody
Antigen/antibody inactivation is directly resulted in, inappropriate position mark can also cause changing for antigen/antibody physical property (such as hydrophobicity)
Become, cause completely unavailable after antigen/antibody marking signal molecule, therefore, optimization labeling process is to control signaling molecule label
The quantity of immunoreagent and site, and keep the activity of antigen or antibody, avoid antigen or affinity of antibody is impaired, stability by
Damage etc., this is most important to the performance of labelled immune reagent.
Invention content
The technical problem to be solved by the present invention is to：To solve above-mentioned technical problem of the existing technology to control signal point
The quantity of sub- labelled immune reagent and site, and the activity of antigen or antibody is kept, avoid antigen or affinity of antibody impaired, steady
It is qualitative impaired etc., a kind of method, labelled immune reagent and the application of fixed point labelled immune reagent are provided.
The technical solution adopted by the present invention to solve the technical problems is：
A method of fixed point labelled immune reagent includes the following steps：
In the polypeptide fragment Leu-Pro-X-Thr-Gly of antibody or the connection of antigen end containing 5 amino acid, (Leu is bright ammonia
Acid, Pro are proline, and X is arbitrary amino acid, and Thr is threonine, and Gly is glycine), obtain engineered antibody or antigen；
The polypeptide fragment that end at least contains 3 glycine is connected on signaling molecule, obtains transformation signaling molecule；
PH, which is added, in engineered antibody or antigen, transformation signaling molecule, transpeptidase Sortase A and polyol is
In 6.0~8.0 buffer solution, signaling molecule is connected to antigen or antibody by hybrid reaction 1-2h at a temperature of 30-45 DEG C
On；
The signaling molecule is biotin, a word used for translation heavy stone used as an anchor ester or acridine sulfonamide；
Preferably, the buffer solution is PB buffer solutions, Tris buffer solutions, HEPES buffer solution, MOPSO buffer solutions, DIPSO
One kind in buffer solution, glycine buffer, acetate buffer solution, a concentration of 0.01mol/L~0.2mol/L.
Preferably, the engineered antibody or antigen, transformation signaling molecule, transpeptidase Sortase A and polyol
By 0.1-5mol:1-100mol:0.01-0.5g:The ratio of 0.045-1g is reacted.
Preferably, the polyol is the mixture of polyethylene glycol and polysaccharide, the polyethylene glycol and polysaccharide
Mass ratio is 1:0.5-2.
Preferably, the polyethylene glycol is at least one of PEG4000, PEG6000, PEG8000, and the polysaccharide is fruit
At least one of sugar, sucrose, trehalose.
Preferably, the polypeptide fragment Leu-Pro-X-Thr-Gly for containing 5 amino acid is connected to antibody Fc end.
Preferably, the method for polypeptide fragment of the signaling molecule connection end at least containing 3 glycine is：By N- hydroxyls
The succinimide activated signaling molecule of base is dissolved in organic solvent, obtains the signaling molecule solution of activation；In the signal point of activation
It is 8-9 that alkaline solution is added in sub- solution and is adjusted to pH, is reacted at room temperature 2-3 hours；It is 6.5-7.5 that acid solution, which is added, and is adjusted to pH,
Sulfo-SMCC is added, is reacted at room temperature 1-2 hours；It is 8-9 that alkaline solution, which is added, and is adjusted to pH, and it is at least sweet containing 3 that end is added
The polypeptide sequence of propylhomoserin reacts at room temperature 8-16 hours.
Preferably, the alkaline solution be Amine Solutions, the organic amine be ethylenediamine, triethylamine, triethylene diamine,
Any one in diethanol amine, triethanolamine, tetramethylethylenediamine.
Preferably, the acid solution be organic acid soln, the organic acid be formic acid, acetic acid, propionic acid, butyric acid, octanoic acid,
Any one in adipic acid, ethanedioic acid, malonic acid, succinic acid, valeric acid, caproic acid, capric acid, acrylic acid.
Preferably, the polypeptide sequence that the end at least contains 3 glycine is Gly-Gly-Gly-Gly-Gly-Cys (Cys
For cysteine).
It is described the present invention also provides a kind of labelled immune reagent that the method by above-mentioned fixed point labelled immune reagent obtains
Labelled immune reagent is the conjugate that antigen or antibody are combined with signaling molecule fixed point, and the signaling molecule is biotin, a word used for translation heavy stone used as an anchor
Ester or acridine sulfonamide.
The present invention also provides a kind of labelled immune reagents that the method by above-mentioned fixed point labelled immune reagent obtains to mark
Remember the application of immunoassay.
The beneficial effects of the invention are as follows：
This application provides a kind of methods of novel and high-efficiency so that signaling molecule is pinpointed labelled antigen or antibody, first anti-
The polypeptide fragment Leu-Pro-X-Thr-Gly of body or the connection of antigen end containing 5 amino acid, then by specific on signaling molecule
Method connects polypeptide fragment of the end at least containing 3 glycine, finally by engineered antibody or antigen, transformation signaling molecule and turns
Peptase Sortase A hybrid reactions make signaling molecule quantify fixed point and are tagged on antigen or antibody, it is worth mentioning at this point that, reaction
In be additionally added polyol as protein protective agent, the activity of can effectively protect antigen or antibody effectively avoids transpeptidase
Antigen caused by Sortase A or affinity of antibody are impaired, stability is impaired etc., and the heat for greatly improving labelled immune reagent is steady
It is qualitative and specific and small using labelled immune reagent differences between batches prepared by this method, considerably reduce enterprise's production examination
The waste for the manpower, material resources and financial resources brought for screening mark of conformity batch when agent, can also be very big when being applied to immunoassay
Save antigen or antibody.
Specific implementation mode
The present invention is described in further detail now.
Embodiment 1
A kind of method for pinpointing label human immunodeficiency virus (HIV) antigen the present embodiment provides acridinium ester obtains acridine
The HIV antigens of ester label, the specific steps are：
It, will in the expression plasmid of the end of HIV antigens clone forward (FWD) Leu-Pro-Ala-Thr-Gly (Ala is alanine)
It is expressed after expression plasmid transfection Escherichia coli, you can in HIV antigens end connecting peptides segment Leu-Pro-Ala-Thr-Gly,
HIV antigens must be transformed；
The a word used for translation heavy stone used as an anchor ester that n-hydroxysuccinimide activates is dissolved with DMF, obtains acridine ester solution；Add in acridine ester solution
It is 8-9 to enter ethylenediamine solution to be adjusted to pH, is reacted at room temperature 3 hours；It is 6.5-7.5 that acetic acid solution, which is added, and is adjusted to pH, is added
Sulfo-SMCC is reacted at room temperature 2 hours；It is 8-9 that ethylenediamine solution, which is added, and is adjusted to pH, and polypeptide sequence Gly-Gly-Gly- is added
Gly-Gly-Cys reacts at room temperature 16 hours, obtains transformation signaling molecule；
50mM is added in transformation HIV antigens, transformation signaling molecule, transpeptidase Sortase A, PEG4000 and fructose, pH is
In 6.0 PB buffer solutions, make the dense of transformation HIV antigens, transformation signaling molecule, transpeptidase Sortase A, PEG4000 and fructose
Degree is respectively 5 μm of ol/L, 100 μm of ol/L, 0.5 μ g/L, 0.5 μ g/L, 0.5 μ g/L, in the shaking table of 50rpm, 45 DEG C of temperature
Signaling molecule is connected on HIV antigens to get labelled immune reagent solution by lower hybrid reaction 1h；
Labelled immune reagent solution is moved into bag filter (molecular cut off 8000-12000), use 0.05mol/L pH for
9.5CB buffer solutions are dialysed 2 times, every time dialysis 3 hours, and equivalent glycerine is added, and are placed -20 DEG C or less and are preserved.
Embodiment 2
The present embodiment provides a kind of methods that acridine sulfonamide pinpoints label HIV antigens to obtain acridine sulfonamide label
HIV antigens, the specific steps are：
In the expression plasmid of the end of HIV antigens clone's forward (FWD) Leu-Pro-Cys-Thr-Gly, expression plasmid is transfected big
It is expressed after enterobacteria, you can in HIV antigens end connecting peptides segment Leu-Pro-Cys-Thr-Gly, HIV antigens must be transformed；
The a word used for translation heavy stone used as an anchor sulfonamide that n-hydroxysuccinimide activates is dissolved with DMF, obtains acridine sulfonamide solution；In acridine sulphur
It is 8-9 that being added in amide solution, which has triethylene diamine solution to be adjusted to pH, is reacted at room temperature 2.5 hours；Solution of adipic acid is added to be adjusted to
PH is 6.5-7.5, adds sulfo-SMCC, is reacted at room temperature 1.5 hours；It is 8-9 that triethylene diamine solution, which is added, and is adjusted to pH, is added
Enter polypeptide sequence Gly-Gly-Gly-Cys, reacts at room temperature 10 hours, obtain transformation signaling molecule；
50mM is added in transformation HIV antigens, transformation signaling molecule, transpeptidase Sortase A, PEG6000 and sucrose, pH is
In 8.0 Tris buffer solutions, make transformation HIV antigens, transformation signaling molecule, transpeptidase Sortase A, PEG6000 and sucrose
Concentration is respectively 0.1 μm of ol/L, 1 μm of ol/L, 0.01 μ g/L, 0.03 μ g/L, 0.015 μ g/L, in the shaking table of 50rpm, 30 DEG C
At a temperature of hybrid reaction 2h, a word used for translation heavy stone used as an anchor sulfonamide is connected on HIV antigens, i.e. labelled immune reagent solution；
Labelled immune reagent solution is moved into bag filter (molecular cut off 8000-12000), use 0.05mol/L pH for
The buffer solution of 9.5CB is dialysed 2 times, every time dialysis 3 hours, and equivalent glycerine is added, and is placed -20 DEG C or less and is preserved.
Embodiment 3
The present embodiment provides a kind of methods that biotin pinpoints label anti-alpha-fetoprotein (AFP) monoclonal antibody specific 1
Biotinylation anti-alpha-fetoprotein monoclonal antibody specific 1 is obtained, the specific steps are：
In the table of the ends Fc of anti-alpha-fetoprotein monoclonal antibody specific 1 clone's forward (FWD) Leu-Pro-Leu-Thr-Gly
It up to plasmid, will be expressed after expression plasmid transfection Escherichia coli, you can in HIV antigens end connecting peptides segment Leu-Pro-Leu-
Anti-alpha-fetoprotein monoclonal antibody specific 1 must be transformed in Thr-Gly；
The biotin that n-hydroxysuccinimide activates is dissolved with DMF, obtains biotin solution；Add in biotin solution
It is 8-9 to enter diethanolamine solution to be adjusted to pH, is reacted at room temperature 2 hours；It is 6.5-7.5 that acrylic acid solution, which is added, and is adjusted to pH, is added
Sulfo-SMCC is reacted at room temperature 1 hour；It is 8-9 that diethanolamine solution, which is added, and is adjusted to pH, and polypeptide sequence Gly-Gly-Gly- is added
Gly-Gly-Cys reacts at room temperature 8 hours, obtains transformation signaling molecule；
Will transformation anti-alpha-fetoprotein monoclonal antibody specific 1, transformation signaling molecule, transpeptidase Sortase A,
PEG6000 and trehalose are added 50mM, in the HEPES buffer solution that pH is 6.8, keep transformation anti-alpha-fetoprotein specific monoclonal anti-
Body 1, transformation signaling molecule, transpeptidase Sortase A, PEG6000 and trehalose concentration be respectively 1 μm of ol/L, 10 μm of ol/L,
0.1 μ g/L, 0.1 μ g/L, 0.2 μ g/L, in the shaking table of 50rpm, hybrid reaction 1.5h, biotin is connected at a temperature of 40 DEG C
Onto anti-alpha-fetoprotein monoclonal antibody specific 1, i.e. labelled immune reagent solution；
Labelled immune reagent solution is moved into bag filter (molecular cut off 8000-12000), use 0.05mol/L pH for
The buffer solution of 9.5CB is dialysed 2 times, every time dialysis 3 hours, and equivalent glycerine is added, and is placed -20 DEG C or less and is preserved.
Comparative example 1
This comparative example provide it is a kind of by n-hydroxysuccinimide by the conventional method of acridinium ester label to HIV antigens with
The HIV antigens for obtaining acridinium ester label, the specific steps are：
By antigen use 0.05mol/L pH for 9.5 CB buffer antigenic solutions；NHS is added in antigenic solution
The a word used for translation heavy stone used as an anchor ester of activation, make antigen, NHS activation a word used for translation heavy stone used as an anchor ester molar ratio be 1:10, it reacts at room temperature 2 hours, obtains reaction solution；It will be anti-
It answers liquid to move to bag filter (molecular cut off 8000-12000), 0.05mol/L pH is used to dialyse 2 times for 9.5 CB buffer solutions,
Equivalent glycerine is added in dialysis 3 hours every time, places -20 DEG C or less and preserves.
Comparative example 2
This comparative example provides a kind of tradition side that acridine sulfonamide is tagged to HIV antigens by n-hydroxysuccinimide
Method with obtain acridine sulfonamide label HIV antigens, the specific steps are：
By antigen use 0.05mol/L pH for 9.5 CB buffer antigenic solutions；NHS is added in antigenic solution
The a word used for translation heavy stone used as an anchor sulfonamide of activation, make antigen, NHS activation a word used for translation heavy stone used as an anchor sulfonamide molar ratio be 1:50, it reacts at room temperature 0.5 hour, obtains
Reaction solution；Reaction solution is moved into bag filter (molecular cut off 8000-12000), 0.05mol/L pH is used to be buffered for 9.5 CB
Liquid is dialysed 2 times, every time dialysis 3 hours, and equivalent glycerine is added, and is placed -20 DEG C or less and is preserved.
Comparative example 3
This comparative example provides a kind of by n-hydroxysuccinimide that biotin labeling is single to anti-alpha-fetoprotein specificity
The conventional method of clonal antibody 1 to obtain biotinylation anti-alpha-fetoprotein monoclonal antibody specific 1, the specific steps are：
By antigen use 0.05mol/L pH for 9.5 CB buffer antigenic solutions；NHS is added in antigenic solution
The biotin of activation, make anti-alpha-fetoprotein monoclonal antibody specific 1, NHS activation biotin molar ratio be 1:50, room temperature
Reaction 0.5 hour, obtains reaction solution；Reaction solution is moved into bag filter (molecular cut off 8000-12000), using 0.05mol/L
The CB buffer solutions that pH is 9.5 are dialysed 2 times, every time dialysis 3 hours, and equivalent glycerine is added, and are placed -20 DEG C or less and are preserved.
Effect example 1
The HIV antigens of three batch acridinium ester labels, respectively leading mark are made using the method for embodiment 1 for this effect example
Object 1-1, leading mark object 2-1, leading mark object 3-1, and three batch acridinium ester labels are made using the method for comparative example 1
HIV antigens, respectively conventional tag object 1-1, conventional tag object 2-1, conventional tag object 3-1, and by all acridinium ester labels
HIV antigens are placed at 4 DEG C and 37 DEG C 3 days respectively, then detect 500 negative blood by chemiluminescence immune analysis method
Sample, the specific steps are：
S1：The biotinylated HIV antigenic solutions of 50 μ L samples to be tested and 100 a concentration of 0.4mg/L of μ L are reacted 10 points
Clock forms antigen-antibody complexes；
S2：The coated magnetic particle reagents of Streptavidin of 20 a concentration of 0.8mg/L of μ L, the Ag-Ab with S1 is added
Compound precursor reactant after ten minutes, forms magnetic antigen-antibody complexes suspension；
S3：The magnetic antigen-antibody complexes suspension of S2 is placed in magnetic field, it is multiple to wash the magnetic Ag-Ab
It is fit；
S4：By the acridinium ester label of magnetic antigen-antibody complexes and 150 a concentration of 0.1mg/L of μ L after the washing of S3
HIV antigenic solutions reaction after twenty minutes, formed magnetic composite suspension；
S5：The magnetic composite suspension of S4 is placed in magnetic field, the magnetic composite is washed；
S6：150 μ L chemiluminescence exciting liquid A (0.1M nitric acid and 1g/ are injected in magnetic composite after being washed to S5
100mL hydrogen peroxide), after 1.5 seconds, inject 150 μ L chemiluminescence exciting liquid B (0.25M sodium hydroxides and 0.1g/
100tritonx-100), the chemiluminescence photon intensity in 5 seconds is collected；
By the chemiluminescence photon intensity compared with Cutoff values, if simple chemical shines, photon intensity is more than cutoff
Value, is judged to the positive, if simple chemical shines, photon intensity is less than cutoff values, is judged to feminine gender, the Cutoff values be 500
Average value+the 2SD of negative sample luminous value, as a result such as table 1.
The different acridinium ester label HIV antigens of table 1 are used for the luminous situation of immunoassay
From upper table, conventional method is prepared (by n-hydroxysuccinimide by acridinium ester label to HIV antigens)
Labelled immune reagent when surveying negative blood sample for chemiluminescence immune assay, the luminous value difference between batch measured is larger, and with
Temperature increases luminous value and significantly increases, and false positive results occurs when using 37 DEG C of labelled immune reagents placed, this may be a word used for translation
The out of contior reason of quantity and site of pyridine ester label HIV antigens causes；It is tried with the labelled immune prepared using conventional method
Agent is compared, and the labelled immune reagent prepared using transpeptidase Sortase A leading mark technologies can control luminous value well
Difference between batch, greatly reduce the wave of manpower, material resources and financial resources brought for screening mark of conformity batch when enterprise's production reagent
Take, and change smaller, non-false positive result as temperature increases luminous value, it is seen that the label prepared using leading mark technology
Immunoreagent thermal stability has obtained very big improvement.
Effect example 2
The HIV antigens of three batch acridine sulfonamide label are made using the method for embodiment 2 for this effect example, respectively orient
Marker 1-2, leading mark object 2-2, leading mark object 3-2, and three batch acridine sulfonamide are made using the method for comparative example 2
The HIV antigens of label, respectively conventional tag object 1-2, conventional tag object 2-2, conventional tag object 3-2, and by all acridine sulphurs
The HIV antigens of amide label are placed 3 days at 4 DEG C and 37 DEG C respectively, then pass through the chemiluminescence immune assay side of effect example 1
The positive blood sample of method detection, the results are shown in Table 2.
The different acridine sulfonamide label HIV antigens of table 2 are used for the luminous situation of immunoassay
From upper table as it can be seen that by conventional method (acridine sulfonamide is tagged to HIV antigens by n-hydroxysuccinimide)
When the labelled immune reagent of preparation surveys positive blood sample for chemiluminescence immune assay, the luminous value difference between batch measured is larger, and
Obviously become smaller as temperature increases luminous value, the labelled immune reagent placed using 37 DEG C is than the labelled immune using 4 DEG C of placements
Reagent luminous value reduces 34.3%-41.1%, this may be that acridine sulfonamide marks the quantity of HIV antigens and site that cannot control
The reason of cause；Compared with the labelled immune reagent prepared using conventional method, using transpeptidase Sortase A leading mark skills
Labelled immune reagent prepared by art can control the difference between batch of luminous value well, be sieve when greatly reducing enterprise's production reagent
The waste of manpower, material resources and financial resources selected mark of conformity batch and brought, and as to increase luminous value variation smaller for temperature, use
The labelled immune reagent of 37 DEG C of placements using the labelled immune reagent luminous value of 4 DEG C of placements than reducing 5.6%-10.0%, it is seen that
The labelled immune reagent thermal stability prepared using leading mark technology has obtained very big improvement.
Effect example 3
Three batch biotinylation anti-alpha-fetoprotein monoclonal antibody specifics are made using the method for embodiment 3 in this effect example
1, respectively leading mark object 1-3, leading mark object 2-3, leading mark object 3-3, and it is made three batches using the method for comparative example 3
Secondary pollutant element anti-alpha-fetoprotein monoclonal antibody specific 1, respectively conventional tag object 1-3, conventional tag object 2-3, tradition
Marker 3-3, by chemiluminescence immune analysis method detect a concentration of 0IU/mL, 0.5IU/mL of alpha-fetoprotein, 1.5IU/mL,
The step of standard items of 5IU/mL, 15IU/mL, 50IU/mL, chemiluminescence immune analysis method, is as follows：
S1：(referred to as by 1 solution of 50 μ L samples to be tested and 100 μ L biotinylation anti-alpha-fetoproteins monoclonal antibody specific
1 solution of biotinylated antibody) and 50 a concentration of 0.1mg/L of μ L acridinium ester label anti-alpha-fetoprotein monoclonal antibody specific
2 reactions 10 minutes, form antibody-antigen-antibody sandwich complex；
S2：The coated magnetic particle reagents of Streptavidin of 20 a concentration of 0.8mg/L of μ L, the antibody-antigene-with S1 is added
The compound precursor reactant of antibody sandwich after ten minutes, forms magnetic coupling liquid suspension；
S3：The magnetic coupling liquid suspension of S2 is placed in magnetic field, the magnetic compound is washed；
S4：150 μ L chemiluminescence exciting liquid A (0.1M nitric acid and 1g/ are injected in magnetic compound after being washed to S3
100mL hydrogen peroxide), after 1.5 seconds, inject 150 μ L chemiluminescence exciting liquid B (0.25M sodium hydroxides and 0.1g/
100tritonx-100), the chemiluminescence photon intensity in 5 seconds is collected, the results are shown in Table 3.
Luminous situation of the different biotinylation anti-alpha-fetoprotein antibodies 1 of table 3 for immunoassay
From the point of view of upper table, the biotinylation anti-alpha-fetoprotein prepared using transpeptidase Sortase A leading mark technologies is special
Specific monoclonal antibodies 1 mark polypeptide fragment Leu-Pro-Leu-Thr-Gly in the ends Fc of monoclonal antibody 1, with tradition
The biology that method is prepared (by n-hydroxysuccinimide by biotin labeling to anti-alpha-fetoprotein monoclonal antibody specific 1)
Elementization anti-alpha-fetoprotein monoclonal antibody specific 1 is compared, when being applied to immunoassay, the biotin of leading mark technology preparation
The utilization rate for changing anti-alpha-fetoprotein monoclonal antibody specific 1 is more preferable, only can reach with tradition side with 1/5th concentration
The luminous value of biotinylation anti-alpha-fetoprotein monoclonal antibody specific 1 prepared by method, good linearity, isolabeling batch is not luminous
It is small to be worth difference between batch, this is because the method for embodiment 3, which can pinpoint biotin, is tagged to anti-alpha-fetoprotein specific monoclonal
On antibody 1 so that anti-alpha-fetoprotein monoclonal antibody specific 1, which can effectively stretch in solution, removes capture antigen, and traditional
Biotinylation anti-alpha-fetoprotein monoclonal antibody specific 1 prepared by method, biotin labeling anti-alpha-fetoprotein specificity Dan Ke
The quantity of grand antibody 1 and site cannot control, and each position of antibody 1 may be crosslinked with biotin and cannot effectively capture anti-
It is former.As it can be seen that the not isolabeling of biotinylation anti-alpha-fetoprotein monoclonal antibody specific 1 prepared by the leading mark method of the present invention
The difference between batch of batch is small, and antibody can be greatly saved when being applied to immunoassay.
It is enlightenment with above-mentioned desirable embodiment according to the present invention, through the above description, relevant staff is complete
Various changes and amendments can be carried out without departing from the scope of the technological thought of the present invention' entirely.The technology of this invention
Property range is not limited to the contents of the specification, it is necessary to determine its technical scope according to right.
Claims (10)
1. a kind of method of fixed point labelled immune reagent, which is characterized in that include the following steps：
The polypeptide fragment Leu-Pro-X-Thr-Gly containing 5 amino acid in antibody or the connection of antigen end, obtains engineered antibody or anti-
It is former；
The polypeptide fragment that end at least contains 3 glycine is connected on signaling molecule, obtains transformation signaling molecule；
By engineered antibody or antigen, transformation signaling molecule, transpeptidase Sortase A and polyol be added pH be 6.0~
In 8.0 buffer solution, signaling molecule is connected on antigen or antibody by hybrid reaction 1-2h at a temperature of 30-45 DEG C；
The signaling molecule is biotin, a word used for translation heavy stone used as an anchor ester or acridine sulfonamide.
2. the method for fixed point labelled immune reagent according to claim 1, which is characterized in that the buffer solution buffers for PB
In liquid, Tris buffer solutions, HEPES buffer solution, MOPSO buffer solutions, DIPSO buffer solutions, glycine buffer, acetate buffer solution
One kind, a concentration of 0.01mol/L~0.2mol/L.
3. it is according to claim 1 or 2 fixed point labelled immune reagent method, which is characterized in that the engineered antibody or
Antigen, transformation signaling molecule, transpeptidase Sortase A and polyol press 0.1-5mol:1-100mol:0.01-0.5g:
The ratio of 0.045-1g is reacted.
4. the method for pinpointing labelled immune reagent according to claim 1-3 any one of them, which is characterized in that the polyhydroxy
Compound is the mixture of polyethylene glycol and polysaccharide, and the mass ratio of the polyethylene glycol and polysaccharide is 1:0.5-2, the poly- second two
Alcohol is at least one of PEG4000, PEG6000, PEG8000, and the polysaccharide is at least one in fructose, sucrose, trehalose
Kind.
5. the method for pinpointing labelled immune reagent according to claim 1-4 any one of them, which is characterized in that described to contain 5
The polypeptide fragment Leu-Pro-X-Thr-Gly of amino acid is connected to antibody Fc end.
6. the method for pinpointing labelled immune reagent according to claim 1-5 any one of them, which is characterized in that the signal point
The method of polypeptide fragment that son connection end at least contains 3 glycine is：The signaling molecule that n-hydroxysuccinimide is activated
It is dissolved in organic solvent, obtains the signaling molecule solution of activation；Alkaline solution is added in the signaling molecule solution of activation and is adjusted to pH
For 8-9, react at room temperature 2-3 hours；It is 6.5-7.5 that acid solution, which is added, and is adjusted to pH, adds sulfo-SMCC, reacts at room temperature 1-
2 hours；It is 8-9 that alkaline solution, which is added, and is adjusted to pH, and the polypeptide sequence that end at least contains 3 glycine is added, reacts at room temperature 8-16
Hour.
7. the method for fixed point labelled immune reagent according to claim 6, which is characterized in that the alkaline solution is organic
Amine aqueous solution, the organic amine are in ethylenediamine, triethylamine, triethylene diamine, diethanol amine, triethanolamine, tetramethylethylenediamine
Any one；
The acid solution is organic acid soln, and the organic acid is formic acid, acetic acid, propionic acid, butyric acid, octanoic acid, adipic acid, second two
Any one in acid, malonic acid, succinic acid, valeric acid, caproic acid, capric acid, acrylic acid.
8. the method for pinpointing labelled immune reagent according to claim 1-7 any one of them, which is characterized in that the end is extremely
The polypeptide sequence for containing 3 glycine less is Gly-Gly-Gly-Gly-Gly-Cys.
9. the labelled immune reagent that a kind of method by pinpointing labelled immune reagent obtains, the labelled immune reagent are antigen
Or the conjugate that antibody is combined with signaling molecule fixed point, the signaling molecule are biotin, a word used for translation heavy stone used as an anchor ester or acridine sulfonamide.
10. labelled immune reagent the answering in label immunoassay technology that a kind of method by pinpointing labelled immune reagent obtains
With.
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