CN106405087A - Goat anti-HBsAg polyclonal antibody-alkaline phosphatase conjugate, and preparation method and detection kit thereof - Google Patents
Goat anti-HBsAg polyclonal antibody-alkaline phosphatase conjugate, and preparation method and detection kit thereof Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention discloses a goat anti-HBsAg polyclonal antibody-alkaline phosphatase conjugate, and a preparation method and detection kit thereof. The conjugate is composed of a goat anti-HBsAg polyclonal antibody, a difunctional cross-linking agent as shown in a formula I which is defined in the specification and alkaline phosphatase. In the formula I, R is a substituted or unsubstituted C2-10 alkyl chain, preferably a C2-6 alkyl chain, and more preferably a C2 alkyl chain; and or R is a substituted or unsubstituted cyclohexyl group, a phenyl group or polyethylene glycol (PEG)n, wherein n may be 2, 4, 6, 8, 12 or 24. The goat anti-HBsAg polyclonal antibody-alkaline phosphatase conjugate provided by the invention has the advantages of high cross-linking efficiency, high detection sensitivity, short preparation period, simple preparation and the like; and the polyclonal antibody is not influenced in capability of recognizing and capturing antigen, and the activity and specificity of each component in the conjugate are well retained.
Description
Technical field
The present invention relates to in-vitro diagnosis immunoassay field.Specifically, the present invention relates to goat-anti HBsAg Anti-TNF-α
Cross-linking agent of body-alkaline phosphatase and preparation method thereof and the chemiluminescence detection kit comprising described cross-linking agent.
Background technology
Immunoassay be using Ag-Ab the various material of specific binding reaction detection (for example medicine, hormone,
Protein, microorganism etc.) analysis method.
The method clinically carrying out immunoassay at present mainly has colloidal gold method, immunoturbidimetry, immunofluorescence technique, chemistry
Luminescent immunoassay etc..The shortcoming of colloidal gold method is accurately to carry out quantitation, and differences between batches are larger；Immunoturbidimetry
Shortcoming be that reagent sensitivity is poor；It is longer to there is the reaction time in immunofluorescence technique, higher to environmental requirement, is easily subject to air dirt
Angstrom impact, the shortcomings of using rare earth label.Chemiluminescence immunoassay is a kind of relatively advanced immunological method, has
Reaction time is short, sensitivity is high, specificity good, range of linearity width, reproducible the advantages of, be therefore that market application foreground is best
And diagnose most reliable methodology for chemiluminescence immunoassay.
Chemiluminescence immunoassay needs using antibody and enzyme conjugate, the cross-linking agent of such as alkaline phosphatase, including
Enzyme labeled immunoassay Lysozyme (ImmunoglobulinG, IgG) polyclonal antibody, enzyme mark IgG monoclonal antibody, enzyme mark are exempted from
Epidemic disease globulin M (ImmunoglobulinM, IgM) monoclonal antibody or the antigen of enzyme mark.Relatively common in enzyme marker it is
Enzyme marks IgG, and it is by suitable crosslinking agent and suitable chemically or physically processing method, IgG to be tagged to above enzyme.Enzyme
Labelled antibody be mainly used in enzyme linked immunosorbent assay (ELISA) (enzyme linked immunosorbent assay, ELISA) and
Quantitative and qualitative point of chemiluminescence enzyme immunoassay (chemiluminescence enzyme immunoassay, CLIA)
Analysis.
At present enzyme mark the method for IgG mainly have following three kinds (BioconjugateTechnoqies, 2013 the 3rd
Version)：
1. Over-voltage protection：For marking the higher enzyme of sugar content, this method is applied to horseradish peroxidase and sugary
Recombinant basic phosphatase.Cardinal principle is the oxidisability using sodium periodate, and the polysaccharide group of oxidizing ferment molecular surface is formed
Easily by the aldehyde groups of nucleopilic reagent attack, in enzyme labelling experiment, commonly referred nucleopilic reagent is exactly to rely on albumen to be marked
Primaquine group on histidine residue is that is to say, that form schiff base (Schiff after the enzyme that aoxidized by sodium periodate and IgG crosslinking
Base), thus reaching cross-linking effect.The method shortcoming is that the operation cycle is long, and process is complicated, right in labeling process
Enzymatic activity has certain damage, and the schiff base (Schiff base) being cross-linked to form is unstable, needs to add reducing agent could be formed
Stable comparative chemistry key.
2. carbodiimide/N-hydroxy-succinamide method：Carboxyl can be cross-linked with each other with amino by the method using one kind
Zero-length cross-linkers (carbodiimide class hydrochloride), carbodiimide first with immunoglobulin G to be marked on carboxyl base
Group's reaction forms one can be with the O- acyl isourea intermediate of amino reaction, and this intermediate can rapidly and an amino group shape
Become amido link and discharge an isourea by-product not having reactivity.Intermediate is very unstable in aqueous, needs
N-hydroxy-succinamide is depended on to be stabilized.The intermediate not reacted with amino can hydrolyze generation carboxyl and N- replaces urine
Element.The shortcoming of the method is that do not have the zero-length cross-linkers of brachium because labeling process uses, and leads to formed enzyme to resist
Body cross-linking agent meeting Existential Space steric hindrance, so that the antibody of marker enzyme is necessarily hindered for the specific reaction of determinand,
The experiment of mark polyclonal antibody affect particularly evident, thus leading to the sensitivity that immunoassay is tested low.
3. glutaraldehyde method：The method is also relatively common in alkali phosphatase enzyme mark experiment, and glutaraldehyde contains two easily quilts
The aldehyde groups of nucleopilic reagent attack, in labeling process, with glutaraldehyde as bridge, make on enzyme and IgG molecule to be marked respectively
Amino group combines, thus forming conjugate.The shortcoming of glutaraldehyde method is the self-crosslinking phenomenon being susceptible to albumen, can form list
The polymer of one component, leads to the activity of enzyme marker and labeling effciency relatively low, the sensitivity of impact immunoassay experiment.
Therefore, the quality of enzymic-labelled antibody directly affects the last measurement result of immunoassay, is that impact chemiluminescence is exempted from
The key technology of epidemic disease reagent detection sensitivity.The quality of enzyme conjugate depends on the activity of enzyme itself, the specificity of IgG and advanced
Preparation technology.
Chemical illuminating reagent quality on the market is uneven at present, affects clinical detection.
Therefore, this area be badly in need of that sensitivity is high, the activity of each component of cross-linking agent after mark and specificity keep good and
And the enzyme labeled immunoassay globulin of short preparation period.
Content of the invention
It is an object of the invention to provide a kind of goat-anti HBsAg polyclonal antibody crosslinking alkaline phosphatase enzyme conjugate, described
Cross-linking agent should possess cross-linking efficiency height, and the ability of polyclonal antibody identification capture antigen is unaffected, and detection sensitivity is high, wherein
The activity of each component and specificity keep good, and short preparation period, easy and simple to handle the advantages of.
It is a further object of the present invention to provide comprising described goat-anti HBsAg polyclonal antibody crosslinking alkaline phosphatase enzyme conjugate
Detection kit.
In a first aspect, the present invention provides a kind of antibody-alkaline phosphatase enzyme conjugate, described cross-linking agent is by goat-anti HBsAg
Bi-functional cross-linking agent shown in polyclonal antibody, formula I or its salt and alkaline phosphatase are constituted,
In Formulas I, R is substituted or unsubstituted C2-10Alkyl chain, preferably C2-6Alkyl chain, more preferably C2Alkyl chain, alkyl by
Optionally substituted selected from following substituent：Halogen, hydroxyl, sulfenyl, cyano group, nitro, alkyl, aryl, alkoxyl, heterocyclic radical, halogen
Substituted alkyl and carboxyl；
Or, in Formulas I, R is substituted or unsubstituted cyclohexyl, phenyl, polyethylene glycol (PEG)n, wherein n can be
2nd, 4,6,8,12,24, preferably n are 2,4,6, and more preferably n is 2.
In a particular embodiment, R is polyethylene glycol (PEG)2.
In second aspect, the present invention provides the preparation of the antibody-alkaline phosphatase enzyme conjugate described in first aspect present invention
Method, including：
1) bi-functional cross-linking agent shown in Formulas I is used to mark alkaline phosphatase enzyme molecule；
2) use protease hydrolyzed goat-anti HBsAg polyclonal antibody molecule, thus obtaining Fab fragment and Fc fragment；
3) by step 1) the alkaline phosphatase enzyme molecule of the bi-functional cross-linking agent that obtains mark and step 2) obtain through albumen
The goat-anti HBsAg polyclonal antibody molecular docking reaction of enzyme enzymolysis, the goat-anti HBsAg that formation is marked with alkaline phosphatase is polyclonal
Antibody.
In a particular embodiment, described protease is papain.
In a preferred embodiment, the preparation method of the antibody of the present invention-alkaline phosphatase enzyme conjugate is in following technique
Implement in parameter area：
In step 1) in, the molal quantity of described bi-functional cross-linking agent is 5-500 times of alkaline phosphatase enzyme molecule molal quantity, excellent
Select 5-50 times；
In step 2) in, described protease, such as Papain enzyme molecule molal quantity are that goat-anti HBsAg polyclonal antibody divides
0.1-100 times of sub- molal quantity, preferably 0.1-10 times；
In step 3) in, the alkaline phosphatase enzyme molecule molal quantity of described bi-functional cross-linking agent mark and through papain enzyme
The ratio of the goat-anti HBsAg polyclonal antibody molecule molal quantity of solution is 1:1-20:1, preferably 1:1-5:1.
In the third aspect, the present invention provides the antibody-alkaline phosphatase enzyme conjugate described in first aspect present invention in preparation
Purposes in hepatitis b virus s antigen (HBsAg) detection kit.
In fourth aspect, the present invention provides kind of hepatitis b virus s antigen (HBsAg) detection kit, described reagent
Box is equipped with the antibody described in first aspect present invention-alkaline phosphatase enzyme conjugate.
In a particular embodiment, described kit, equipped with reagent 1 (R1), comprises anti-HBs list
Anti- coated magnetic particle, buffer solution, preservative；Reagent 2 (R2), comprises the antibody-alkali described in calf serum, claim 1 or 2
Acid phosphatase cross-linking agent, buffer solution, preservative；With calibration product A, comprise calf serum, hepatitis b virus s antigen, buffering
Liquid, preservative.
At the 5th aspect, the present invention provides the preparation method of the detection kit described in fourth aspect present invention, described side
Method includes：
Antibody described in first aspect present invention-alkaline phosphatase enzyme conjugate is made detection kit.
At the 6th aspect, the present invention provides a kind of detection method, and described detection method includes utilizing first aspect present invention
Detection kit vitro detection human serum described in described antibody-alkaline phosphatase enzyme conjugate or third aspect present invention or blood
Hepatitis b virus s antigen (HBsAg) in slurry sample.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and having in below (eg embodiment)
Can be combined with each other between each technical characteristic of body description, thus constituting new or preferred technical scheme.As space is limited, exist
This no longer repeats one by one.
Brief description
Fig. 1 shows and marks alkaline phosphatase using bi-functional cross-linking agent (succinimide ester-maleimide crossing linking reagent)
The process of enzyme molecule；Wherein, molecular formula 1 represents succinimide ester-maleimide bi-functional cross-linking agent；In molecular formula 2, R1
Represent alkaline phosphatase enzyme molecule, NH2It is the primaquine group on alkaline phosphatase molecular surface lysine residue；Molecular formula 3 is N-
HOSu NHS, the accessory substance of cross-linking reaction, it can be removed by desalting steps；Molecular formula 4 is to be marked with Malaysia acyl
The alkaline phosphatase enzyme molecule of imine group, this group can be reacted with mercapto groups；
Fig. 2 shows the goat-anti HBsAg polyclonal antibody through protease hydrolyzed, respectively Fab section (containing mercapto groups)
With Fc section；
Fig. 3 shows and will mark alkaline phosphatase through bi-functional cross-linking agent (succinimide ester-maleimide crossing linking reagent)
Enzyme molecule is reacted with the polyclonal antibody molecular docking through protease hydrolyzed, thus obtaining the goat-anti being marked with alkaline phosphatase
HBsAg polyclonal antibody；Wherein molecular formula 4 is the alkaline phosphatase enzyme molecule being marked with maleimide base group, and this group can
React with mercapto groups；In molecular formula 5, R2Represent the Fab section of the goat-anti HBsAg polyclonal antibody through protease hydrolyzed, it
Containing the mercapto groups (- SH) being exposed to molecular surface；Molecular formula 6 represents many grams of the goat-anti HBsAg that marked alkaline phosphatase
Grand antibody, R1Represent alkaline phosphatase enzyme molecule, R2Represent the Fab fragment of goat-anti HBsAg polyclonal antibody；
Fig. 4 is the structural representation of the succinimide ester-maleimide crossing linking reagent used by the present invention.
Specific embodiment
Inventor through extensively in-depth study, it was unexpectedly found that using specific bi-functional cross-linking agent and specific
Protease hydrolyzed technology combine, when being marked alkaline phosphatase enzyme molecule and polyclonal antibody molecule, cross-linking efficiency is high, obtains
To be marked with alkaline phosphatase goat-anti HBsAg polyclonal antibody is stable, detection specificity and sensitivity are high and react behaviour
Make easy, the activity of enzyme marker and labeling effciency are remarkably improved.Complete the present invention on this basis.
The bi-functional cross-linking agent of the present invention
As described above, inventor is it was unexpectedly found that utilize specific bi-functional cross-linking agent and specific protease hydrolyzed
Technology combines, and when being marked alkaline phosphatase enzyme molecule with polyclonal antibody molecule, can produce significant technique effect.
Bi-functional cross-linking agent used by the present invention is succinimide ester-maleimide crossing linking reagent, and it is shown in formula I：
In Formulas I, R is substituted or unsubstituted C2-10Alkyl chain, preferably C2-6Alkyl chain, more preferably C2Alkyl chain；Alkyl by
Optionally substituted selected from following substituent：Halogen, hydroxyl, sulfenyl, cyano group, nitro, alkyl, aryl, alkoxyl, heterocyclic radical, halogen
Substituted alkyl and carboxyl；
Or, in Formulas I, R is substituted or unsubstituted cyclohexyl, phenyl, polyethylene glycol (PEG)n, wherein n can be
2nd, 4,6,8,12,24, preferably n are 2,4,6, and more preferably n is 2.
The common knowledge based on the teachings of the present invention and this area for the those skilled in the art, can determine R as needed
Specifically chosen.For example, in a particular embodiment, the present invention using bi-functional cross-linking agent be the succinyl shown in Formulas I
Imines ester-maleimide bi-functional cross-linking agent, wherein R is polyethylene glycol (PEG)2.
Goat-anti HBsAg polyclonal antibody-alkaline phosphatase enzyme conjugate and preparation method thereof
The antibody of the present invention-alkaline phosphatase enzyme conjugate is by goat-anti HBsAg polyclonal antibody, above-mentioned bi-functional cross-linking agent
Constitute with alkaline phosphatase.
The goat-anti HBsAg polyclonal antibody-alkaline phosphatase enzyme conjugate of the present invention is prepared as described below：
Step one：
Using bi-functional cross-linking agent (succinimide ester-polyethylene glycol (PEG)2- maleimide crossing linking reagent) mark alkali
Acid phosphatase molecule, one end succinimide ester of this crosslinking agent can be with lysine residue on alkaline phosphatase molecular surface
Primary amine group reacts, thus forming the alkaline phosphatase enzyme molecule being marked with maleimide base group.
As shown in figure 1, wherein, molecular formula 1 represents the difunctional crosslinking of succinimide ester-maleimide to above-mentioned steps one
Agent；R in molecular formula 21Represent alkaline phosphatase enzyme molecule, NH2It is exactly the primaquine on alkaline phosphatase molecular surface lysine residue
Group；Molecular formula 3 is N-hydroxy-succinamide, and it is the accessory substance of cross-linking reaction, can be removed by desalting steps；Molecule
Formula 4 represents the alkaline phosphatase enzyme molecule being marked with maleimide base group, and this group can be reacted with mercapto groups；
Step 2：
Antibody molecule can be digested with suitable protease (preferably papain), by goat-anti HBsAg polyclonal antibody
Obtain Fab fragment and Fc fragment after enzymolysis, Fab fragment contains complete antigen recognition region thus not impact is last
The activity of cross-linking products.Meanwhile, the polyclonal antibody molecule through protease hydrolyzed contains the sulfydryl base being exposed to molecular surface
Group, this group has the characteristic that can react with maleimide base group.
Fig. 2 shows the goat-anti HBsAg polyclonal antibody through protease hydrolyzed, and respectively Fab section is (containing sulfydryl base
Group) and Fc section.
Step 3：
Finally, will be through bi-functional cross-linking agent (succinimide ester-polyethylene glycol (PEG)2- maleimide crossing linking reagent)
The alkaline phosphatase enzyme molecule of mark, is reacted with the polyclonal antibody molecular docking through papain enzymolysis, through certain
Mixed in molar ratio, forms the goat-anti HBsAg polyclonal antibody being marked with alkaline phosphatase under specific reaction condition.
As shown in figure 3, wherein, molecular formula 4 is that the alkaline phosphatase being marked with maleimide base group divides to above-mentioned steps three
Son, this group can be reacted with mercapto groups；R in molecular formula 52Represent polyclonal through the goat-anti HBsAg of protease hydrolyzed
The Fab section of antibody, it contains the mercapto groups (- SH) being exposed to molecular surface；Molecular formula 6 expression marked alkaline phosphatase
Goat-anti HBsAg polyclonal antibody, wherein R1Represent alkaline phosphatase enzyme molecule, R2Represent the Fab piece of goat-anti HBsAg polyclonal antibody
Section.
The preparation method of goat-anti HBsAg polyclonal antibody-alkaline phosphatase enzyme conjugate mentioned above can be in following work
Implement in skill parameter area：
Step one：Succinimide ester-polyethylene glycol (PEG) is added in containing a certain amount of alkaline phosphatase enzyme solutions2-
Maleimide crossing linking reagent, its alkaline phosphatase solution concentration is 1mg/mL-10mg/mL, preferably 2mg/mL-5mg/mL；Alkali
Acid phosphatase solution need to be saved in PBS cushioning liquid, and pH value of buffer solution is 7.0-7.5 (preferably pH7.2-7.3)；This crosslinking
The molal quantity ratio of the molal quantity of agent molecule and alkaline phosphatase enzyme molecule is 2:1-30:1, preferably 10:1-20:1；Reaction condition is
Room temperature；In 1 hour after adding crosslinking agent, (preferably 15-30 minute) need to carry out purification process, and the mode of purifying can be pure for ultrafiltration
Change, desalting column purifies or dialysis purification, preferably ultrafiltration purification；Alkaline phosphatase enzyme solutions after the completion of purifying can be in 2-8 DEG C of environment
Middle preservation.
Step 2：Papain, wherein goat-anti is added in containing a certain amount of goat-anti HBsAg Anti-TNF-α liquid solution
The concentration of HBsAg Anti-TNF-α liquid solution is 1mg/mL-10mg/mL, preferably 2mg/mL-3mg/mL；Goat-anti HBsAg Anti-TNF-α
Body need to be saved in sodium-acetate buffer, and pH value of buffer solution is 4.0-5.5 (preferably pH4.0-4.5)；Papain enzyme molecule
Molal quantity and goat-anti HBsAg polyclonal antibody molecule molal quantity ratio be 1:10-100:1, preferably 1:10-10:1；Reaction
Condition is 37 DEG C；In 12 hours after adding pepsin, (preferably 8-12 hour) need to carry out purification process, and way of purification can be
Ultrafiltration purification, desalting column purify or dialysis purification, preferably ultrafiltration purification；Goat-anti HBsAg polyclonal antibody after the completion of purifying can
2-8 DEG C of environment preserves.
Step 3：The alkaline phosphatase enzyme solutions that obtain above-mentioned steps one and step 2 and goat-anti HBsAg polyclonal antibody
Reacted after mixing, alkaline phosphatase enzyme molecule molal quantity and goat-anti HBsAg polyclonal antibody molecule molal quantity ratio are 1:1-
20:1, preferably 1:1-5:1；Reaction temperature is 4-37 DEG C；Reaction time is 1.5-18 hour；Reaction time can get after terminating
Goat-anti HBsAg polyclonal antibody-alkaline phosphatase enzyme conjugate.
The detection kit of the present invention
The goat-anti HBsAg polyclonal antibody-alkaline phosphatase enzyme conjugate of the present invention can be used for preparing HBsAg detection reagent
Box or for enzyme linked immunosorbent assay (ELISA) or be used for chemiluminescence immune assay.Therefore, the present invention further provides a kind of detect
Kit, described kit is equipped with antibody mentioned above-alkaline phosphatase enzyme conjugate.
In view of the teachings of the present invention and prior art, those of ordinary skill in the art can determine detection examination as herein described
Other components in agent box, for example, in a particular embodiment, the detection kit component of the present invention includes：Reagent 1 (R1)
(the coated magnetic particle of anti-HBs monoclonal antibody, buffer solution, preservative)；Reagent 2 (R2) (calf serum, right
Require the antibody-alkaline phosphatase enzyme conjugate described in 1 or 2, buffer solution, preservative)；Calibration product A (calf serum, hepatitis B
Viral surface antigen, buffer solution, preservative).
The detection kit of the present invention can be prepared as described below, including：By the goat-anti HBsAg polyclonal antibody of the present invention-
Alkaline phosphatase enzyme conjugate makes detection kit.In a particular embodiment, the preparation side of the detection kit of the present invention
Method includes for following reagent making detection kit：(the coated magnetic of anti-HBs monoclonal antibody is micro- for reagent 1 (R1)
Grain, buffer solution, preservative)；Reagent 2 (R2) (calf serum, the antibody described in claim 1 or 2-alkaline phosphatase enzyme conjugate,
Buffer solution, preservative)；Calibration product A (calf serum, hepatitis b virus s antigen, buffer solution, preservative).Described detection
Operation instructions and the appropriate device for liquid relief is can further be provided with kit.
The detection method of the present invention
Goat-anti HBsAg polyclonal antibody-alkaline phosphatase enzyme conjugate and the detection kit comprising it in the present invention
On the basis of, the present invention also provides a kind of hepatitis b virus s antigen clearly or in plasma sample for detection detection Human Blood In Vitro
(HBsAg) method, methods described utilizes the goat-anti HBsAg polyclonal antibody-alkaline phosphatase of the present invention to hand over mark cross-linking agent,
Or the goat-anti HBsAg polyclonal antibody-alkaline phosphatase enzyme conjugate of the present invention and detection kit detection Human Blood In Vitro clear or
Hepatitis b virus s antigen (HBsAg) in plasma sample.
Those skilled in the art know, the detection method of the present invention can be used for diagnostic purpose, but is not limited to diagnostic purpose,
For example, can be used for scientific research purpose etc..
Advantages of the present invention：
1. the method for the crosslinked alkaline phosphatase of the present invention and goat-anti HBsAg polyclonal antibody does not result in each albumen itself
It is produced from crosslinking phenomena, thus improve both cross-linking efficiencies；
2. will not occur to identify capture because of the sterically hindered polyclonal antibody leading to using the bi-functional cross-linking agent of the present invention
The defect that the ability of antigen reduces；
3. to possess excellent detection sensitive for the goat-anti HBsAg polyclonal antibody crosslinking alkaline phosphatase enzyme conjugate of the present invention
Degree；
4. the present invention goat-anti HBsAg polyclonal antibody crosslinking alkaline phosphatase enzyme conjugate in, each component active, special
Property and stability keep good；
5. the short preparation period of goat-anti HBsAg polyclonal antibody crosslinking alkaline phosphatase enzyme conjugate of the present invention, operation letter
Just.
Embodiment
Embodiment 1
10mg alkaline phosphatase is dissolved in 4mL 10Mm PBS (pH7.5), makes alkaline phosphatase enzyme solutions eventually
Concentration is 2.5mg/mL；Simultaneously by 5mg succinimide ester-polyethylene glycol (PEG)2- maleimide crossing linking reagent is dissolved in 1mL
So that the final concentration of 5mg/mL of crosslinking agent in ultra-pure water；150 μ l succinimides are added in above-mentioned alkaline phosphatase enzyme solutions
Ester-polyethylene glycol (PEG)2- maleimide amine aqueous solution.After 25 DEG C are reacted 15 minutes, with the ultrafiltration of 15mL 10KD molecular cut off
Pipe, using 10Mm PBS (pH7.5) as changing liquid buffer solution, 5000rpm ultrafiltration 3 times, removes free crosslinking agent and friendship
Connection byproduct of reaction, obtains marking succinimide ester-polyethylene glycol (PEG)2The alkaline phosphatase enzyme solutions of-maleimide.
10mg goat-anti HBsAg polyclonal antibody is dissolved in 3mL 15mM sodium-acetate buffer (pH4.0), in above-mentioned goat-anti
2mg papain is added in HBsAg Anti-TNF-α liquid solution.After 37 DEG C of reactions 8 as a child, retain molecule with 15mL 10KD
The super filter tube of amount, using 10Mm PBS (pH7.5) as changing liquid buffer solution, 5000rpm ultrafiltration 3 times, obtains through enzymolysis
Goat-anti HBsAg polyclonal antibody.
After goat-anti HBsAg Anti-TNF-α liquid solution after above-mentioned process and alkaline phosphatase enzyme solutions are mixed, you can obtain
Cross-linking agent.
Embodiment 2
With reference to BioconjugateTechnoqies, glutaraldehyde method in the third edition in 2013, Over-voltage protection and carbon two
Imines/N-hydroxy-succinamide method, prepares the goat-anti HBsAg Anti-TNF-α liquid solution of alkali phosphatase enzyme mark.By embodiment 1
In goat-anti HBsAg polyclonal antibody-alkaline phosphatase enzyme conjugate solution of obtaining and with glutaraldehyde method, Over-voltage protection and carbon
The goat-anti HBsAg polyclonal antibody that diimine/N-hydroxy-succinamide method obtains-alkaline phosphatase enzyme conjugate solution is used for second
The chemiluminescence enzyme immunoassay detection of HBsAg B.
The sample to be tested of this experiment is the hepatitis b virus s antigen sample of 5IU/mL, the hepatitis B of 0IU/mL
Malicious surface antigen sample, solid phase is to be coated with the magnetic particle of anti-HBsAg, and enzyme marker is the goat-anti of above-mentioned each method preparation
HBsAg polyclonal antibody-alkaline phosphatase enzyme conjugate, chemical luminous substrate is that Lumiphos530 is (public purchased from Beckman Kurt
Department), carry out chemiluminescence enzyme with C1800 type Full-automatic chemiluminescence immunoassay analysis meter (Shanghai Kehua Experimental System Co., Ltd.)
Immunoassay detects；Instrument arrange parameter is sample application amount 100uL, magnetic particle sample-adding amount 50uL, enzyme marker solution 50uL,
Chemical luminous substrate 200uL, instrument is set to one-step method reaction；Measured with 3 parts of samples, average, the results are shown in Table 1.
5IU/mL sample gained luminous value is bigger, and 0IU/mL gained luminous value is less, illustrates that the sensitivity of enzyme marker is high, specificity
Good.
The goat-anti HBsAg polyclonal antibody of table 1. each method mark-alkaline phosphatase enzyme conjugate detection luminous value contrast knot
Really
As seen from the results in Table 1, using the goat-anti HBsAg polyclonal antibody-alkaline phosphatase of the marking process preparation of the present invention
Enzyme conjugate, the activity of reagent is significantly better than the labeling method of routine.
Embodiment 3
The goat-anti HBsAg polyclonal antibody-alkaline phosphatase enzyme conjugate of preparation in embodiment 1 and embodiment 2 is configured to
Reagent 2 (R2) working solution of hepatitis b virus s antigen (HBsAg) detection kit (chemoluminescence method), by above-mentioned work
Make solution to place 6 days under 4 DEG C and 37 DEG C of environment respectively, carry out hepatitis type B virus table also according to the method in embodiment 2
The chemiluminescence enzyme immunoassay detection of face antigen.This experiment sample to be tested is the hepatitis b virus s antigen sample of 5IU/mL
This, measured with 3 parts of samples, average, the results are shown in Table 2.
The thermal acceleration stability comparing result of the enzyme conjugate of table 2. each method mark
As seen from the results in Table 2, using the goat-anti HBsAg polyclonal antibody-alkaline phosphatase of the marking process preparation of the present invention
Enzyme conjugate, the stability of reagent is substantially better than the labeling method of routine.
Therefore, the unexpected of the present invention has the technical effect that using the crosslinked specific enzyme of specific bi-functional cross-linking agent,
That is, alkaline phosphatase and specific antibody, i.e. the goat-anti HBsAg of goat-anti HBsAg polyclonal antibody, preferably Papain ferment treatment
Obtain under polyclonal antibody.
The all documents referring in the present invention are all incorporated as reference in this application, independent just as each document
It is incorporated as with reference to like that.In addition, it is to be understood that after the above-mentioned instruction content having read the present invention, those skilled in the art can
To make various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Claims (9)
1. a kind of antibody-alkaline phosphatase enzyme conjugate, described cross-linking agent is double shown in goat-anti HBsAg polyclonal antibody, formula I
Functional cross-link agent or its salt and alkaline phosphatase are constituted,
In Formulas I, R is substituted or unsubstituted C2-10Alkyl chain, preferably C2-6Alkyl chain, more preferably C2Alkyl chain, alkyl is by being selected from
Following substituent is optionally substituted：Halogen, hydroxyl, sulfenyl, cyano group, nitro, alkyl, aryl, alkoxyl, heterocyclic radical, alkyl halide
Base and carboxyl；
Or, in Formulas I, R is substituted or unsubstituted cyclohexyl, phenyl, polyethylene glycol (PEG)n, wherein n can for 2,4,6,
8th, 12,24, preferably n are 2,4,6, and more preferably n is 2.
2. antibody as claimed in claim 1-alkaline phosphatase enzyme conjugate is it is characterised in that R is polyethylene glycol (PEG)2.
3. the preparation method of antibody as claimed in claim 1 or 2-alkaline phosphatase enzyme conjugate, including：
1) bi-functional cross-linking agent shown in Formulas I is used to mark alkaline phosphatase enzyme molecule；
2) use protease hydrolyzed goat-anti HBsAg polyclonal antibody molecule, thus obtaining Fab fragment and Fc fragment；
3) by step 1) the alkaline phosphatase enzyme molecule of the bi-functional cross-linking agent that obtains mark and step 2) obtain through protease enzyme
The goat-anti HBsAg polyclonal antibody molecular docking reaction of solution, forms the goat-anti HBsAg Anti-TNF-α being marked with alkaline phosphatase
Body.
4. preparation method as claimed in claim 3 is it is characterised in that described protease is papain.
5. antibody as claimed in claim 1 or 2-alkaline phosphatase enzyme conjugate is preparing hepatitis b virus s antigen
(HBsAg) purposes in detection kit.
6. a kind of hepatitis b virus s antigen (HBsAg) detection kit, described kit is equipped with claim 1 or 2 institute
The antibody stated-alkaline phosphatase enzyme conjugate.
7. detection kit as claimed in claim 5 is it is characterised in that described kit, equipped with reagent 1 (R1), comprises B-mode
The coated magnetic particle of hepatitis viruse surface antibody monoclonal antibody, buffer solution, preservative；Reagent 2 (R2), comprises calf serum, right will
Ask antibody-alkaline phosphatase enzyme conjugate described in 1 or 2, buffer solution, preservative；With calibration product A, comprise calf serum, B-mode liver
Scorching viral surface antigen, buffer solution, preservative.
8. the preparation method of the detection kit as described in claim 5 or 6, methods described includes：
Antibody described in claim 1 or 2-alkaline phosphatase enzyme conjugate is made detection kit.
9. a kind of detection method, described detection method is included using the antibody described in claim 1 or 2-alkaline phosphatase enzyme crosslinking
Detection kit vitro detection human serum described in thing or claim 5 or 6 or the hepatitis B virus surface in plasma sample
Antigen (HBsAg).
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| CN111721931A (en) * | 2020-06-23 | 2020-09-29 | 广州万孚生物技术股份有限公司 | Solid-phase time-resolved fluorescence immune marker and preparation method and application thereof |
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