CN110133271A

CN110133271A - A method of antibody or its antigen-binding fragment are covalently bond to particle surface

Info

Publication number: CN110133271A
Application number: CN201810133293.9A
Authority: CN
Inventors: 朱驰
Original assignee: Beijing Heroic Bioengineering Ltd By Share Ltd
Current assignee: Beijing Heroic Bioengineering Ltd By Share Ltd
Priority date: 2018-02-09
Filing date: 2018-02-09
Publication date: 2019-08-16
Anticipated expiration: 2038-02-09
Also published as: CN115639361A; CN110133271B

Abstract

This application involves a kind of methods that antibody or its antigen-binding fragment are covalently bond to particle surface.Specifically, the structure of the particle is shown below this application involves the particle that a kind of surface is combined with antibody: antibody-bridging albumen-particle.This structure be conducive to maintain antibody activity, while bridging albumen be formed by it is long-armed facilitate reduce antibody steric effect, convenient for and antigen combination.Particle prepared by the application and its reagent make accuracy and sensitivity be improved.

Description

A method of antibody or its antigen-binding fragment are covalently bond to particle surface

Technical field

This application involves clinical examination fields.More particularly to a kind of antibody or its antigen-binding fragment are covalently bond to The method on grain surface.

Background technique

Anti- Miao Leguan hormone (anti-Mullerian hormone, AMH) is transforming growth factor β (TGF-β) superfamily Member.AMH is as secreted by the granular cell of ovarian follicle and little Dou ovarian follicle before the immature Sertoli cell of testis and ovary sinus A kind of glycoprotein.Entovarial small Antral follicles amount is more, and the concentration of AMH is just higher；Conversely, when ovarian follicle is with age and various Factor gradually uses up, and AMH concentration can also decrease；When close to menopause, AMH is just gradually in 0.In consideration of it, AMH can be used as it is pre- Survey the marker of ovarian reserve.

The clinical meaning that AMH is examined is:

(1) Ovary reserve is evaluated: when women is born, AMH concentration level is very low in serum, AMH concentration after puberty It peaks, and maintains high level in the entire childbearing age, later as age and various factors gradually use up, AMH concentration It can decrease, until post menopausal will be unable to measure.As it can be seen that AMH value can be in fluctuations with the variation of ovarian function.AMH Index is higher, illustrates that the quantity in stock of ovum is bigger, and fecundity is naturally just relatively strong.When AMH is reduced, ovary is represented just in aging, Indicate the decline of female fertility.It can the more acurrate decline for reflecting age correlation Ovary reserve earlier by detection AMH.

(2) Stein-Leventhal syndrome (PCOS): PCOS is the highest reproductive endocrine disorders of incidence in crowd, but Current diagnosis method is inaccurate.Sinus ovarian follicle dyscalculia, is affected by subjective factor under traditional ultrasound.Clinical research hair Existing, PCOS patients serum's AMH level is higher than 2 to 3 times of normal level, and serum AMH can be used to diagnose PCOS and assess its curative effect, Its sensitivity 67%, specificity 92%.Therefore, AMH concentration is that ovary Antral follicles purpose accurately reflects, serum AMH level takes Small antral follicle count counting can be used as one of PCOS diagnostic criteria under generation ultrasound.

(3) premature ovarian failure (POF): POF patients serum's AMH level is significantly lower than normal women of the same age, with menopause Women is on close level.And since AMH is not only generated by little Dou ovarian follicle, even if the small ovarian follicle that colorful ultrasonic can't detect equally may be used With secretion, and it is aperiodic dependence, is not involved in the negative-feedback regu- lation of hypothalamic pituitary gonadal axis.Therefore AMH is expected into For the good clinical indices of early prediction POF morbidity.

(4) assisted reproductive technology: AMH level can predict ovary responsiveness, and identification has ovarian hyperstimulation syndrome wind The women of danger, can judge the dosage using promoting ovulation drug according to AMH numerical value.Research finds to receive the trouble of IVF/ICSI treatment The more high then rate of fertilization of AMH level is higher in person's serum and liquor folliculi, and AMH is likely to become the index of prediction rate of fertilization.In addition to this, AMH predicts that OHSS is better than age and BMI, and the basic AMH that OHSS patient occurs is 6 times high compared with normal person, prompts AMH pre- in advance Survey OHSS.

(5) granulosa cell tumor of ovary: because only being expressed by granular cell due to AMH, expression is by ovary primary follicle Determine that this illustrates that AMH can be used as a marker of granulosa cell tumor of ovary with the granular cell of ovarian follicle before sinus.It is reported that AMH expression is the positive in 76% to 93% granulosa cell tumor of ovary, and the tumor size of AMH and granulosa cell tumor of ovary and Differentiation degree has independent correlates, and has certain predictive value to the recurrence of granulosa cell tumor of ovary.

(6) age that prediction menopause occurs: AMH level and age of meuopause are closely related, and serum AMH level is premenopausal 5 Year gradually decreases, therefore it is now recognized that serum AMH can predict age of meuopause.

(7) sexual development abnormelity: diagnosis infant can be assisted with the presence or absence of the development such as Gonad dysplasia, sex premature, cryptorchidism Disorder disease.If serum AMH level is very low or inspection does not measure, height prompts anorchia tissue.Studies have shown that is in cryptorchidism In patient, it is horizontal normal there is 50% patients serum AMH, and cryptorchidism patients serum's AMH level is significantly lower than monorchism patient.

(8) related disease assistant diagnosis: serum AMH level is also related to a series of autoimmune diseases, because such The ovarian function of patient is often also damaged.Such as autoimmune thyroid disease, systemic loupus erythematosus and Crohn disease Deng.In addition to this, the detection of AMH can be used for predicting and assessing the treatment such as radiotherapy, chemotherapy and operation on ovary to her ovaries' function The influence of energy, the selection of guiding treatment scheme.

Chinese patent application CN107192827A discloses a kind of colloidal gold detection device of anti-Miao Leguan hormone.Detection dress Nitrocellulose filter is equipped in setting, one end of nitrocellulose filter connects sample pad, and the other end connects water absorption pad.Position on the shell It is equipped with sample aperture in the position of sample pad, peep hole is equipped with positioned at the position of nitrocellulose filter, on the nitrocellulose filter Equipped with detection line and quality inspection line.The AMH monoclonal antibody of label colloidal gold is coated in the sample pad；It is fixed in detection line There is the 2nd AMH monoclonal antibody.Colloidal gold detection device in CN107192827A is conducive to rapidly preliminary interpretation.This side Preliminarily qualitative or half-quantitative detection may be implemented in method, but can not achieve accurate quantitative analysis.

Currently, the quantitative detecting method of AMH is based primarily upon magnetic microparticle chemiluminescence analytic approach in this field, this is a kind of The detection method that sandwich method chemiluminescence immunoassay is combined with magnetic particle isolation technics.Its testing principle is: in sample The AMH monoclonal that quantitative enzyme mark AMH monoclonal antibody is added in this (standard items, calibration object or quality-control product), is bound to magnetic particle Antibody is after 37 DEG C are incubated for, and the AMH monoclonal antibody and enzyme mark AMH monoclonal antibody combined on magnetic particle is respectively and in sample The different epitopes of AMH molecule combine, and form " sandwich " structure；It is i.e. separable to be not required to centrifugation for the Direct precipitation in externally-applied magnetic field； Supernatant is removed, the compound of precipitating is cleaned, enzyme-catalyzed chemical luminescence substrate is then added；Substrate is split under enzyme effect by catalysis Solution, forms unstable excitation state intermediate, and photon is just issued when excitation state intermediate returns to ground state, forms luminescence-producing reaction； The luminous intensity that light-emitting appearance detection reaction can be used, the AMH content in sample can be calculated according to standard curve.In detection model In enclosing, luminous intensity is directly proportional to the AMH concentration in sample.

A kind of method that AMH is quantitative in human serum is disclosed in IN2015CH00633A Indian patent application.This method relates to And it is based on graphene oxide (graphene (oxide)) chemoluminescence method and/or ELISA diagnostic techniques.In the method, stone Black olefinic oxide has the abilities for retaining a large amount of capture antibody (anti-AMH is mostly anti-), to improve the accuracy of detection and sensitive Degree.The detection device according to prepared by this method includes: porous plate, the graphene oxygen for being conjugated with 3- aminopropyl triethoxysilane Compound；And it is conjugated with the capture antibody of EDC.

European patent application discloses the sidestream immune analytical equipments of AMH in detection whole blood a kind of by EP2817621.Passing through will Gold particle or fluorescent grain are conjugated on antibody, allow the device to generate color development or fluorescence signal, and this signal Intensity and AMH concentration are in proportionate relationship.

Currently, the method for antibody coupling to antibody is had very much.However, these coupling methods can more or less exist in this way Or such some problems, such as be directly coupled to magnetic bead and will lead to steric effect, cause sensitivity and antibody dosage to increase； It then will lead to the patient for taking biotin treatment by biotin-labeled pentylamine system and the inaccurate situation of testing result occur.

A kind of method by antibody coupling to latex particle is disclosed in CN101625366A.The latex particle of surface modification And the combination of antibody is the aminoterminal covalent bond of the carboxyl or amino and antibody by its surface, is had between microballoon and antibody One bridging chemistry arm, reduces steric effect.Which not only improves the Percentage bounds of antibody, but also provide suitably for antibody Three-dimensional space stereochemical structure is effectively protected the active region of antibody and antigen binding.

Another method by antibody coupling to latex particle is provided in CN102161716A, wherein first passing through chemical bond By latex nothing to do with protein binding, glutaraldehyde is reused by antibody linked on unrelated protein, to make latex sensitization.Use this The sensitivity of reagent can be improved in method.

CN101625366A is coupled using EDC, therefore it does not play the role of increasing brachium, and EDC is made For a simple chemical cross-linking agent.CN102161716A the method will lead to crosslinking in practical applications, using glutaraldehyde Low efficiency, and crosslinking time is too long.The method has been eliminated substantially in commercial applications.

In view of the above problems, there is still a need for a kind of new coupling process for this field, it is desirable that is guaranteeing antibody coupling to magnetic bead After maintain original activity, while also avoiding the interference in sample.

Summary of the invention

Therefore, according to some embodiments of the application, a kind of bridging albumen is provided, it includes NHS-PEG and unrelated Albumen；NHS-PEG and unrelated protein are covalently attached in a linear fashion.Wherein, distinguish in the c-terminus of unrelated protein and aminoterminal It is covalently bonded with NHS-PEG.The PEG is selected from: PEG500, PEG1000, PEG2000；The NHS is N- hydroxy amber Imide ester or N-hydroxy-succinamide ester.

In some embodiments, the total arms of bridging albumen is a length ofExtremelyIt is preferred thatExtremely

Total arm length refers to: the distance that particle and antibody (or its antigen-binding fragment) are separated.Total arm length can pass through ammonia Base acid structure is as measured by automatic amino acid analyzer.

In some embodiments, the molecular weight of unrelated protein in 8kDa between 12kDa, preferably 9kDa to 11kDa it Between.Molecular weight is determined according to any known method in this field.

In some embodiments, the unrelated protein is selected from: irrelevant antibody or its segment, chicken egg white or its piece Section, gelatin or its segment, keyhole limpet hemocyanin or its segment, bovine serum albumin or its segment, human serum albumins or its segment, Albumin rabbit serum or its segment, poly-D-lysine.

Technical solution involved in the application will be applied to clinical examination field, thus be related to determinand in sample Detection.In consideration of it, in this context, irrelevant antibody refers to such a antibody, not significant shadow statistically Ring identification or combination of the antibody (or its antigen-binding fragment) on (or not interfering significantly with) coating particle to determinand.The application Technical solution do not depend on the particular sequence of unrelated protein, actually unrelated protein, which at least plays, separates particle and antibody Effect, to avoid steric hindrance.Therefore, other than the above-mentioned unrelated protein type referred to, it might even be possible to artificial synthesized nonfunctional Linear polypeptide.In the present context, " nonfunctional " refers to and does not significantly affect statistically (or significantly not dry Disturb) to the detection of determinand in sample, nor affect on the preparation of coating particle.This " nonfunctional " technical staff has the ability to pass through Experiment is prejudged, for example technical staff will prepare the reagent of detection A under the application introduction, thus needs to borrow anti-A antibody Bridging albumen is helped to be coated with to particle；The prior synthesizing linear polypeptide of technical staff, tests whether it influences between A and anti-A antibody In conjunction with.It is therefore not necessary to carry out the specific restriction in sequence to artificial synthesized nonfunctional linear polypeptide.It is illustrative real at one It applies in scheme, artificial synthesized non-functional linear polypeptide is a segment in thymopeptide-5, sequence Arg-Lys-Asp- Val-Tyr, this structure have no effect to object to be checked.In other embodiments, artificial synthesized non-functional linear polypeptide The glutathione segment containing anti-oxidation characteristics, or the segment therein using MDpep9 antibacterial peptide can be selected.

According to some embodiments of the application, a kind of reagent is provided, it includes the bridging albumen of the application.

According to some embodiments of the application, use of the bridging albumen of the application in preparation coating particle is provided On the way.

According to some embodiments of the application, the particle that a kind of surface is combined with antibody is provided.

In some embodiments, the particle that a kind of surface is combined with antibody is provided, it includes following or by following Composition:

Particle；

Antibody or its antigen-binding fragment；And

Bridging albumen.

In some specific embodiments, particle is combined by bridging albumen and antibody (or its antigen-binding fragment).

In some specific embodiments, particle is covalently tied by bridging albumen and antibody (or its antigen-binding fragment) It closes.

In some specific embodiments, the antibody is selected from: monoclonal antibody, polyclonal antibody, recombinant antibodies, embedding Close antibody, humanized antibody, camel antibodies, or combinations thereof.

In some specific embodiments, the antigen-binding fragment is selected from: Fv, sc-Fv, Fab, F (ab ')₂、 Fab ', scFv-Fc segment, bispecific antibody.

In some specific embodiments, bridging albumen refers to is covalently bonded with NHS- in c-terminus and aminoterminal respectively The unrelated protein of PEG.

In some embodiments, the molecular weight of unrelated protein is in 8kDa between 12kDa.In some specific embodiment party In case, unrelated protein does not influence the combination of the antibody (or its antigen-binding fragment) and its antigen.

In some embodiments, the unrelated protein molecular weight is in 9kDa between 11kDa.

In some embodiments, the PEG is selected from: PEG500, PEG1000, PEG2000.

In some embodiments, the NHS is N- hydroxy thiosuccinimide ester or n-hydroxysuccinimide Ester.

In some embodiments, the total arms of bridging albumen is a length ofSuch asExtremely

In some embodiments, unrelated protein is selected from: irrelevant antibody or its segment (such as Fab), chicken egg white or Its segment, gelatin or its segment, keyhole limpet hemocyanin or its segment, bovine serum albumin or its segment, human serum albumins or its Segment, albumin rabbit serum or its segment, poly-D-lysine, artificial synthesized nonfunctional linear polypeptide.

In a specific embodiment, unrelated protein is BSA or its segment.BSA stable in physicochemical property, it is not variable Property, cheap and easy to get, it is more that molecule includes free amino, is able to maintain under larger pH value range and different ionic strength biggish molten Xie Du is conducive to the progress of coupling reaction.

In a specific embodiment, unrelated protein is artificial synthesized nonfunctional linear polypeptide.

With broadest sense, term " antibody ", " ab " or " immunoglobulin " be may be used interchangeably, and including Dan Ke Grand antibody, engineered antibody, chemically synthesized antibody or recombinant antibodies, polyclonal antibody, multivalent antibody, multi-specificity antibody. In one embodiment, this application involves a kind of monoclonal or polyclonal antibodies.

As used herein term " monoclonal antibody " or " Mab " refer to obtained anti-from the antibody population of generally homogeneity Body；In other words, in addition to may be with minimum other than existing natural mutation, it is believed that the single antibody in antibody population is identical 's.Monoclonal antibody is high degree of specificity for single epitope.Such monoclonal antibody can by monoclonal B cell or Hybridoma is produced.Monoclonal antibody is also possible to recombination, i.e., is produced by protein engineering.Monoclonal antibody can also Being separated from phage antibody library.

In this application, " antigen-binding fragment ", it is intended that it is (also commonly referred to as anti-to indicate to remain the combination of antibody its target It is former) any peptides of ability, more peptide or proteins.In one embodiment, such " antigen-binding fragment " is selected from: Fv, scFv (sc indicates single-stranded), Fab, F (ab ')₂, Fab ', scFv-Fc segment or bispecific antibody.The antigen-binding fragment has source From at least one CDR of its complete antibody.

Term " recombinant antibodies " refers to the antibody obtained by the expression of recombinant DNA.

In some embodiments, particle is selected from: latex particle, gold particle, magnetic particle, plastic grains.Preferred real It applies in scheme, particle is magnetic particle.

In some embodiments, the antibody is the antibody for being specific to small molecule determinand, such as, but not limited to hormone Antibody or hapten antibody.

In preferred embodiments, haptens is selected from Miao Leguan hormone, corticotropin, aldosterone, blood vessel Angiotensin Converting Enzyme, feritin, osteocalcin, parathormone, vitamin.

In some embodiments, the partial size of particle is 50 to 200nm；Such as 80 to 150nm.

In some embodiments, particle is the particle with surface modification group, and modification group is selected from: amino, carboxyl, Hydroxyl, sulfydryl；It is preferred that the particle is amido modified particle.

According to some embodiments, additionally provides and a kind of antibody or its antigen-binding fragment are covalently bond to particle surface Method comprising step:

1) particle is provided；

2) antibody or its antigen-binding fragment are provided；

3) bridging albumen is provided；

4) so that the bridging albumen and the particle is carried out covalent bond, form bridging albumen-particle composites；

5) the bridging albumen-particle composites and the antibody or its antigen-binding fragment are subjected to covalent bond, shape At antibody or its antigen-binding fragment-bridging albumen-particle composites；

6) optionally, the antibody or its antigen-binding fragment-bridging albumen-particle composites are closed；

Wherein, the step 1), 2) or 3) between can be interchangeable with one another.

In some embodiments, using one or more closings for carrying out step 6) selected from the following: PEG1000, PEG2000, PEG500, casein, BSA.

In some embodiments, covalent bond is carried out in the PB buffer that the pH containing NaCl is 7 to 9.

In some embodiments, covalent bond be the NaCl containing 0.15M pH be 8 0.01M PB buffer in into Capable.

In some embodiments, covalent bond be 28 to 40 degrees Celsius carry out, such as, but not limited to 30,31,32, 33,34,35,36,37,38 degrees Celsius.

In some embodiments, covalent bond continues 20 to 60 minutes, such as, but not limited to 25,26,27,28,29, 30,31,32,33,34,35,36,37,38,39,40 minutes.

According to some embodiments, provides and a kind of antibody or its antigen-binding fragment are covalently bond to particle surface Method comprising step:

1) in the 0.01M PB buffer that the pH of the NaCl containing 0.15M is 8, the NH of 100nm is provided₄Modify magnetic particle；

2) in the 0.01M PB buffer that the pH of the NaCl containing 0.15M is 8, antibody (or its antigen-binding fragment) is provided；

3) in the 0.01M PB buffer that the pH of the NaCl containing 0.15M is 8, bridging albumen is provided；

4) so that the bridging albumen and the particle is carried out covalent bond at 37 degrees Celsius, continue 30 minutes, form bridge Even albumen-particle composites；

5) by the bridging albumen-particle composites and the antibody or its antigen-binding fragment, at 37 degrees Celsius into Row covalent bond continues 30 minutes, forms antibody or its antigen-binding fragment-bridging albumen-particle composites；

6) optionally, the antibody or its antigen-binding fragment-bridging albumen-particle composites are closed；Wherein, The step 1), 2) or 3) between can be interchangeable with one another.

According to some embodiments, a kind of detection reagent is provided, it includes:

- the first reagent, it includes the first antibodies and buffer of enzyme label；Or it includes the antigens and buffering of enzyme label Liquid；

- the second reagent, it includes coating particles of the invention；

Optional dilution, it includes BSA and buffer；

Optional calibration object and

Optional quality-control product.

In some embodiments, the dilution includes BSA and phosphate buffer；Or include BSA and physiology salt Water；

In some embodiments, the first antibody of enzyme label is the first antibody of alkali phosphatase enzyme mark.

In some embodiments, the first reagent includes the first antibody (or antigen) and buffer (Tris, vinegar of enzyme label Phthalate buffer), pH 6 to 8.

In some embodiments, the second reagent is combined with comprising 0.2mg/L to 2.0mg/L according to the surface of the application anti- The particle of body (or its antigen-binding fragment).Concentration of the particle in the second reagent, such as, but not limited to 0.3mg/L, 0.7mg/ L、0.8mg/L、0.9mg/L、1.1mg/L、1.5mg/L。

Specific embodiment

Embodiment 1: the preparation method of bridging albumen

1. selecting unrelated protein:

Before formally being prepared, first have to select a kind of unrelated protein.

According to wanting covalently bound antibody (for example, source of species of the type of antibody, antibody), antibody on particle most Reagent environment locating for whole purposes (what is, what sample detected for which kind of detection, the antigen combined), particle/antibody (other compositions in kit locating for particle/antibody), select a kind of unrelated protein, so that this nothing in final detection Closing albumen does not influence the detection effect of antibody, such as unrelated protein discord detection architecture (sample, determinand, antibody, its in reagent Its ingredient) occur to influence the reaction of testing goal.

The molecular weight of unrelated protein is about 10kDa.

Unrelated protein can be naturally occurring albumen, be also possible to purified albumen, be also possible to the piece of albumen Section, is also possible to artificial synthesized nonfunctional linear polypeptide.For example, in subsequent embodiment, unrelated protein it is non-limiting Example is linear polypeptide (c-terminus by chemical modification company of the artificial synthesized both ends with reactive group (such as amino) Reactive group is connected to facilitate and connect with NHS group).In consideration of it, without containing this reactive base in subsequent buffer Group.

2. the preparation of bridging albumen:

Being covalently attached the PEG (polyethylene glycol) 2000 with NHS group section respectively in the N-terminal and C of unrelated protein (can also be with Using PEG500 or PEG1000, as a result without significant difference), it is named as bridging albumen.

Embodiment 2: the coupling method of particle

The magnetic bead of 80nm, 100nm, 150nm are selected, surface modification has NH₄Group is purchased in merck.

Embodiment 1 is prepared resulting bridging albumen to be first covalently attached on magnetic bead；Then, various lists are covalently attached to then On clonal antibody (commercial antibody, is detailed in subsequent embodiment, and brand is unlimited), magnetic bead-bridging Protein-antibody complexes are formed；

Above-mentioned magnetic bead is closed using PEG1000 (or PEG2000, PEG500, casein).

Specific step is as follows:

(1) 0.05ml amino magnetic bead is taken, is cleaned 3 times with the solution of the 0.01M PB of PH8.0,0.15M NaCl, finally will The 0.01M PB of magnetic bead 1ml PH8.0, the solution of 0.15M NaCl are spare after mixing；

(2) by the antibody-solutions of 0.2mg, volume is not more than 100 μ l, and 0.01M PB, the 0.15M of 2ml PH8.0 is added The solution of NaCl, after mixing well, be added in Centricon-10 concentration tube be then placed in sigma 2-16k high speed freeze from About 30min is concentrated in scheming under the centrifugal force of 3000g to volume as no more than 0.5ml；

(3) after redissolving the solution of the 0.01M PB of bridging albumen PH8.0,0.15M NaCl, 0.1mg, body are taken out Product is not more than 100 μ l, is then added into the magnetic particle suspension liquid of step (1), and rotation mixes 30 points in 37 degree of baking ovens Clock；

(4) it is cleaned again magnetic bead 3 times with 0.01M PB of PH8.0,0.15M NaCl solution, finally by magnetic bead 1ml After the 0.01M PB of PH8.0, the solution of 0.15M NaCl mix, the antibody that step (2) prepare is added, equally in 37 degree of baking ovens Rotation mixes 30 minutes；

(5) it is cleaned again magnetic bead 3 times with 0.01M PB of PH8.0,0.15M NaCl solution, obtains antibody-bridging albumen- Particle composites；

(6) optionally, antibody-bridging albumen-particle composites are added to 0.01M TRIS, the 0.15M of 2ml PH8.0 NaCl, 1%PEG1000,1%PEG2000,1%PEG500 and 0.2% casein, 0.5%BSA solution keep mixing state 6 Hour, to close magnetic bead surfaces；

(7) gained particle can be stand-by (e.g., but being not limited to, use after 50 times of dilution) after closing.

The preparation of embodiment 3.AMH kit

1. embodiment 2 is prepared gained particle to prepare according to the constituent of following table, be assembled into kit.

Table 1.AMH kit forms

Voluntarily prepare calibration object and quality-control product, it is possible to use commercial calibration product and quality-control product.

2. kit working principle:

The AMH antibody that quantitative enzyme mark AMH monoclonal antibody (R1) is added in sample, calibration object and quality-control product, is bound to magnetic grain (separation agent) and R2.

After 37 DEG C are incubated for, the AMH monoclonal antibody that is combined on magnetic grain and monoclonal antibody linked with peroxidase respectively with AMH molecule Different epitopes combine, and form " sandwich " structure.

It is i.e. separable to be not required to centrifugation for the Direct precipitation in externally-applied magnetic field.Supernatant is removed, cleans the compound of precipitating, so After enzyme-catalyzed chemical luminescence substrate is added.

Substrate, by catalytic pyrolysis, forms unstable excitation state intermediate under enzyme effect, when excitation state intermediate returns to Just photon is issued when ground state, forms luminescence-producing reaction, that is, the luminous intensity of light-emitting appearance detection reaction can be used, be according to standard curve The AMH content in sample can be calculated.In detection range, luminous intensity is directly proportional to the AMH concentration in sample.

The preparation of 4. comparative particle of embodiment and its kit

Using the method in CN101625366A, using EDC by carboxyl microballoon directly and antibody coupling.Specific coupling method Position:

(1) 0.05ml carboxyl microballoon (80,100 or 150nm) is taken, cleans 3 times with the 0.05M MES solution of PH6.0, most It is mixed afterwards with the 0.05M MES solution of 1ml PH6.0；

(2) concentration is 50mg/ml after taking 10mg EDC to dissolve it with the 0.05M MES of PH6.0；

(3) it takes the 50mg/ml EDC solution of 0.02ml to add it in 1, while the antibody of 0.2mg is added, mix anti- It answers 30 minutes；Then it is cleaned and is mixed at least 30 minutes with the TRIS solution of pH7.4,0.01M；

(4) after cleaning 2 times with PB, 1%BSA, 0.01%TWEEN-20 of PH7.4,0.01M, 2ml step (1) is added Magnetic bead mixes；

(5) stand-by after diluting.

The preparation of 5. corticotropin of embodiment (ACTH) kit

Particle is prepared according to the method for embodiment 2, and according to the constituent of following table, is assembled into kit.

Table 2.ACTH kit forms

The preparation of 6. aldosterone of embodiment (ALD) kit

Table 3.ALD kit forms

7. Angiotensin II of embodiment (ANGIOTENSIN II) assay kit

Table 4.ANGII kit forms

8. osteocalcin of embodiment (BGP) assay kit

Table 5.BGP kit forms

The preparation of 9. parathormone of embodiment (PTH) assay kit

Table 6.PTH kit forms

10. feritin of embodiment (RENIN) assay kit

Table 7.RENIN kit forms

11. vitamin D of embodiment (V-D) assay kit

8. vitamin D kit forms of table

Test case

Test case 1: stability verifying

The present inventor further compares embodiment 3 (particle and its kit of embodiment 2) and (comparison of embodiment 4 Grain and its kit) in prepared magnetic bead and its performance in kit.

The specific method is as follows:

Prepare AMH autogamy calibration point (S0, S1, S2, S3, S4, S5), by magnetic bead according to same formula both PH7.4 PB, 0.5%BSA (1%BSA), the 0.01%TWEEN-20 of 0.01M is configured.Every kind of BSA concentration of every kind of magnetic bead configures altogether Then 10ml is divided into 5ml pipe, totally 2 pipe, a pipe are put into 37 degree of baking ovens, are placed 4 days, another pipe is put into refrigerator cold-storage, and temperature is 2 degree to 8 degree.

It is taken out after 4 days, operation to specifications, the luminous value of comparative determination calibration point S1, S3, S5.The result shows that making The luminous value ratio for magnetic bead its measuring point being marked with bridging albumen close to 1, show the potency of entire compound substantially without Loss.Using the magnetic bead of EDC label under high protein concentration, ratio is close to 1；But at low protein concentrations, potency decays The magnetic bead being significantly greater than marked using bridging albumen.

9. magnetic bead stability contrast of table

Reagent preparation when, those skilled in the art would generally be added in agent formulations a certain amount of protein protective agent with Keep the biological activity of albumen (enzyme, antibody etc.).Applicants have discovered that even if the protein protection of extremely low concentration is added in reagent Agent (such as BSA, trehalose etc.), the antibody prepared by 2 method of embodiment-bridging albumen-particle still can maintain albumen Activity.However, the particle (antibody is directly coupled to particle) prepared with conventional method (embodiment 4) is then in higher concentration protective agent In the presence of, just it is able to maintain stabilization.

2. sensitivity of test case

It uses zero-dose calibration object or Sample dilution to be detected as sample, replication 20 times, obtains 20 measurements As a result RLU value (relative light unit) calculates its average value (M) and standard deviation (SD), obtains M-2SD.

Two o'clock regression fit is carried out according to the concentration-RLU value result between zero-dose calibration object and adjacent calibration object to obtain The RLU value of M-2SD is brought into above-mentioned equation, finds out corresponding concentration value, as minimum detection limit, as a result such as by linear function Under.

The comparison of 10. reagent sensitivity of table

	Sensitivity
		Embodiment 3	0.01ng/ml
Embodiment 4	0.06ng/ml

This has used particle obtained by the preparation method of embodiment 2 that can obtain higher detection sensitivity as the result is shown.

The correlation of test case 3. and commercially available mature commodity

The present inventor compared the test result of embodiment 3, the reagent of embodiment 4 and Roche AMH kit respectively.

The comparison of 11. correlation of table

Concentration of specimens	Embodiment 3	Embodiment 4 (comparison)
			0ng/ml-0.2ng/ml	R²=0.94	R²=0.85
0.2ng/ml-1ng/ml	R²=0.93	R²=0.88
			1ng/ml-5ng/ml	R²=0.96	R²=0.94
5ng/ml-10ng/ml	R²=0.92	R²=0.93
			10ng/ml-25ng/ml	R²=0.90	R²=0.91

It is compared on the serum sample of various concentration range.Inventor discovery: even if 0 to 1ng/ml it is low dense It spends in sample range, the reagent prepared using 3 method of embodiment still keeps superior correlation with commercially available mature kit.

The performance indicator of the AMH kit of 4. the application of test case

1. anti-interference:

If containing the chaff interferent of following concentration in sample to be tested, do not make significant difference to testing result: hemoglobin≤160g/ L, triglycerides≤150mg/dl, bilirubin≤17g/dl, alkaline phosphatase≤150U/L.

2. detection limit:

Minimum detectability: it is not more than 0.03ng/ml.

3. accuracy:

Recovery experiment is done with sterling (being purchased from ORIGENE company outside), the rate of recovery is in 85% to 115% range.

4. repeatability:

It is detected with two samples of various concentration, each to repeat detection 10 times, coefficient of variation CV is not more than 10%.

5. difference between batch:

With three lot number kit detections with a sample, then the interassay coefficient of variation CV between three batches of kits is not more than 15%.

6. the range of linearity:

In 0.03ng/ml to 25ng/ml measurement range, dose-response curve linearly dependent coefficient R²≥0.9900。

The detection of test case 5.ACTH kit

1. the kit working principle of embodiment 5:

Quantitative enzyme mark ACTH monoclonal antibody is added in sample, standard items, calibration object and quality-control product, combines magnetic particle ACTH monoclonal antibody and ACTH reagent 2.After 37 DEG C are incubated for, the ACTH monoclonal antibody and enzyme mark Dan Ke that are combined on magnetic particle Grand antibody in conjunction with the different epitopes of ACTH molecule, forms " sandwich " structure respectively.The Direct precipitation in externally-applied magnetic field, is not required to Centrifugation is i.e. separable.Supernatant is removed, the compound of precipitating is cleaned, enzyme-catalyzed chemical luminescence substrate is then added.Substrate is made in enzyme With lower by catalytic pyrolysis, unstable excitation state intermediate is formed, photon, shape are just issued when excitation state intermediate returns to ground state At luminescence-producing reaction, that is, the luminous intensity of light-emitting appearance detection reaction can be used, the ACTH in sample can be calculated according to standard curve Content.In detection range, luminous intensity is directly proportional to the ACTH concentration in sample.

2. performance:

Accuracy: make recovery experiment with sterling, the rate of recovery is in 85% to 115% range.

Minimum detectability: it is not more than 3pg/ml.

Repeatability: being detected with two samples of various concentration, and each to repeat detection 10 times, coefficient of variation CV is not more than 10%.

Difference between batch: same a samples are detected with three lot number kits, then the interassay coefficient of variation between three batches of kits CV is not more than 15%.

The range of linearity: in 3pg/ml to 1500pg/ml measurement range, dose-response curve linearly dependent coefficient R²≥ 0.9900。

If containing the chaff interferent of following concentration in sample, do not make significant difference to testing result: hemoglobin≤160g/l, it is sweet Oil three esters≤150mg/dl, bilirubin≤17g/dl, alkaline phosphatase≤150U/L.

When concentration of specimens is when within 3000pg/ml, there is not hook effect.

3. magnetic bead stability contrast:

12. magnetic bead stability contrast of table

* antibody used is ACTH antibody in comparative example embodiment 4.

4. reagent sensitivity compares:

The comparison of 13. reagent sensitivity of table

	Sensitivity
		Embodiment 5	1pg/ml
Embodiment 4	4pg/ml

5. the correlation with commercially available mature commodity:

The comparison of 14. correlation of table

Concentration of specimens	Embodiment 5	Embodiment 4 (comparison)
			0pg/ml-30pg/ml	R²=0.92	R²=0.81
30pg/ml-75pg/ml	R²=0.95	R²=0.85
			75pg/ml-150pg/ml	R²=0.93	R²=0.92
150pg/ml-500pg/ml	R²=0.94	R²=0.94
			500pg/ml-1500pg/ml	R²=0.98	R²=0.95

The test of test case 6.ALD kit

1. the kit of embodiment 6 is what competing method chemiluminescence immune analysis method was combined with magnetic particle isolation technics A kind of detection method.The ALD that quantitative enzyme mark ALD antigen is added in sample, calibration object and quality-control product, combines magnetic particle Monoclonal antibody and ALD reagent 2.After 37 DEG C are incubated for, in enzyme mark ALD antigen and sample, calibration object and quality-control product ALD competition with It is specifically bound in conjunction with the ALD monoclonal antibody of magnetic particle.The Direct precipitation in externally-applied magnetic field, is not required to centrifugation Separation.Supernatant is removed, the compound of precipitating is cleaned, enzyme-catalyzed chemical luminescence substrate is then added.Substrate is urged under enzyme effect Change cracking, form unstable excitation state intermediate, photon is just issued when excitation state intermediate returns to ground state, is formed and shone instead It answers, that is, the luminous intensity of light-emitting appearance detection reaction can be used.In detection range, the dimension in the luminous intensity and sample of reaction is raw Plain D (ALD) concentration is inversely proportional.

2. performance:

If containing the chaff interferent of following concentration in sample, do not make significant difference to testing result: hemoglobin≤160g/l, it is sweet Oil three esters≤150mg/dl, bilirubin≤17g/dl, alkaline phosphatase≤150U/L.The intersection for measuring high concentration 100ng/mL is anti- Substance C ortisol is answered, degree of disturbance is less than 0.1%.

Accuracy: make recovery experiment with sterling (being purchased from sigma company outside), the rate of recovery is in 85% to 115% range.

Minimum detectability: it is not more than 5pg/ml.

The range of linearity: in 5pg/ml to 1000pg/ml measurement range, dose-response curve linearly dependent coefficient R²≥ 0.9900。

3. magnetic bead stability contrast:

15. magnetic bead stability contrast of table

4. reagent sensitivity compares

The comparison of 16. reagent sensitivity of table

	Sensitivity
		Embodiment 6	3pg/ml
Embodiment 4	7pg/ml

5. the correlation with commercially available mature commodity:

The comparison of 17. correlation of table

The test of test case 7.ANG II kit

1. 7 kit working principle of embodiment: competing method chemiluminescence immune analysis method and magnetic particle isolation technics phase In conjunction with a kind of detection method.Quantitative enzyme mark ANGIOTENSIN II is added in sample, standard items, calibration object and quality-control product Antigen, the ANGIOTENSIN II monoclonal antibody for combining magnetic particle and ANGIOTENSIN II reagent 2.37 DEG C of incubations Afterwards, enzyme-labelled antigen, ANGIOTENSIN II molecule compete in conjunction with the ANGIOTENSIN II monoclonal antibody of magnetic particle. It is i.e. separable to be not required to centrifugation for the Direct precipitation in externally-applied magnetic field.Supernatant is removed, the compound of precipitating is cleaned, enzyme is then added Promote chemiluminescent substrate.Substrate, by catalytic pyrolysis, forms unstable excitation state intermediate, among excitation state under enzyme effect Body just issues photon when returning to ground state, form luminescence-producing reaction, that is, the luminous intensity of light-emitting appearance detection reaction can be used, according to standard Curve can calculate the ANGIOTENSIN II content in sample.In detection range, in luminous intensity and sample ANGIOTENSIN II concentration is inversely proportional.

2. performance:

The chaff interferent for containing following concentration in sample, does not make significant difference to testing result: hemoglobin≤160g/l, glycerol Three esters≤150mg/dl, bilirubin≤17g/dl, alkaline phosphatase≤150U/L.

Minimum detectability: it is not more than 2ng/ml.

The range of linearity: in 2ng/ml to 180ng/ml measurement range, dose-response curve linearly dependent coefficient R²≥ 0.9900。

3. magnetic bead stability contrast:

18. magnetic bead stability contrast of table

* antibody used is ANG II antibody in comparative example embodiment 4.

4. reagent sensitivity compares:

The comparison of 19. reagent sensitivity of table

	Sensitivity
		Embodiment 7	1ng/ml
Embodiment 4	4ng/ml

5. the correlation with commercially available mature commodity:

The comparison of 20. correlation of table

Concentration of specimens	Embodiment 7	Embodiment 4 (contrast agent)
			0ng/ml-24ng/ml	R²=0.91	R²=0.84
24ng/ml-50ng/ml	R²=0.96	R²=0.86
			50ng/ml-130ng/ml	R²=0.94	R²=0.91
130ng/ml-250ng/ml	R²=0.97	R²=0.90
			250ng/ml-360ng/ml	R²=0.92	R²=0.95

The detection of test case 8.BGP kit

1. 8 kit working principle of embodiment:

A kind of detection method that sandwich method chemiluminescence immunoassay is combined with magnetic particle isolation technics.It is marking The BGP monoclonal antibody that quantitative enzyme mark BGP monoclonal antibody is added in sheet, standard items, calibration object and quality-control product, combines magnetic particle And BGP reagent 2.After 37 DEG C are incubated for, the BGP monoclonal antibody that is combined on magnetic particle and monoclonal antibody linked with peroxidase respectively with BGP The different epitopes of molecule combine, and form " sandwich " structure.It is i.e. separable to be not required to centrifugation for the Direct precipitation in externally-applied magnetic field.? Supernatant is removed, the compound of precipitating is cleaned, enzyme-catalyzed chemical luminescence substrate is then added.Substrate under enzyme effect by catalytic pyrolysis, Unstable excitation state intermediate is formed, photon is just issued when excitation state intermediate returns to ground state, forms luminescence-producing reaction Using the luminous intensity of light-emitting appearance detection reaction, the BGP content in sample can be calculated according to standard curve.In detection range Interior, luminous intensity is directly proportional to the BGP concentration in sample.

2. performance:

When hook effect does not occur when within 1000ng/ml in concentration of specimens.

Accuracy: make recovery experiment with sterling (being purchased from abcam company outside), the rate of recovery is in 85% to 115% range.

Minimum detectability: it is not more than 1ng/ml.

The range of linearity: in 1ng/ml to 300ng/ml measurement range, dose-response curve linearly dependent coefficient R2 >= 0.9900。

3. magnetic bead stability contrast:

21. magnetic bead stability contrast of table

* antibody used is BGP antibody in comparative example embodiment 4.

4. Sensitivity comparison:

The comparison of 22. reagent sensitivity of table

	Sensitivity
		Embodiment 8	1ng/ml
Embodiment 4	2.5ng/ml

5. the correlation with commercially available mature commodity:

The comparison of 23. correlation of table

Concentration of specimens	Embodiment 8	Embodiment 4 (contrast agent)
			0ng/ml-10ng/ml	R²=0.92	R²=0.83
10ng/ml-50ng/ml	R²=0.93	R²=0.86
			50ng/ml-100ng/ml	R²=0.96	R²=0.94
100ng/ml-150ng/ml	R²=0.92	R²=0.94
			150ng/ml-300ng/ml	R²=0.97	R²=0.95

The detection of test case 9.PTH kit

1. the working principle of 9 kit of embodiment:

A kind of detection method that sandwich method chemiluminescence immunoassay is combined with magnetic particle isolation technics.It is marking The PTH monoclonal antibody that quantitative enzyme mark PTH monoclonal antibody is added in sheet, standard items, calibration object and quality-control product, combines magnetic particle And PTH reagent 2.After 37 DEG C are incubated for, the PTH monoclonal antibody that is combined on magnetic particle and monoclonal antibody linked with peroxidase respectively with PTH The different epitopes of molecule combine, and form " sandwich " structure.It is i.e. separable to be not required to centrifugation for the Direct precipitation in externally-applied magnetic field.? Supernatant is removed, the compound of precipitating is cleaned, enzyme-catalyzed chemical luminescence substrate is then added.Substrate under enzyme effect by catalytic pyrolysis, Unstable excitation state intermediate is formed, photon is just issued when excitation state intermediate returns to ground state, forms luminescence-producing reaction Using the luminous intensity of light-emitting appearance detection reaction, the PTH content in sample can be calculated according to standard curve.In detection range Interior, luminous intensity is directly proportional to the PTH concentration in sample.

2. performance:

Measure the cross reacting material osteocalcin of 300ng/mL high concentration, degree of disturbance < 0.1%.

When hook effect does not occur when within 6000pg/ml in concentration of specimens.

Minimum detectability: it is not more than 2pg/ml.

The range of linearity: in 2pg/ml to 3000pg/ml measurement range, dose-response curve linearly dependent coefficient R²≥ 0.9900。

3. magnetic bead stability contrast

24. magnetic bead stability contrast of table

* antibody used is PTH antibody in comparative example embodiment 4.

4. Sensitivity comparison:

The comparison of 25. reagent sensitivity of table

	Sensitivity
		Embodiment 9	1pg/ml
Embodiment 4	3pg/ml

5. the correlation with commercially available mature commodity:

The comparison of 26. correlation of table

Concentration of specimens	Embodiment 9	Embodiment 4 (contrast agent)
			0pg/ml-13pg/ml	R²=0.93	R²=0.85
13pg/ml-70pg/ml	R²=0.97	R²=0.91
			70pg/ml-300pg/ml	R²=0.94	R²=0.95
300pg/ml-1500pg/ml	R²=0.99	R²=0.93
			1500pg/ml-3000pg/ml	R²=0.93	R²=0.92

The detection of test case 10.RENIN kit

1. the working principle of 10 kit of embodiment:

A kind of detection method that sandwich method chemiluminescence immunoassay is combined with magnetic particle isolation technics.It is marking This, standard items, the RENIN monoclonal for quantitative enzyme mark RENIN monoclonal antibody being added, combines magnetic particle in calibration object and quality-control product Antibody and RENIN reagent 2.After 37 DEG C are incubated for, the RENIN monoclonal antibody and monoclonal antibody linked with peroxidase point that are combined on magnetic particle Not in conjunction with the different epitopes of RENIN molecule, " sandwich " structure is formed.The Direct precipitation in externally-applied magnetic field is not required to centrifugation i.e. It is separable.Supernatant is removed, the compound of precipitating is cleaned, enzyme-catalyzed chemical luminescence substrate is then added.Substrate quilt under enzyme effect Catalytic pyrolysis forms unstable excitation state intermediate, and photon is just issued when excitation state intermediate returns to ground state, is formed and is shone Reaction can be used the luminous intensity of light-emitting appearance detection reaction, the RENIN content in sample can be calculated according to standard curve. In detection range, luminous intensity is directly proportional to the RENIN concentration in sample.

2. performance:

When hook effect does not occur when within 500pg/ml in concentration of specimens.

Accuracy: make recovery experiment with sterling (being purchased from sigma company outside), the rate of recovery should be in 85%-115% range It is interior.

Minimum detectability: it is not more than 1pg/ml.

The range of linearity: in 1pg/ml-150pg/ml (1.44 μ IU/ml-216 μ IU/ml) measurement range, dose-response Curve linear coefficient R²≥0.9900。

3. magnetic bead stability contrast

27. magnetic bead stability contrast of table

* antibody used is renin antibody in comparative example embodiment 4.

4. Sensitivity comparison:

The comparison of 28. reagent sensitivity of table

	Sensitivity
		Embodiment 10	0.8pg/ml
Embodiment 4	3pg/ml

5. the correlation with commercially available mature commodity:

The comparison of 29. correlation of table

Concentration of specimens	Embodiment 10	Embodiment 4 (contrast agent)
			0pg/ml-3pg/ml	R²=0.88	R²=0.75
3pg/ml-36pg/ml	R²=0.92	R²=0.80
			36pg/ml-48pg/ml	R²=0.91	R²=0.79
48pg/ml-75pg/ml	R²=0.93	R²=0.83
			75pg/ml-150pg/ml	R²=0.95	R²=0.91

The detection of test case 11.V-D kit

1. the working principle of 11 kit of embodiment:

A kind of detection method that competing method chemiluminescence immune analysis method is combined with magnetic particle isolation technics.In sample Quantitative enzyme mark microorganism D antigen is added in sheet, calibration object and quality-control product, combines the microorganism D monoclonal of magnetic particle anti- Body and microorganism D reagent 2.After 37 DEG C are incubated for, microorganism D is competed in enzyme mark microorganism D antigen and sample, calibration object and quality-control product Specifically bound with the microorganism D monoclonal antibody of magnetic particle is combined.The Direct precipitation in externally-applied magnetic field, is not required to Centrifugation is i.e. separable.Supernatant is removed, the compound of precipitating is cleaned, enzyme-catalyzed chemical luminescence substrate is then added.Substrate is made in enzyme With lower by catalytic pyrolysis, unstable excitation state intermediate is formed, photon, shape are just issued when excitation state intermediate returns to ground state At luminescence-producing reaction, that is, the luminous intensity of light-emitting appearance detection reaction can be used.In detection range, the luminous intensity and sample of reaction In vitamin D concentrations be inversely proportional.

2. performance:

Accuracy: make recovery experiment with sterling (being purchased from sigma company outside), the rate of recovery is within the scope of 85%-115%.

Minimum detectability: it is not more than 5ng/ml.

The range of linearity: in 5ng/ml-100ng/ml measurement range, dose-response curve linearly dependent coefficient R²≥ 0.9900。

3. magnetic bead stability contrast

30. magnetic bead stability contrast of table

* antibody used is V-D antibody in comparative example embodiment 4.

4. Sensitivity comparison:

The comparison of 31. reagent sensitivity of table

	Sensitivity
		Embodiment 11	3ng/ml
Embodiment 4	6ng/ml

5. the correlation with commercially available mature commodity:

The comparison of 32. correlation of table

Concentration of specimens	Embodiment 11	Embodiment 4 (contrast agent)
			0ng/ml-4.5ng/ml	R²=0.95	R²=0.89
4.5ng/ml-20ng/ml	R²=0.94	R²=0.91
			20ng/ml-40ng/ml	R²=0.95	R²=0.92
40ng/ml-70ng/ml	R²=0.90	R²=0.84
			70ng/ml-100ng/ml	R²=0.89	R²=0.88

The improvement of the application is:

1) using bridging albumen composition (total arm length >), it is avoided that steric effect；

2) bridging albumen can guarantee the stability of entire conformation；

3) bridging albumen both ends have NHS group, can control the time of entire coupling reaction in 60 minutes；

4) Fab fragments of non-object to be checked be may be incorporated on bridging albumen to play the work for eliminating specificity interference With.

Claims

1. a kind of surface is combined with the particle of antibody, it includes following:

Particle；

Antibody or its antigen-binding fragment；And

Bridging albumen；

Wherein, the particle is by bridging albumen in conjunction with the antibody or its antigen-binding fragment；The combination is covalently to tie It closes；

Wherein, the bridging albumen refers to is covalently bonded with the unrelated protein of NHS-PEG in c-terminus and aminoterminal respectively；

The molecular weight of the unrelated protein in 8kDa between 12kDa,

The total arms of the bridging albumen is a length ofExtremelyIt is preferred thatExtremely

The unrelated protein is selected from: irrelevant antibody or its segment, chicken egg white or its segment, gelatin or its segment, keyhole blood Azurin or its segment, bovine serum albumin or its segment, human serum albumins or its segment, albumin rabbit serum or its segment, Poly-D-lysine, artificial synthesized nonfunctional linear polypeptide；

The unrelated protein does not influence the combination of the antibody or its antigen-binding fragment and antigen；

The antibody is selected from: monoclonal antibody, polyclonal antibody, recombinant antibodies, chimeric antibody, humanized antibody, camel antibodies, Or combinations thereof；

The antigen-binding fragment is selected from: Fv, sc-Fv, Fab, F (ab ')₂, Fab ', scFv-Fc segment, bispecific antibody；

The particle is selected from: latex particle, gold particle, magnetic particle, plastic grains；It is preferred that magnetic particle；

Preferably, the antibody is the antibody for being specific to haptens,

Preferably, the haptens is hormone, and the more preferable haptens is selected from: anti-Miao Leguan hormone, corticotrophins Element, aldosterone, angiotensins, feritin, osteocalcin, parathormone, vitamin.

2. the particle that surface according to claim 1 is combined with antibody, in which:

The partial size of the particle is 50nm to 200nm；It is preferred that 80nm to 150nm；

The particle is the particle with surface modification group, and the modification group is selected from: amino, carboxyl, hydroxyl, sulfydryl；It is excellent Selecting the particle is amido modified particle；

The molecular weight of the unrelated protein is in 8kDa between 12kDa, and preferably 9kDa is between 11kDa；

The PEG is selected from: PEG500, PEG1000, PEG2000；

The NHS is N- hydroxy thiosuccinimide ester or N-hydroxy-succinamide ester.

3. a kind of method that antibody or its antigen-binding fragment are covalently bond to particle surface comprising step:

1) particle is provided；

2) antibody or its antigen-binding fragment are provided；

3) bridging albumen is provided；

5) the bridging albumen-particle composites and the antibody or its antigen-binding fragment are subjected to covalent bond, are formed anti- Body or its antigen-binding fragment-bridging albumen-particle composites；

Wherein, the step 1), 2) or 3) between can be interchangeable with one another.

4. according to the method described in claim 3, wherein:

The bridging albumen refers to is covalently bonded with the unrelated protein of NHS-PEG in c-terminus and aminoterminal respectively；

The molecular weight of the unrelated protein in 8kDa between 12kDa, preferably 9kDa between 11kDa,

The particle is selected from: latex particle, gold particle, magnetic particle, plastic grains；It is preferred that magnetic particle.

5. according to the method described in claim 4, wherein:

The particle is the particle with surface modification group, and modification group is selected from: amino, carboxyl, hydroxyl, sulfydryl；It is preferred that institute Stating particle is amido modified particle；

The PEG is selected from: PEG500, PEG1000, PEG2000；

The NHS is N- hydroxy thiosuccinimide ester or N-hydroxy-succinamide ester.

6. according to the method described in claim 4, being closed using selected from the following a kind of or combination: PEG500, PEG1000, PEG2000, casein, BSA.

7. according to the method described in claim 3, wherein the covalent bond is carried out under the following conditions:

The PB buffer that pH containing NaCl is 7 to 9, the 0.01M PB buffer that the pH of the preferably NaCl containing 0.15M is 8；

28 to 40 degrees Celsius, preferably 30,31,32,33,34,35,36,37,38 degrees Celsius；

Continue 20 to 60 minutes, preferably 25,26,27,28,29,30,31,32,33,34,35,36,37,38,39 or 40 minutes.

8. a kind of bridging albumen, it includes NHS-PEG and unrelated protein, in which:

It is covalently bonded with NHS-PEG respectively in the c-terminus and aminoterminal of unrelated protein；

The PEG is selected from: PEG500, PEG1000, PEG2000；

The NHS is N- hydroxy thiosuccinimide ester or N-hydroxy-succinamide ester.

9. purposes of the bridging albumen according to any one of claims 8 in preparation coating particle, in which:

The coating particle surface is combined with antibody or its antigen-binding fragment.

10. a kind of reagent, it includes:

Bridging albumen according to any one of claims 8；Or

Surface described in claim 1 is combined with the particle of antibody.