CN110669134A

CN110669134A - IgM-FC fragment, IgM-FC antibody, preparation method and application

Info

Publication number: CN110669134A
Application number: CN201910981165.4A
Authority: CN
Inventors: 韩玉婷; 柯拓; 唐佳
Original assignee: Guangdong Peng Peng Biological Co Ltd
Current assignee: Guangdong Peng Peng Biological Co Ltd
Priority date: 2019-10-15
Filing date: 2019-10-15
Publication date: 2020-01-10

Abstract

The invention provides an IgM-FC fragment, an IgM-FC antibody, a preparation method and an application thereof, relates to the technical field of biochemistry, and provides an application of the IgM-FC fragment as an immunogen in preparation of an antibody capable of being specifically combined with IgM, wherein the IgM-FC fragment is used as the immunogen to immunize an animal to generate the antibody capable of being specifically combined with IgM.

Description

IgM-FC fragment, IgM-FC antibody, preparation method and application

Technical Field

The invention relates to the technical field of biochemistry, in particular to an IgM-FC fragment, an IgM-FC antibody, a preparation method and an application.

Background

Immunoglobulin m (IgM) is one of the human immunoglobulins, which are classified into five types, IgA, IgG, IgD, IgE and IgM, according to their structures. IgM accounts for 5-10% of the total amount of serum immunoglobulin, the serum concentration is about 1mg/ml, and the Ig with the largest molecular weight in the immunoglobulin generally cannot pass through the blood vessel wall and is mainly present in blood. It is the antibody secreted first in immune response, is the first army against infection of the body, and once infected, it is produced rapidly, and over a period of time, the amount of IgM antibody gradually decreases and disappears. The inability of maternal IgM to pass through the placenta indicates that an intrauterine infection has occurred if IgM is found in the blood of the fetus or neonate. IgM was detected in serum, suggesting that infection occurred recently, and was useful for early diagnosis of infection.

At present, monoclonal antibodies or polyclonal antibodies of anti-human IgM are generally used for early diagnosis of infectious diseases in China, and the monoclonal antibodies are more specific than the polyclonal antibodies, so that false positives are reduced. Mu chain is used as a special heavy chain of IgM, the anti-mu chain antibody is more specific, the mu chain is divided into a constant region and a variable region, the sequence of the constant region is relatively conserved and has an antigen binding site, and the antibody produced by animals immunized by taking the constant region as an antigen can be specifically combined with most IgM, so that the peptide segment sequence of the constant region is selected as a substitute of the whole-molecule IgM, and the anti-IgM mu chain antibody specifically combined with the IgM can be obtained.

Although the monoclonal antibody group of the anti-IgM mu chain is an enzyme-labeled secondary antibody, which is widely used in the aspects of early diagnosis of infectious diseases, immunological research and the like, the process for preparing the monoclonal antibody of the anti-IgM mu chain is complicated and has high cost.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

A first object of the present invention is to provide the use of IgM-FC fragments as immunogens for the preparation of antibodies capable of specifically binding to IgM, to alleviate at least one of the technical problems of the prior art.

The second purpose of the invention is to provide a preparation method of IgM-FC fragments, which has the advantages of simple process, convenient operation of the used purification process and enzyme digestion process, wide universality, no need of expensive experimental instruments, low cost and suitability for large-scale popularization and application.

The third purpose of the invention is to provide the IgM-FC fragment prepared by the above preparation method, wherein the IgM-FC fragment is an IgM specific fragment and has weak cross reaction with full-length IgM than other immunoglobulin antibodies.

The fourth purpose of the invention is to provide a preparation method of the IgM-FC antibody, which has simple process and convenient operation and is suitable for large-scale popularization and application.

The fifth object of the present invention is to provide an IgM-FC antibody which can specifically bind to IgM.

The sixth purpose of the invention is to provide the application of the IgM-FC antibodies in a latex enhancement platform as a blocking agent.

The invention provides application of IgM-FC fragments as immunogens in preparing antibodies capable of being specifically combined with IgM;

preferred IgM-FC fragments are pentameric proteins.

The invention also provides a preparation method of the IgM-FC fragment, which comprises the following steps:

and (3) carrying out rough separation treatment and protein impurity removal treatment on the pretreated negative serum in sequence to obtain IgM, and carrying out enzyme digestion on the IgM to obtain IgM-FC fragments.

Further, the pretreatment comprises removal of lipoproteins in negative serum.

Further, the crude separation treatment comprises a precipitation method treatment;

preferably, the precipitation method comprises an ammonium sulfate precipitation method, a polyethylene glycol precipitation method, an acetone precipitation method, a picric acid precipitation method, a phosphotungstate precipitation method or a trichloroacetic acid precipitation method.

Further, the protein impurity removal treatment comprises chromatography treatment;

preferably, the chromatographic treatment comprises an affinity chromatographic treatment and an ion exchange chromatographic treatment;

preferably, the affinity chromatography treatment comprises removal of IgG using Protein G;

preferably, the ion exchange chromatography treatment comprises removing impure protein using hydroxyapatite;

preferably, Protein G is used to remove IgG, and then hydroxyapatite is used to remove impure Protein.

Further, carrying out enzyme digestion on the IgM by using trypsin to obtain an IgM-FC fragment;

preferably, the trypsin digestion is performed at 55 ℃ to 60 ℃ for 1 hour.

Further, after the IgM is subjected to enzyme digestion, the method also comprises the step of purifying the obtained IgM-FC fragment;

preferably, the IgM-FC fragments are purified by chromatography;

preferably, the chromatography comprises ion exchange chromatography and gel filtration chromatography;

preferably, the ion exchange chromatography comprises chromatography using a DEAE anion exchange medium;

preferably, the gel filtration chromatography comprises chromatography using superdex200 gel media;

preferably, the IgM-FC fragments are first purified once using DEAE anion exchange medium and then secondarily purified using superdex200 gel medium.

The invention also provides the IgM-FC fragment prepared by the preparation method.

The invention also provides a preparation method of the IgM-FC antibody, which is used for immunizing experimental animals by using the IgM-FC fragment to obtain the IgM-FC antibody.

The invention also provides the IgM-FC antibody prepared by the preparation method.

In addition, the invention also provides application of the IgM-FC antibody in a biochemical detection kit as a blocking agent and/or application in an immunodiagnosis kit.

The invention provides application of an IgM-FC fragment as an immunogen in preparation of an antibody capable of being specifically combined with IgM, wherein the IgM-FC fragment belongs to one part of an IgM mu chain and is an IgM specific fragment, and an antibody generated by an animal immunized by taking the IgM-FC fragment as the immunogen can be specifically combined with IgM, so that severe cross reaction between the IgM fragment as the immunogen and other Ig antibodies is avoided; the IgM mu chain contains Fab components and may bind with some foreign proteins which are difficult to remove, thereby causing a problem of interfering with the specificity of the IgM mu chain. Moreover, the IgM-FC fragment has relatively simple preparation process and low cost, and is suitable for large-scale popularization and application.

The preparation method of the IgM-FC fragment provided by the invention comprises the steps of carrying out rough separation treatment and protein impurity removal treatment on the pretreated negative serum in sequence to obtain IgM, and carrying out enzyme digestion on the IgM to obtain the IgM-FC fragment. The method comprises the steps of carrying out reasonable selection and combination on purification technologies, determining the sequence of the purification technologies to purify a serum raw material, obtaining IgM with the purity of more than 95%, and then carrying out enzyme digestion on the IgM to prepare IgM-FC fragments, so as to obtain pentamer IgM-Fc protein. In addition, the method has the advantages of simple process, convenience in operation of the used purification process and enzyme digestion process, wide universality, no need of expensive experimental instruments, low cost and suitability for large-scale popularization and application.

The IgM-FC fragment prepared by applying the preparation method provided by the invention is an IgM specific fragment, compared with full-length IgM, the IgM-FC fragment has only weak cross reaction with other immunoglobulin antibodies, and has strong antibody specificity and good immune effect.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. In this application, unless otherwise indicated, the use of the term "including" and other forms is not limiting.

Generally, the nomenclature used, and the techniques thereof, in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly employed in the art.

At present, anti-IgM antibody products at home and abroad are mostly monoclonal antibodies prepared by taking full-molecular IgM as immunogen. The screening process of the specific IgM monoclonal antibody is complicated, so that the cost is high; the preparation of IgM mu chain needs to undergo a strong reduction reaction to dissociate IgM, which not only destroys the natural polymer structure of IgM, but also destroys the intrachain-S-S-structure of mu chain, which seriously affects its immune effect (the affinity of antibody and natural IgM is weaker).

Based on the IgM-FC fragments, the invention provides the application of the IgM-FC fragments as immunogens in the preparation of antibodies capable of specifically binding to IgM.

The IgM-FC fragment belongs to a part of an IgM mu chain and is an IgM specific fragment, and an antibody generated by an animal immunized by taking the IgM-FC fragment as an immunogen can be specifically combined with the IgM, so that the severe cross reaction between the IgM fragment used as the immunogen and other Ig antibodies is avoided; the IgM mu chain contains Fab components and may bind with some foreign proteins which are difficult to remove, thereby causing a problem of interfering with the specificity of the IgM mu chain. Moreover, the IgM-FC fragment has relatively simple preparation process and low cost, and is suitable for large-scale popularization and application.

The preferable IgM-FC fragments are pentameric proteins, and antibodies generated by animals immunized by using the pentameric IgM-FC fragments as immunogens are more specifically combined with IgM.

For IgM-FC fragments, at present, IgM-FC fragments can be synthesized in vitro by a genetic engineering method and efficiently expressed in Escherichia coli, but most IgM-FC fragments are expressed by inclusion bodies, and protein renaturation is a difficult problem. And the recombinant protein of each IgM-FC peptide segment obtained by recombinant expression has poorer immunogenicity than that of each natural IgM-FC peptide segment.

Based on the above, the invention also provides a preparation method of the IgM-FC fragment, which comprises the following steps:

And the IgM can be primarily separated from the pretreated negative serum through the coarse separation treatment, and then other proteins in the IgM can be removed through the protein impurity removal treatment. According to the preparation method of the IgM-FC fragments, crude separation treatment and protein impurity removal treatment are reasonably selected as purification technologies, the sequence of the crude separation treatment and the protein impurity removal treatment is determined, serum raw materials are purified, IgM with the purity of more than 95% is obtained, and then IgM-FC fragments are prepared through IgM enzyme digestion, so that pentamer IgM-Fc proteins are obtained. In addition, the method has the advantages of simple process, convenience in operation of the used purification process and enzyme digestion process, wide universality, no need of expensive experimental instruments, low cost and suitability for large-scale popularization and application.

In the present invention, "negative serum" refers to serum of a healthy person.

The invention does not limit the specific mode of the rough separation treatment, and all methods can play a role in primarily separating IgM; the invention also does not limit the specific way of protein impurity removal treatment, and all methods can play a role in removing impurity proteins.

In some preferred embodiments, the pretreatment comprises removing lipoproteins from negative serum.

The lipoprotein in the serum is a heterogeneous compound consisting of lipid in the blood and certain specific protein, and the existence of the lipoprotein has great influence on the experiment, so that the removal of the lipoprotein in the negative serum by pretreatment can ensure the accuracy of the subsequent experiment and the purity of the product.

The method for removing lipoproteins in this embodiment is not limited, and any method can be used as long as lipoproteins in negative serum can be effectively removed, and examples thereof include, but are not limited to, centrifugation, precipitation, electrophoresis, and the like.

In some preferred embodiments, the crude separation treatment comprises a precipitation treatment;

It should be understood that the above precipitation methods are all known precipitation methods in the art, and the above precipitation method is not particularly limited, and in principle, the precipitant used does not chemically react with each component, and the excess precipitant does not interfere with the measurement and can completely precipitate.

For example, in some embodiments, a crude separation treatment may be performed to precipitate IgM whole molecules using a 50% saturated ammonium sulfate precipitation method; in other embodiments, a crude separation treatment using PEG6000 can be used to precipitate IgM whole molecules.

In some preferred embodiments, the protein decontamination treatment comprises a chromatography treatment.

It is understood that, in the present invention, the target protein is IgM, and therefore, other proteins than IgM need to be subjected to protein decontamination treatment. Chromatography is a separation technique established by using the difference of physicochemical properties of different components in a substance to be separated, and in the embodiment, the mode of chromatography is not limited, and any chromatography method capable of achieving a protein impurity removal effect can be used.

Preferably, the chromatographic treatment comprises an affinity chromatographic treatment and an ion exchange chromatographic treatment.

Affinity chromatography is a chromatographic method for separating substances by specific interaction between biomolecules, such as recognition binding of an antibody to an antigen, recognition binding of a receptor to a ligand, and the like, and in a preferred embodiment of the present invention, the affinity chromatography process includes removing IgG using Protein G; ion exchange chromatography uses the interaction between the charge of the substance and the charge of the chromatographic carrier to achieve the separation and purification, and in a preferred embodiment of the invention, the ion exchange chromatography comprises removing impure proteins by using hydroxyapatite. The affinity chromatography treatment and the ion exchange chromatography treatment can effectively realize protein impurity removal.

In this embodiment, the order of the affinity chromatography and the ion exchange chromatography is not limited, and preferably, Protein G is used to remove IgG, and then hydroxyapatite is used to remove impure proteins. After IgG is removed, hydroxyapatite is used for removing impurity proteins, the impurity removal efficiency is higher, and the purity of the obtained product is higher.

In some preferred embodiments, the IgM is cleaved with trypsin, the IgM is cleaved into Fab and FC fragments, and the FC fragments are removed to obtain IgM-FC fragments.

Preferably, the trypsin is digested at 55 ℃ to 60 ℃ for 1 hour, and the digestion temperature may be, but is not limited to, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, or 60 ℃. The trypsin enzyme digestion is carried out under the enzyme digestion conditions, so that the cost can be effectively saved on the basis of ensuring the complete enzyme digestion.

In some preferred embodiments, the method further comprises a step of purifying the obtained IgM-FC fragments after the enzyme digestion of the IgM. After purification, the obtained IgM-FC fragment has higher purity and stronger antibody specificity.

Preferably, the IgM-FC fragments are purified by chromatography.

In the present embodiment, the type of chromatography is not limited, and any chromatography that can achieve the effect of purifying IgM-FC fragments may be used.

Preferably, the chromatography comprises ion exchange chromatography and gel filtration chromatography.

Ion exchange chromatography, which utilizes the interaction between the charge of a substance and the charge of a chromatographic carrier to achieve separation and purification, in a preferred embodiment of the invention comprises chromatography using a DEAE anion exchange medium. Gel filtration chromatography is a chromatography using the porous network of the gel as a molecular sieve, and in a preferred embodiment of the invention, comprises a chromatography using superdex200 gel media. The IgM-FC fragments can be effectively purified by both ion exchange chromatography and gel filtration chromatography.

In this embodiment, the order of the ion exchange chromatography and the gel filtration chromatography is not limited, and preferably, the IgM-FC fragments are first purified once using DEAE anion exchange medium and then secondarily purified using superdex200 gel medium.

The DEAE anion exchange medium can effectively remove the Fab fragments after enzyme digestion, and after the Fab fragments are removed, the Fab fragments are purified by superdex200, so that residual foreign proteins can be further removed, the purification efficiency is higher, and the effect is better.

The complete IgM has severe cross reaction with other Ig antibodies; the IgM mu chain contains Fab components and may bind to some foreign proteins which are difficult to remove, thus interfering with the specificity of the IgM mu chain, resulting in poor antibody specificity. The IgM-FC fragment prepared by applying the preparation method provided by the invention is pure pentameric IgM-Fc protein, belongs to a part of IgM mu chain, is an IgM specific fragment, and only has weak cross reaction with other immunoglobulin antibodies by using antibodies generated by animals immunized by taking the IgM-FC fragment as immunogen, so that the antibody specificity is strong and the immune effect is good.

The method has the advantages of simple process, convenient operation and low cost, and is suitable for large-scale popularization and application.

The IgM-FC antibody has strong specificity and can be specifically combined with IgM.

The IgM-FC antibody provided by the invention is applied to a biochemical detection kit as a blocking agent and/or in an immunodiagnosis kit, and can eliminate the false positive effect.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

The main reagents and instrument information used in the embodiment of the invention are as follows:

example 1

This example provides a method for preparing IgM-FC fragments, comprising the steps of:

(a) pretreatment of negative serum: adding solid potassium bromide into the negative blood sample to adjust the density, and centrifuging through an ultracentrifuge; the running parameters were 25000rpm, 10 ℃ and 23 hours. The protein forms a gradient according to different densities, and the lipoprotein with relatively low density is distributed on the uppermost layer; the upper layer of lipoproteins was collected and discarded to obtain pretreated negative serum.

(b) Rough separation treatment: precipitation with PEG 6000: slowly adding PEG6000 (prepared into 20% mother liquor in mass-volume ratio) with the final concentration of 4-8% into the pretreated negative serum, uniformly mixing, standing for 1h at room temperature, centrifuging, and re-dissolving and precipitating with 1 × PBS.

(c) Protein impurity removal treatment: and (3) passing the redissolved solution through a Protein G column to remove IgG, collecting the flow-through solution, and then passing through a hydroxyapatite column to further remove foreign Protein, thereby obtaining IgM.

(d) The IgM fragment was cleaved into Fab and Fc fragments by trypsin digestion, and the Fc fragment was taken.

(e) And purifying the Fc fragment by using a DEAE anion exchange column, and then purifying by using Superdex200 to obtain the IgM-FC fragment.

Example 2

This example provides a method for producing IgM-FC fragments, which is different from example 1 in that, in the step (b) crude separation treatment, ammonium sulfate precipitation is used: slowly adding saturated ammonium sulfate while stirring until the final concentration of ammonium sulfate is 45-50%, uniformly mixing, standing at room temperature for 1h, centrifuging, and redissolving and precipitating with 1 × PBS.

Example 3

(a) pretreatment of negative serum: 1/5 vol of 55% CaCl were added with stirring₂Then, dextran sulfate (0.3g/ml mother liquor) is added dropwise according to the volume of 0.2 percent of the original blood serum, and the stirring is continued for 20 min; centrifuging at 9000rpm for 20min, and removing precipitate to obtain pretreated negative serum.

(c) Protein impurity removal treatment: and (3) passing the redissolved solution through a Protein A column to remove IgG, collecting the flow-through solution, and then passing through a hydroxyapatite column to further remove foreign Protein, thereby obtaining IgM.

Experimental example 1 Activity and specificity detection

Purpose of the experiment: IgM full length, IgM mu chain, IgM-FC antigen Activity specificity assay provided in example 1 of the present invention.

Numbering	Antigens	Concentration of	Batch number
				1	IgM-FC	2.12mg/ml	20190425
2	IgM full Length	1.6mg/ml	20180730
				3	IgM mu chain	1.84mg/ml	20190523-1

Concentration: three times of gradient dilution is carried out on 3 batches of antigen from 10 mu g/mL to the back;

activity: three-fold gradient dilutions were started from 10, 5, 1.66667 μ g/ml and back.

Sample treatment:

active antibody sample:

IgM goat anti (ADVY 020/0113)1:20 k.

Specific antibody sample:

IgA goat resistance (Capricorn 160/902) diluted 5K load;

IgG (ABMM 001179 BLR/1116) diluted 5K loading;

ALB goat anti (11327/71901 goat anti) diluted 5K loading;

ADVY-TF (ABMM 007034/1213) diluted 5K loading;

ADVY-C3(ABMM 004025/1217) diluted 5K loading;

CTNI-1 goat antibody (4.47mg/mL) diluted 5K loading;

diluting MB sheep anti-IIC by 5K and loading;

OYC-CRP Rabbit anti-70510.2 mg/ml diluted 5K loading;

CYSC-DAKO rabbit anti-dilution 5K loading.

(1) And (3) detecting IgM full length, IgM mu chain and IgM-FC enzyme immunity activity:

dilution gradient	IgM-FC	IgM full Length	IgM mu chain
				10	4.0543	3.8986	3.8732
5	3.8654	3.5741	3.8331
				1.6667	3.6183	2.9686	3.4751
0.5556	2.5794	1.5632	2.2653
				0.1852	1.2309	0.5277	1.0352
0.0617	0.4522	0.1915	0.3444
				0.0206	0.1687	0.0733	0.1143
0.0069	0.1124	0.0623	0.0789

(2) IgM full length, IgM mu chain and IgM-FC enzyme immunity specificity detection:

from the above results, it can be seen that:

the activity of IgM-FC and IgM mu chain antigen is slightly better than that of IgM full length;

the cross reaction of the IgM-FC antigen to IgG, ALB, TF, C3 and other antibodies is obviously lower than that of the IgM full length;

and 3, the IgM-FC antigen has no cross reaction to antibodies such as CTNI, MB, CRP, CYSC and the like, IgM mu chains have slight cross, and the cross reaction of the IgM full length is slightly obvious.

Experimental example 2 blocking Effect of IgM-FC sheep polyclonal antibody as blocking agent

Purpose of the experiment: the IgM-FC goat antiserum provided by the embodiment 1 of the invention blocks the RF effect on MB and CTNI goat antiserum kits after purification.

(1) Blocking the RF effect on the MB sheep antiserum kit after purification of IgM-FC sheep antiserum:

the detection method comprises the following steps: the blocking effect of the MB goat anti-latex kit R1 with and without the addition of blocking agent was compared.

Sample treatment:

sample 1: the MB concentration in the RF high value 2000IU/ml is 40 ng/ml;

sample 2: the concentration of MB contained in human serum was 163.58 ng/ml.

Sample 1 was diluted with physiological saline to a concentration of RF 1500(IU/mL), 900(IU/mL), 300(IU/mL), and the diluted sample was diluted 3-fold with human serum of sample 2 to a final RF concentration of 100, 300, 500 (IU/mL); sample 1 was diluted with sample 2 at equal fold to 1000(IU/mL) RF; according to the physiological saline solution: sample 2 was scaled to 1:2 to contain a RF concentration of 0 (IU/mL).

Wherein the reagent A is an MB goat anti-latex reagent R1 without IgM-FC antibody as a blocking agent; the reagent B is MB sheep anti-latex reagent R1 added with purified IgM-FC antibody containing 1% as a blocking agent.

From the above results, it can be seen that:

after 1% of IgM-FC goat antiserum purified samples are added into the MB goat anti-latex reagent R1, the sample has a good RF blocking effect on 1000 IU/mL.

(2) The IgM-FC goat antiserum provided in the embodiment 1 of the present invention is purified and then the RF effect is blocked on the CTNI goat antiserum kit:

sample treatment:

sample 1: the high RF value of 2000IU/ml contains CTNI with concentration of 3.6 ng/ml;

sample 2: human serum contained CTNI at a concentration of about 0.3 ng/ml.

Sample 1 was diluted with physiological saline to have RF contents of 1500(IU/mL), 900(IU/mL) and 300(IU/mL), and the diluted sample was diluted 3-fold with human serum of sample 2 to have final RF concentrations of 100, 300 and 500 (IU/mL); sample 1 was diluted with sample 2 at equal fold to 1000(IU/mL) RF; according to the physiological saline solution: sample 2 was scaled to 1:2 to contain a RF concentration of 0 (IU/mL).

Wherein the reagent A is CTNI goat anti-latex reagent R1 without IgM-FC antibody as a blocking agent; the reagent B is CTNI goat anti-latex reagent R1 added with purified IgM-FC antibody containing 1% as a blocking agent.

From the above results, it can be seen that:

after 1% of IgM-FC goat antiserum purified samples are added into the CTNI goat anti-latex reagent R1, the sample has a good effect of blocking RF of 1000 IU/mL.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

Use of an IgM-FC fragment as an immunogen for the preparation of an antibody capable of specifically binding to IgM;

preferred IgM-FC fragments are pentameric proteins.
2. A method for producing IgM-FC fragments, comprising:

and (3) carrying out rough separation treatment and protein impurity removal treatment on the pretreated negative serum in sequence to obtain IgM, and carrying out enzyme digestion on the IgM to obtain IgM-FC fragments.
3. The production method according to claim 2, wherein the pretreatment comprises removing lipoproteins in negative serum;

preferably, the crude separation treatment comprises a precipitation treatment;

preferably, the precipitation method comprises an ammonium sulfate precipitation method, a polyethylene glycol precipitation method, an acetone precipitation method, a picric acid precipitation method, a phosphotungstate precipitation method or a trichloroacetic acid precipitation method.
4. The method according to claim 2, wherein the protein impurity removal treatment comprises a chromatography treatment;

preferably, the chromatographic treatment comprises an affinity chromatographic treatment and an ion exchange chromatographic treatment;

preferably, the affinity chromatography treatment comprises removal of IgG using Protein G;

preferably, the ion exchange chromatography treatment comprises removing impure protein using hydroxyapatite;

preferably, Protein G is used to remove IgG, and then hydroxyapatite is used to remove impure Protein.
5. The method of claim 2, wherein the IgM is cleaved with trypsin to yield IgM-FC fragments;

preferably, the trypsin digestion is performed at 55 ℃ to 60 ℃ for 1 hour.
6. The method according to any one of claims 2 to 5, wherein the method further comprises a step of purifying the obtained IgM-FC fragments after the digestion of the IgM;

preferably, the IgM-FC fragments are purified by chromatography;

preferably, the chromatography comprises ion exchange chromatography and gel filtration chromatography;

preferably, the ion exchange chromatography comprises chromatography using a DEAE anion exchange medium;

preferably, the gel filtration chromatography comprises chromatography using superdex200 gel media;

preferably, the IgM-FC fragments are first purified once using DEAE anion exchange medium and then secondarily purified using superdex200 gel medium.
7. IgM-FC fragments produced by the process according to any one of claims 2 to 6.
8. A method for producing an IgM-FC antibody, characterized by immunizing an experimental animal with the IgM-FC fragment of claim 7 to obtain said IgM-FC antibody.
9. The IgM-FC antibody produced by the method according to claim 8.
10. The IgM-FC antibody according to claim 9 for use in diagnosis,

preferably the use of an IgM-FC antibody for the preparation of an immunodiagnostic kit;

preferably the IgM-FC antibody is used for preparing a blocking agent.