CN109613242A - A kind of creatine kinase isozyme detection kit and preparation method thereof - Google Patents
A kind of creatine kinase isozyme detection kit and preparation method thereof Download PDFInfo
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- CN109613242A CN109613242A CN201811298267.8A CN201811298267A CN109613242A CN 109613242 A CN109613242 A CN 109613242A CN 201811298267 A CN201811298267 A CN 201811298267A CN 109613242 A CN109613242 A CN 109613242A
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Abstract
The invention belongs to vitro detection technical fields, and in particular to a kind of creatine kinase isozyme detection kit and preparation method thereof.Creatine kinase isozyme detection kit provided by the present invention, comprising: Magneto separate reagent and enzymic-labelled antibody；Magneto separate reagent includes: the magnetic particle that the first anti-CK-MB monoclonal antibody and the second anti-CK-MB monoclonal antibody are combined on surface；Enzymic-labelled antibody includes: the anti-CK-MB monoclonal antibody of third of enzyme label；And/or the 4th anti-CK-MB monoclonal antibody of enzyme label；Wherein, the first, second, third and fourth anti-CK-MB monoclonal antibody is specific to the different epitopes of creatine kinase isozyme respectively.The the first anti-CK-MB monoclonal antibody and the second anti-CK-MB monoclonal antibody of the different epitopes for being specific to creatine kinase isozyme are combined on the surface of magnetic particle in Magneto separate reagent of the present invention, the CK-MB antigen in multidigit point identification sample to be tested can be achieved, enhance the combination of magnetic bead antibody and antigen, the case where can avoid missing inspection, high sensitivity, target user are extensive.
Description
Technical field
The invention belongs to vitro detection technical fields, and in particular to a kind of creatine kinase isozyme detection kit and its system
Preparation Method.
Background technique
Cardiovascular disease is to endanger the great fatal disease of the health of our people and life, is counted according to the Ministry of Public Health,
In China city, just there are 235 people to die of cardiovascular and cerebrovascular disease in every 100,000 people, accounts for dead caused by various diseases
First place, and every year with 2% speed increase.Acute myocardial infarction AMI (Acute Myocardial Infarction, AMI) refers to
The part cardiac muscle acute necrosis caused by lasting and serious myocardial ischemia is the main reason for causing cardiovascular patient dead.
Clinically often show as pectoralgia, acute circulatory dysfunction and the series of features such as reflecting myocardium damage, ischemic and necrosis
The ECG change of property.Clinical manifestation often has severe pain, acute circulatory dysfunction, arrhythmia cordis, the heart after lasting breastbone
Functional failure, fever, the raising of white blood cell count(WBC) and serum cardiac damage score enzyme and myocardium acute injury and necrosis the heart
Electrograph progressive develops.The acute myocardial infarction rate of China is in obvious ascendant trend in recent years, is newly sent to few 500,000 every year,
Now suffer from least 2,000,000.
Creatine kinase (CK) is primarily present in vertebrate, exists in the groups such as the heart, skeletal muscle and brain of animal
In the cytoplasm and mitochondria knitted.CK exists wide in human body, and half-life period is about 6-8 hours in blood.CK is for being catalyzed
The reversible reaction that phosphoric acid shifts between creatine and ATP or phosphocreatine and ADP, to maintain the physiological concentration of intracellular ATP.CK is urged
Change effect is reversible, i.e., can be catalyzed creatine in pH9.0 and ATP generates phosphocreatine and ADP, be positive reaction；It can also be
Catalytic phosphatase creatine and ADP generate creatine and ATP when pH6.7, are back reaction.Wherein, in neutral conditions, back reaction is positive and negative
2-6 times answered, i.e., to generate based on ATP, with energy needed for guaranteeing histocyte from ATP.Positive reaction is conducive to mitochondria
The ATP that internal oxidition phosphorylation generates, cell liquid is entered in the form of phosphocreatine, supplies the needs of cellular physiological events.Therefore,
Creatine kinase is the important kinases that one and intracellular energy transhipment, the regeneration of contraction of muscle and ATP have direct relation.
CK is a dimer, is existed in the form of 3 kinds of isodynamic enzymes: 2 B monomers (CK-BB), 2 M monomers (CK-MM),
The monomer mixture (CK-MB) of MB.This 3 kinds of isodynamic enzyme molecular weight are identical, are catalyzed identical chemical reaction, but its molecular structure and
Source is different: CK-BB derives from striated muscle, and CK-MM derives from brain and alimentary canal, and CK-MB is from cardiac muscle.In cardiac muscle
CK-MB accounts for the 15%~25% of CK total amount, so in myocardial damage, when especially acute myocardial infarction AMI (AMI), measures CK-MB
There is great clinical meaning to the diagnosis of AMI.Related experiment shows the sampling analysis in 12~48d after symptom occurs, CK-MB
The Clinical Sensitivity and clinical specificity of quality are respectively 96.8% and 89.6%, this takes off it in numerous Applications of Cardiac Markers
Grain husk and go out, become the index to play an important role to AMI clinical diagnosis.Actual clinical diagnosis in, can by combine cTnI,
The testing results of the cardiac muscle specific markers such as Myo further increases the accuracy of clinical diagnosis.
The conventional determining method of CK-MB is mainly immunodepression, measures the vigor of CK-MB.The same work of CK in normal serum
Enzyme is mainly CK-MM, CK-MB and minimal amount of CK-BB, since the content of CK-BB is atomic, be can be neglected, therefore the method
Assuming that there was only CK-MM and CK-MB in serum, the polyclonal antibody of anti-CK-M subunit is added in reagent, to inhibit CK-MM
It with M subunit active in CK-MB, measures result and is equivalent to B subunit active in CK-MB, by result multiplied by 2 that is, CK-
MB vigor.The method is rapid, brief introduction, time saving, there is higher sensibility.However, the influence factor and defect of the method are numerous:
1) enzyme is easy to aging
The active enzyme of CK-MB is easy to aging, is mainly shown as enzyme conformational change.Its cause CK-MB Activity determination reduce or
Normal reason may be due to having crossed detection time so that enzyme aging reduces or loses the catalytic activity of enzyme in serum, from
And caused by causing the activation energy of enzyme to increase.
2) interference of huge CK
Huge CK is Macro-enzyme, has macromolecule group than corresponding enzyme under normal circumstances, is not pressed down by anti-CK-M antiserum
System shows as in serum " CK-MB " and accounts for 50% or more CK vigor, and CK vigor is normal or slight or moderate increases.The huge big portion CK
It is the compound of enzyme Yu a kind of immunoglobulin in the case of point, claims huge CK1, usual immunoglobulin is IgG and IgA, accidental
IgM.When forming compound, CK-MB reagent cannot not only inhibit CK-M, and result is more expanded multiplied by 2 when because calculating
Error；And huge CK2 is a kind of abnormal oligomeric mitochondria CK, also known as CK-Mt, CK-Mt antigenicity is different from CK-M, and anti-M is anti-
Body cannot inhibit CK-Mt, therefore form the false positive of CK-MB.
3) interference of CK-BB
The active method of immunosuppression assay for detecting CK-MB assumes that in sample without CK-BB activity.Isodynamic enzyme CK-BB is main
It is present in brain tissue, its activity in serum of normal condition is very low, and the tissue tumors such as part brain disorder, prostate occur
When, CK-BB discharges into peripheral blood in tissue, due to the active raising of CK-BB, must influence the accurate of CK-MB measurement result
Property, cause the false positive of CK-MB.
4) interference of haemolysis
Measurement CK, CK-MB vigor does not use haemolysis sample as far as possible.It is well known that being free of CK in red blood cell, but in red blood cell
Containing adenosine acid kinase (AK), AK can directly participate in the second step reaction of CK performance rate method, and in the case where no CK, AK can be direct
NAD is set to be reduced into NADH through gl ucose-6-phasphate dehydrogensae catalysis under adenosine diphosphate effect with -6 phosphatase reaction of glucose,
Rise so as to cause absorbance at 340nm, can be seen that from the reactions above, even if being free of CK in serum, the AK in haemolysis sample
The reaction of 2ADP → ATP+AMP can be directly participated in, therefore certain influence is also brought on the measurement result of CK-MB.
Thus, a kind of those skilled in the art's urgently creatine kinase isozyme CK-MB detection that can be quick, highly sensitive
Method.
Summary of the invention
The main purpose of the present invention is to provide a kind of creatine kinase isozyme detection kits, it is intended to solve existing CK-
The technical issues of disturbing factor of MB detection method excessive bring muting sensitivity.
Another object of the present invention is to provide a kind of preparation methods of creatine kinase isozyme detection kit, to provide
Easy to operate, the quick method of one kind goes to prepare above-mentioned creatine kinase isozyme detection kit.
In order to achieve the above-mentioned object of the invention, an aspect of of the present present invention provides a kind of creatine kinase isozyme detection reagent
Box, comprising: Magneto separate reagent and enzymic-labelled antibody；
The Magneto separate reagent includes: that the first anti-CK-MB monoclonal antibody and the second anti-CK-MB Dan Ke are combined on surface
The magnetic particle of grand antibody；
The enzymic-labelled antibody includes: the anti-CK-MB monoclonal antibody of third of enzyme label；And/or the 4th of enzyme label is anti-
CK-MB monoclonal antibody；
Wherein, the described first, second, third and fourth anti-CK-MB monoclonal antibody is specific to the same work of creatine kinase respectively
The different epitopes of enzyme.
Compared with prior art, magnetic particle in Magneto separate reagent of the present invention is combined with the first anti-CK- simultaneously on the surface
MB monoclonal antibody and the second anti-CK-MB monoclonal antibody, the first and second anti-CK-MB monoclonal antibodies are specific to creatine kinase
The different epitopes of isodynamic enzyme are, it can be achieved that multidigit point identifies the CK-MB antigen in sample to be tested, the knot of enhancing magnetic bead antibody and antigen
The case where closing, improve sensitivity, can avoid missing inspection in actual application, target user is extensive.
Another aspect of the present invention provides a kind of preparation method of creatine kinase isozyme detection kit, comprising:
A) by magnetic particle and the first anti-CK-MB monoclonal antibody, the second anti-CK-MB monoclonal antibody reactive, surface is prepared
On be combined with the magnetic particle of the first anti-CK-MB monoclonal antibody and the second anti-CK-MB monoclonal antibody；
B) the anti-CK-MB monoclonal antibody of third or the 4th anti-CK-MB monoclonal antibody are coupled marker enzyme, prepare enzyme label
Detection antibody；
Wherein, the described first anti-CK-MB monoclonal antibody and the second anti-CK-MB monoclonal antibody and the magnetic particle
Covalent bond.
Preparation method provided by the present invention, easy to operate, exploitativeness is strong.By above-mentioned preparation method, can quickly make
It is standby to obtain a kind of creatine kinase isozyme detection kit of high sensitivity.
Detailed description of the invention
Fig. 1 is line of the creatine kinase isozyme detection kit in serum detection with existing product in test case of the present invention
Property correlation fitted figure.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
The quality of each component noted in the disclosure of the embodiment of the present invention not only may refer to specifically containing for each component
Amount, can also indicate the proportionate relationship of quality between each component, therefore, as long as each according to specification composition of the embodiment of the present invention
The content of component is scaled up or is reduced within specification of embodiment of the present invention scope of disclosure.Specifically, of the invention
Embodiment quality described in the specification can be unit of weight well known to the field of medicaments such as μ g, mg, g, kg.
On the one hand, the embodiment of the invention provides a kind of creatine kinase isozyme detection kits, comprising: Magneto separate reagent
And enzymic-labelled antibody；
Magneto separate reagent includes: that the first anti-CK-MB monoclonal antibody and the second anti-CK-MB monoclonal are combined on surface is anti-
The magnetic particle of body；
Enzymic-labelled antibody includes: the anti-CK-MB monoclonal antibody of third of enzyme label；And/or the 4th anti-CK- of enzyme label
MB monoclonal antibody；
Wherein, the first, second, third and fourth anti-CK-MB monoclonal antibody is specific to creatine kinase isozyme respectively
Different epitopes.
In the above-mentioned technical solutions, the first anti-CK-MB monoclonal antibody and the second anti-CK-MB monoclonal are combined on surface
The magnetic particle of antibody plays very important effect, the first and second anti-CK-MB monoclonal antibodies on improving detection sensitivity
The different epitopes of creatine kinase isozyme are specific to, they are combined with the surface of magnetic particle, in magnetic particle antibody and antigen
, it can be achieved that multidigit point identifies the CK-MB antigen in sample to be tested in conjunction with during, enhance the combination of magnetic bead antibody and antigen,
And then the case where improving detection efficiency, avoiding missing inspection, high sensitivity, target user are extensive.
Preferably, in Magneto separate reagent, the first anti-CK-MB monoclonal antibody, the second anti-CK-MB monoclonal antibody and
The mass ratio of magnetic particle is (1~2): (2~4): 20.When the first anti-CK-MB monoclonal antibody, the second anti-CK-MB monoclonal are anti-
The mass ratio of body and magnetic particle is (1~2): (2~4): when 20, coated two kinds of antibody being enabled to be uniformly distributed in magnetic particle
Surface enhances detection sensitivity.
Preferably, in enzymic-labelled antibody, the mass ratio of the anti-CK-MB monoclonal antibody of third and marker enzyme be 1:(1~
5)；Or, the mass ratio of the 4th anti-CK-MB monoclonal antibody and the marker enzyme is 1:(1~10).In this way, may make third,
The potency of four CK-MB monoclonal antibodies is similar, and not in proportional region, the potency difference of different enzyme labelled antibodies is larger, reagent spirit
Sensitivity decline.In enzymic-labelled antibody, the mass ratio of marker enzyme and the 3rd CK-MB monoclonal antibody is greater than 5, and marker enzyme is excessive,
Lead to wastage of material；When marker enzyme and the mass ratio of the 3rd CK-MB monoclonal antibody are less than 1, label enzyme amount is inadequate, causes
The mark rate of antibody is low, influences reagent sensitivity.4th CK-MB monoclonal antibody is similarly.In embodiments of the present invention, the 4th
CK-MB monoclonal antibody needs detection sensitivity more higher than third antibody, in general, the 4th CK-MB monoclonal antibody of coupling
Marker enzyme should be 1~2 times of marker enzyme of the 3rd CK-MB monoclonal antibody of coupling.
Preferably, the first anti-CK-MB monoclonal antibody, the second anti-CK-MB monoclonal antibody and the anti-CK-MB Dan Ke of third
The mass ratio of grand antibody is 1:(1~2): (2~4)；Or
First anti-CK-MB monoclonal antibody, the second anti-CK-MB monoclonal antibody and the 4th anti-CK-MB monoclonal antibody
Mass ratio is 1:(1~2): (2~4).
First, second anti-CK-MB monoclonal antibody coupling magnetic particle passes through magnetic for the antigen in capture reaction system
Isolation technics gets up the antigen enrichment of capture, its general usage amount can be less than the usage amount of enzyme labelled antibody, can achieve best
Sexual valence ratio.Magnetic particle answers priority flag affinity and the higher antibody of potency, promotes capture rate.Third, the 4th anti-CK-MB
Monoclonal antibody enzyme labelled antibody needs to complete the antigen of capture almost 100% identification, needs for the sensitivity for promoting reagent
Want enzyme labelled antibody much excessive, to sacrifice into the original sensitivity for obtaining reagent.In embodiments of the present invention, the amount of enzyme labelled antibody
1~2 multiple proportions for the first, second anti-CK-MB monoclonal antibody is convenient.
Preferably, the first, second anti-CK-MB monoclonal antibody is specific to the M subunit of creatine kinase isozyme, third and
4th anti-CK-MB monoclonal antibody is specific to the B subunit of creatine kinase isozyme, it is ensured that the compound analyte detection of CK-MB it is special
Property, improve the sensitivity of reagent.
Preferably, marker enzyme is alkaline phosphatase or horseradish peroxidase in stating enzymic-labelled antibody.
Preferably, the partial size of magnetic particle is 0.5~3.5 μm.
When the partial size of magnetic particle is greater than 3.5 μm, magnetic particle specific surface area is reduced, and reagent sensitivity becomes smaller, is not achieved super
Quick effect；When the partial size of magnetic particle is less than 0.5 μm, Magneto separate effect is influenced.
In the embodiment of the present invention, above-mentioned creatine kinase isozyme detection kit further include: chemiluminescent substrate.In this hair
In bright embodiment, as long as can be suitable for this hair by the chemiluminescent substrate of the enzyme marker catalytic luminescence of the embodiment of the present invention
It is bright.
Preferably, chemiluminescent substrate includes dioxane, dioxane analog, luminol and Rumi
At least one of promise derivative.
Preferably, Magneto separate reagent further include: reagent R1；
The formula of the reagent R1 be 5~8 parts of Tris, 6~10 parts of sodium chloride, 4~6 parts of bovine serum albumin(BSA), tween
0.8~1.2 part, water complements to 1000 parts, and salt acid for adjusting pH to 7.3~7.5, the number is parts by weight.Tris provides best
PH reaction environment, and be able to maintain pH and keep stablizing, fluctuation is not generated；Sodium chloride provides salt ionic concentration, keeps life macromolecule
Natural conformation；Bovine serum albumin(BSA) provides protein protective agent；Tween, less non-specific adsorption improve magnetic bead dispersion degree.
Preferably, being combined with the magnetic of the first anti-CK-MB monoclonal antibody and the second anti-CK-MB monoclonal antibody on surface
Particle is mixed with the reagent R1, and working concentration is 10~50 μ g/mL.In this way, may make the antibody coating rate on magnetic particle
Highest.When the excessive concentration of magnetic particle, the peridium concentration for being easily reduced antibody is low；It is a large amount of free when the concentration of magnetic particle is too low
Antibody can not be coated with magnetic particle surface, cause wastage of material.
On the other hand, the embodiment of the invention also provides the preparations of creatine kinase isozyme detection kit described above
Method, comprising:
A) by magnetic particle and the first anti-CK-MB monoclonal antibody, the second anti-CK-MB monoclonal antibody reactive, surface is prepared
On be combined with the magnetic particle of the first anti-CK-MB monoclonal antibody and the second anti-CK-MB monoclonal antibody；
B) the anti-CK-MB monoclonal antibody of third or the 4th anti-CK-MB monoclonal antibody are coupled marker enzyme, prepare enzyme label
Detection antibody；
Wherein, the second anti-CK-MB monoclonal antibody and the combination activity of magnetic particle are better than the first anti-CK-MB monoclonal antibody
And the combination activity of magnetic particle；
First anti-CK-MB monoclonal antibody and the second anti-CK-MB monoclonal antibody and magnetic particle covalent bond.
In embodiments of the present invention, micro- to the first anti-CK-MB monoclonal antibody and the second anti-CK-MB monoclonal antibody and magnetic
The reaction sequence of grain is not specifically limited.It, can be with it is, in embodiments of the present invention are as follows: the first anti-CK-MB monoclonal antibody
With the second anti-CK-MB monoclonal antibody simultaneously with magnetic particle hybrid reaction, prepares and be combined with the first anti-CK-MB monoclonal on surface
The magnetic particle of antibody and the second anti-CK-MB monoclonal antibody；Or: the first anti-CK-MB monoclonal antibody is first micro- with magnetic
Grain hybrid reaction, prepares the magnetic particle that the first anti-CK-MB monoclonal antibody is combined on surface, and the second anti-CK-MB is then added
The reaction was continued for monoclonal antibody, and then prepares and be combined with the first anti-CK-MB monoclonal antibody and the second anti-CK-MB Dan Ke on surface
The magnetic particle of grand antibody.
As further preferred embodiment, the preparation side of creatine kinase isozyme detection kit of the embodiment of the present invention
Method, comprising:
A) it by magnetic particle and the first anti-CK-MB monoclonal antibody reactive, prepares and is combined with the first anti-CK-MB Dan Ke on surface
The magnetic particle of grand antibody；The magnetic particle and the second anti-CK-MB monoclonal of the first anti-CK-MB monoclonal antibody will be combined on surface
Antibody response prepares and is combined with the first anti-CK-MB monoclonal antibody and the magnetic of the second anti-CK-MB monoclonal antibody is micro- on surface
Grain；
B) the anti-CK-MB monoclonal antibody of third or the 4th anti-CK-MB monoclonal antibody are coupled marker enzyme, prepare enzyme label
Detection antibody；
Wherein, the second anti-CK-MB monoclonal antibody and the combination activity of magnetic particle are better than the first anti-CK-MB monoclonal antibody
And the combination activity of magnetic particle；
First anti-CK-MB monoclonal antibody and the second anti-CK-MB monoclonal antibody and magnetic particle covalent bond.
In the above-mentioned technical solutions, by first reacting the first anti-CK-MB monoclonal antibody with magnetic particle, second is added
Anti- CK-MB monoclonal antibody is reacted, and enables the first anti-CK-MB monoclonal antibody and the second anti-CK-MB monoclonal antibody
Coating arrives the surface of magnetic particle simultaneously, avoids causing it in the unevenness on magnetic particle surface due to the activity between antibody, conformational difference
Even distribution, stability is high, easy to operate.
Specifically, by magnetic particle and the first anti-CK-MB monoclonal antibody reactive, main purpose is elder generation in step a)
First anti-CK-MB monoclonal antibody is coated on to the surface of magnetic particle, which is chemical coating.
By magnetic particle and the first anti-CK-MB monoclonal antibody reactive, the specific implementation process of the step includes:
S11, magnetic particle is provided, centrifugation is added MES buffer and is balanced processing, obtains micro- by the magnetic of Balance Treatment
Grain；
S12, the magnetic particle Jing Guo Balance Treatment is placed in cross-linking reagent, is lived to the surface active groups of magnetic particle
Change, the magnetic particle after being activated；
S13, by after activation magnetic particle and the first anti-CK-MB monoclonal antibody be placed in MES buffer, room temperature is protected from light mixed
Even reaction；It to after reaction, is separated by solid-liquid separation, washing, confining liquid is added and is closed, washs, obtain being combined with the on surface
The magnetic particle of primary antibody CK-MB monoclonal antibody；
S14, it will be combined with the first anti-CK-MB monoclonal antibody on surface is placed in reagent R1 and store.
Further, the magnetic particle and the second anti-CK-MB Dan Ke of the first anti-CK-MB monoclonal antibody will be combined on surface
Grand antibody response, main purpose are that the second anti-CK-MB monoclonal antibody coating is first combined with the first anti-CK-MB on the surface
The surface of the magnetic particle of monoclonal antibody, the coating are chemical coating.
Magnetic particle and the second anti-CK-MB monoclonal antibody that the first anti-CK-MB monoclonal antibody is combined on surface is anti-
It answers, the specific implementation process of the step includes:
S21, take the magnetic particle that the first anti-CK-MB monoclonal antibody is combined on surface, be centrifuged, be added MES buffer into
Row Balance Treatment obtains the magnetic particle that the first anti-CK-MB monoclonal antibody is combined on the surface by Balance Treatment；
S22, the magnetic particle that the first anti-CK-MB monoclonal antibody is combined on the surface Jing Guo Balance Treatment is placed in crosslinking
In reagent, its surface active groups is activated, the first anti-CK-MB monoclonal antibody is combined on the surface after being activated
Magnetic particle；
S23, the magnetic particle and the second anti-CK-MB that the first anti-CK-MB monoclonal antibody will be combined on the surface after activation
Monoclonal antibody is placed in MES buffer, and room temperature is protected from light mixing reaction；To after reaction, be separated by solid-liquid separation, envelope is added in washing
It closes liquid to be closed, obtains the magnetic for being combined with the first anti-CK-MB monoclonal antibody and the second anti-CK-MB monoclonal antibody on surface
Particle.
S24, take the magnetic for being combined with the first anti-CK-MB monoclonal antibody and the second anti-CK-MB monoclonal antibody on surface micro-
Grain is added reagent R1 and is stored, and obtains Magneto separate reagent.
In the above-mentioned technical solutions, the present invention passes through and first reacts the first anti-CK-MB monoclonal antibody with magnetic particle, then plus
Enter the second anti-CK-MB monoclonal antibody to be reacted, so that the first anti-CK-MB monoclonal antibody and the second anti-CK-MB monoclonal
Antibody can be coated with the surface of magnetic particle simultaneously, avoid causing it on magnetic particle surface due to the activity between antibody, conformational difference
Uneven distribution, stability is high, easy to operate.
Preferably, the preparation of reagent R1 includes: to take 5~8 parts and 6~10 parts of sodium chloride of Tris to be dissolved in portion of pure water
In, 4~6 parts and 0.8~1.2 part of tween of bovine serum albumin(BSA) are added after salt acid for adjusting pH to 7.3~7.5, water complements to 1000
Part.
Preferably, the preparation of MES buffer includes: to take 20~22g MES that about 800mL pure water is added to be allowed to dissolve, later
With NaOH tune pH to 5.0, it is settled to 1L.In this way, the coating for magnetic particle provides a buffer environment, suitable pH environment can promote
Into reaction rate, and MES buffer (pH4.5~5.5) within the scope of the pH can provide powerful buffer capacity, i.e. reactant
Non- strong alkaline substance is largely added in system, will not change this pH range.Suitable ionic strength can keep oneself of large biological molecule
Right folded conformation, antibody molecule generally keep conformation in the case where cation is 50mM concentration, and ion concentration is excessively high to make molecule
Shrinkage, it is too low molecule to be made excessively to unfold, all it is unfavorable for that the immune association reaction of antibody and ligand occurs.
Preferably, cross-linking reagent is 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide salt containing 9~11mg/mL
The MES buffer of hydrochlorate (EDC) or n-hydroxysuccinimide (NHS).Cross-linking reaction efficiency is promoted in this way, improving, is shortened real
It tests the time, improves the degree of reaction of magnetic particle and antibody.
Preferably, for washing cleaning solution preparation include: weigh 2.8~3.2g of Tris, 8~10g of NaCl adds
About 800mL pure water is allowed to dissolve, and later with after HCl tune pH to 7.2, after addition 0.5g Tween-20 is mixed, is settled to 1L.
Preferably, the preparation for closed confining liquid includes: to weigh 5~10g of Tris 5~7g and NaCl addition
80ml pure water is allowed to dissolve, and later with after HCl tune pH to 6~8, it is mixed that 4.5~5.5g BSA, 0.8~1.2g Tween-20 is added
After even, it is settled to 1000mL.
As the preferred embodiment of the embodiment of the present invention, in step a), the first anti-CK-MB monoclonal antibody, second
The mass ratio of anti-CK-MB monoclonal antibody and magnetic particle is (1~2): (2~4): 20.
In step b), the anti-CK-MB monoclonal antibody of third or the 4th anti-CK-MB monoclonal antibody are coupled marker enzyme,
Its object is to prepare enzyme marker, so that the anti-CK-MB monoclonal antibody of third or the 4th anti-CK-MB monoclonal antibody are labeled
Enzyme label.
The anti-CK-MB monoclonal antibody of third is coupled marker enzyme, the step for specific implementation process include:
The anti-CK-MB monoclonal antibody of third and marker enzyme of S31, offer Jing Guo dialysis treatment, by the anti-CK-MB Dan Ke of third
Grand antibody, marker enzyme and the mixing of the first enzyme labelled reagent, room temperature are protected from light；
S32, to which second enzyme labelled reagent after reaction, is added, the reaction was continued, and desalting and purifying obtains the third of enzyme label
Anti- CK-MB monoclonal antibody.
In one embodiment, the mass ratio of the anti-CK-MB monoclonal antibody of third and marker enzyme is preferably 1:(1~5).
As the preferred embodiment of the embodiment of the present invention, the first anti-CK-MB monoclonal antibody, the second anti-CK-MB Dan Ke
Grand antibody and the mass ratio of the anti-CK-MB monoclonal antibody of third are 1:(1~2): (2~4).
In another embodiment, in order to guarantee the best coupling state between antibody and enzyme, dialysis treatment is carried out, displacement is anti-
The buffer system of body and enzyme.The detailed process of the dialysis treatment includes: to distinguish the anti-CK-MB monoclonal antibody of third and marker enzyme
It is added in two bag filters, dialyse 1~4h in PBS buffer solution (0.1mol/L, pH7.4).
In another embodiment, the first enzyme labelled reagent is the glutaraldehyde solution that mass percentage concentration is 0.1%, second enzyme
Labelled reagent is ethanol amine.
Preferably, marker enzyme is selected as alkaline phosphatase or horseradish peroxidase.
It is worth noting that, the 4th anti-CK-MB monoclonal antibody is coupled marker enzyme, the step for specific implementation process
Identical with by the anti-CK-MB monoclonal antibody coupling process of marker enzyme of third, difference is only that: by the anti-CK-MB Dan Ke of third
Grand antibody replaces with the 4th anti-CK-MB monoclonal antibody.
In step s 32, desalting and purifying is mainly used for the enzyme marker that purifying reaction obtains, and improves the pure of enzyme marker
Degree.Specifically, the step for implementation process include:
Using 20mM PBS as mobile phase, the HPLC prepacked column of flow velocity 0.5mL/min, TSK3000.Firstly, by the anti-CK- of third
The anti-CK-MB monoclonal antibody of third of MB monoclonal antibody, marker enzyme and enzyme label distinguishes loading, when recording their appearance
Between；Later, by the reaction product loading of step S32, the anti-CK-MB monoclonal antibody appearance time pair of third with enzyme label is collected
The component answered is saved in 4 DEG C after ultrafiltration concentration.
To make, the above-mentioned implementation detail of the present invention and operation can be clearly readily appreciated by one skilled in the art and the present invention is real
The progress performance for applying creatine kinase isozyme detection kit and preparation method thereof embodies significantly, right by the following examples
Implementation of the invention is illustrated.
Magnetic particle employed in following embodiment is selected as the EM1-100140 of Merck company, the first and second anti-CK-MB
Monoclonal antibody is selected as the MAB7501 and MAB7502 of Medix company, the third and fourth anti-CK-MB monoclonal antibody difference respectively
It is selected as the A27060 and A27080 of Biospecific company.
Embodiment 1
A kind of creatine kinase isozyme detection kit is present embodiments provided, preparation method includes:
1, reagent prepares
The preparation of MES buffer: taking 21.32g MES that about 800mL pure water is added to be allowed to dissolve, later extremely with NaOH tune pH
5.0, it is settled to 1L.
The preparation of cross-linking reagent: taking appropriate EDC to be added in MES buffer, and the concentration for adjusting EDC is 10mg/mL.
The preparation of confining liquid: weighing Tris 6.05g, NaCl 9.0g, and 80ml pure water is added and is allowed to dissolve, uses HCl later
After adjusting pH to 7.2,5.0g BSA, 1.0g Tween-20 is added and mixes, is settled to 1000mL.
The preparation of cleaning solution: weighing Tris 3.025g, NaCl 9g, adds about 800mL pure water to be allowed to dissolve, uses HCl later
After adjusting pH to 7.2, after 0.5g Tween-20 mixing is added, it is settled to 1L.
The preparation of reagent R1: taking Tris 6.5g, sodium chloride 9.0g, 80mL pure water added to be allowed to dissolve, and later plus hydrochloric acid is adjusted
PH to 7.2 adds 5.0gBSA and 1.0g tween, adds pure water and complements to 1L.
2, the preparation of Magneto separate reagent
1) 0.3g magnetic particle EM1-100140 (0.93 μm of partial size), centrifugation are added MES buffer and are resuspended, Magneto separate, so
It is repeated 3 times, obtains the magnetic particle by Balance Treatment.
2) magnetic particle in step 1) Jing Guo Balance Treatment is placed in the cross-linking reagent of 1mL, is mixed, room temperature is protected from light
30min removes supernatant after Magneto separate, the magnetic particle after being activated；Wherein, the cross-linking reagent of the present embodiment is containing 10mg/mL
The MES buffer of EDC.
3) magnetic particle after activation is placed in MES buffer with the anti-CK-MB monoclonal antibody of 1.5mg first, room temperature is kept away
Light mixes reaction 3h；To after reaction, remove supernatant after Magneto separate, cleaning solution is added and washes repeatedly 3 times, 5mL envelope is added later
It closes liquid and is protected from light mixing reaction overnight in room temperature, add cleaning solution and wash repeatedly 3 times, obtain being combined with the first anti-CK- on surface
The magnetic particle of MB monoclonal antibody.
4) magnetic particle for being combined with the first anti-CK-MB monoclonal antibody on the surface of step 3) is taken, is centrifuged, it is slow that MES is added
Fliud flushing is resuspended, and Magneto separate is so repeated 3 times, and obtains being combined with the first anti-CK-MB monoclonal on the surface by Balance Treatment anti-
The magnetic particle of body.
5) magnetic particle that the first anti-CK-MB monoclonal antibody is combined on the surface by Balance Treatment of step 4) is set
It in cross-linking reagent, mixes, room temperature is protected from light 30min, removes supernatant after Magneto separate, and the is combined on the surface after being activated
The magnetic particle of primary antibody CK-MB monoclonal antibody；Wherein, the MES that the cross-linking reagent of the present embodiment is the EDC containing 10mg/mL is buffered
Liquid.
6) magnetic particle and 1.5mg the of the first anti-CK-MB monoclonal antibody will be combined on the surface after step 5) activation
Secondary antibody CK-MB monoclonal antibody is placed in MES buffer, and room temperature is protected from light mixing reaction 3h；To after reaction, be separated by solid-liquid separation,
Washing is added confining liquid in room temperature and is protected from light mixing reaction overnight, obtain being combined on surface the first anti-CK-MB monoclonal antibody and
The magnetic particle of second anti-CK-MB monoclonal antibody.
7) magnetic particle that the first anti-CK-MB monoclonal antibody and the second anti-CK-MB monoclonal antibody are combined on surface is taken
Reagent R1 is added to be stored, Magneto separate reagent is obtained.
3, the preparation of enzymic-labelled antibody
1) the anti-CK-MB monoclonal antibody of 2mg third and 2mg alkaline phosphatase are separately added into two bag filters, in PBS
Dialyse 2h in buffer (0.1mol/L, pH7.4), obtains the anti-CK-MB monoclonal antibody of third and the label by dialysis treatment
Enzyme.
2) it takes the anti-CK-MB monoclonal antibody of the third by dialysis treatment and marker enzyme to mix, is slowly added to 0.2mL
0.1wt% glutaraldehyde, room temperature are protected from light 2h；Later, it is slowly added to 0.1mL ethanol amine the reaction was continued 2h, desalting and purifying obtains
The anti-CK-MB monoclonal antibody of third of enzyme label.
4, chemiluminescent substrate is selected as dioxane or its analog.
Comparative example
This comparative example provides a kind of creatine kinase isozyme detection kit, and preparation method includes:
1, reagent prepares
It is identical as the step 1 of embodiment 1, it no longer repeats one by one herein.
2, the preparation of Magneto separate reagent
1) it takes 0.3g magnetic particle EM1-100140 (0.93 μm of partial size), is centrifuged, MES buffer is added and is resuspended, Magneto separate, such as
This is repeated 3 times, and obtains the magnetic particle by Balance Treatment.
2) magnetic particle in step 1) Jing Guo Balance Treatment is placed in the cross-linking reagent of 1mL, is mixed, room temperature is protected from light
30min removes supernatant after Magneto separate, the magnetic particle after being activated；Wherein, the cross-linking reagent of the present embodiment is containing 10mg/mL
The MES buffer of EDC.
3) magnetic particle after activation is placed in MES buffer with the anti-CK-MB monoclonal antibody of 1.5mg first, room temperature is kept away
Light mixes reaction 3h；To after reaction, remove supernatant after Magneto separate, cleaning solution is added and washes repeatedly 3 times, 5mL envelope is added later
It closes liquid and is protected from light mixing reaction overnight in room temperature, add cleaning solution and wash repeatedly 3 times, obtain being combined with the first anti-CK- on surface
The magnetic particle of MB monoclonal antibody.
4) it takes the magnetic particle for being combined with the first anti-CK-MB monoclonal antibody on surface that reagent R1 is added to be stored, obtain
Magneto separate reagent.
3, the preparation of enzymic-labelled antibody
It is identical as the step 3 of embodiment 1, it no longer repeats one by one herein.
4, chemiluminescent substrate is identical as embodiment 1.
Test case
1, the creatine kinase isozyme detection kit for taking the embodiment of the present invention 1 to prepare takes comparative example 1 as sample sets
CK-MB detection kit as a control group, using chemoluminescence method examines the 20 parts of serum samples collected from hospital in parallel
It surveys, and records data, table 1 is testing result.
Minimum detection limit, that is, sensitivity for analysis, also referred to as detection lower bound or detection limit, refer to the minimum of analyte in sample, can
To be detected under defined possible condition.As shown in 1 result of table, using detection reagent provided by the embodiment of the present invention
Box checks the CK-MB in sample to be tested, and calculates its minimum detection limit.It was found that inspection provided by the embodiment of the present invention
The minimum detection limit of test agent box is down to 0.001773, hence it is evident that better than existing product.
Table 1
2, the creatine kinase isozyme detection kit for taking the embodiment of the present invention 1 to prepare takes comparative example 1 as sample sets
CK-MB detection kit as a control group, using chemoluminescence method examines the 87 parts of serum samples collected from hospital in parallel
It surveys, and records data, table 2 is testing result.Linear dependence analysis is carried out to 2 data of table, as a result such as Fig. 1, it is found that it is linear
Relative coefficient is 0.994, slope 1.01, shows that the consistency of detection kit of the present invention and existing product is good.
Table 2
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (10)
1. a kind of creatine kinase isozyme detection kit characterized by comprising Magneto separate reagent and enzymic-labelled antibody；
The Magneto separate reagent includes: that the first anti-CK-MB monoclonal antibody and the second anti-CK-MB monoclonal are combined on surface is anti-
The magnetic particle of body；
The enzymic-labelled antibody includes: the anti-CK-MB monoclonal antibody of third of enzyme label；And/or the 4th anti-CK- of enzyme label
MB monoclonal antibody；
Wherein, the described first, second, third and fourth anti-CK-MB monoclonal antibody is specific to creatine kinase isozyme respectively
Different epitopes.
2. creatine kinase isozyme detection kit according to claim 1, which is characterized in that the first anti-CK-MB
The mass ratio of monoclonal antibody, the second anti-CK-MB monoclonal antibody and the anti-CK-MB monoclonal antibody of the third is 1:(1
~2): (2~4)；Or
The first anti-CK-MB monoclonal antibody, the second anti-CK-MB monoclonal antibody and the 4th anti-CK-MB Dan Ke
The mass ratio of grand antibody is 1:(1~2): (2~4).
3. according to claim 1 to 2 described in any item creatine kinase isozyme detection kits, which is characterized in that the magnetic
The partial size of particle is 0.5~3.5 μm.
4. according to claim 1 to 2 described in any item creatine kinase isozyme detection kits, which is characterized in that described
It states in enzymic-labelled antibody, marker enzyme is alkaline phosphatase or horseradish peroxidase.
5. creatine kinase isozyme detection kit according to any one of claims 1 to 3, which is characterized in that further include:
Chemiluminescent substrate；
The chemiluminescent substrate includes dioxane, dioxane analog, luminol and Derivative of Luminol
At least one of.
6. creatine kinase isozyme detection kit according to any one of claims 1 to 3, which is characterized in that the magnetic
Separation agent further include: reagent R1；
The formula of the reagent R1 be 5~8 parts of Tris, 6~10 parts of sodium chloride, 4~6 parts of bovine serum albumin(BSA), tween 0.8~
1.2 parts, water complements to 1000 parts, and salt acid for adjusting pH to 7.3~7.5, the number is parts by weight.
7. creatine kinase isozyme detection kit according to claim 6, which is characterized in that in the Magneto separate reagent
In, be combined on the surface magnetic particle of the first anti-CK-MB monoclonal antibody and the second anti-CK-MB monoclonal antibody with it is described
Reagent R1 is mixed, and is combined with the first anti-CK-MB monoclonal antibody on the surface and the magnetic of the second anti-CK-MB monoclonal antibody is micro-
The concentration of grain is 10~15 μ g/mL.
8. a kind of preparation method of creatine kinase isozyme detection kit characterized by comprising
A) it by magnetic particle and the first anti-CK-MB monoclonal antibody, the second anti-CK-MB monoclonal antibody reactive, prepares and is tied on surface
Close the magnetic particle for having the first anti-CK-MB monoclonal antibody and the second anti-CK-MB monoclonal antibody；
B) the anti-CK-MB monoclonal antibody of third or the 4th anti-CK-MB monoclonal antibody are coupled marker enzyme, prepare the inspection of enzyme label
Survey antibody；
Wherein, the described first anti-CK-MB monoclonal antibody and the second anti-CK-MB monoclonal antibody and the magnetic particle are covalent
In conjunction with.
9. preparation method according to claim 8, which is characterized in that in step a), the first anti-CK-MB monoclonal
The mass ratio of antibody, the second anti-CK-MB monoclonal antibody and the magnetic particle is (1~2): (2~4): 20.
10. preparation method according to claim 8, which is characterized in that in step b), the anti-CK-MB Dan Ke of third
The mass ratio of grand antibody and marker enzyme is 1:(1~5)；Or, the matter of the 4th anti-the CK-MB monoclonal antibody and the marker enzyme
Amount is than being 1:(1~10).
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