CN108912223A

CN108912223A - Insulin immunogene, preparation method, insulin antibody and kit

Info

Publication number: CN108912223A
Application number: CN201810267089.6A
Authority: CN
Inventors: 饶微; 杜凯; 刘蕾; 高莉; 肖国华; 罗春
Original assignee: Shenzhen New Industries Biomedical Engineering Co Ltd
Current assignee: Shenzhen New Industries Biomedical Engineering Co Ltd
Priority date: 2018-03-28
Filing date: 2018-03-28
Publication date: 2018-11-30

Abstract

The present invention provides a kind of insulin immunogene, preparation method, insulin antibody and kits.The insulin immunogene is insulin crosslinked, is formed for multiple insulin monomers by intramolecular crosslinking and/or intermolecular cross-linking, containing one or moreCrosslink unit, 1. and is 2. each independently selected from A chain or B chain, R1 areR2 and R3 is that empty or R1 and R3 is each independently selected fromOr-S-, R2 are selected from the linear chain or branched chain alkylidene of C2~C74, the part-CH in the linear chain or branched chain alkylidene of C2~C74₂Arbitrarily by-O- ,-S- ,-NH,‑COO‑、‑CO‑、Arlydene, heteroarylidene or sub- cyclic hydrocarbon substitution or R1 and R3 are each independently selected from

Description

Insulin immunogen, preparation method thereof, insulin antibody and kit

Technical Field

The invention relates to the field of insulin antibody detection, and particularly relates to an insulin immunogen, a preparation method thereof, an insulin antibody and a kit.

Background

Insulin is the 11 th human body to the chromosome broken arm insulin gene region DNA to mRNA transcription, mRNA from the nucleus to move to the cytoplasmic endoplasmic reticulum, translated into 105 amino acid residues constituted Preproinsulin, Preproinsulin is proteolytically processed to remove the propeptide, to generate 86 amino acid long peptide chain, Proinsulin (Proinsulin), Proinsulin enters Golgi along with the microvesicles in the cytoplasm, is connected with disulfide bonds, and is processed by the action of proteolytic enzyme to cut off 31, 32 and 60 arginine-linked chains, the broken chain generates C peptide without action, forms α chain (also called A peptide) and β chain (also called B peptide) insulin, and is secreted to β cells and enters the blood circulation, mature insulin has the following structure:

anti-Human Insulin antibodies (IAAs) are important immunological markers of type 1 diabetes mellitus (or Insulin-dependent diabetes mellitus), and are generally produced by patients who use Insulin preparations and stimulate immune responses of the patients due to the heterogeneity or immunogenicity of exogenous Insulin.

IAA is predominantly IgG, and IgM, IgA, IgD and IgE are detectable in a small number of drug recipients. The majority of insulin antibodies in serum are combined with insulin, and a small part of insulin antibodies are in a free state, so that the method has very important significance for diagnosis, differential diagnosis and treatment of diabetes and hypoglycemia.

The IAA is usually used as calibration sample including patient serum, monoclonal antibody and polyclonal antibody. The patient serum has the problems of unfixed source, limited yield and unstable calibration products of the kit in each batch; the insulin monoclonal antibody is difficult to obtain a high-specificity antibody aiming at insulin in the preparation process, simultaneously, the insulin is used as an immunogen, the animal can be caused to die due to hypoglycemia, the immunity success rate is not high, and the single antibody preparation cost is high, so that the cost of a calibration product is high; the insulin polyclonal antibody has low immune efficiency and low antibody titer.

Therefore, it is necessary to develop an antibody having a very high titer for measurement. Most of the currently commercialized insulin antibody titers are low in titer, so that the reliability of IAA determination is reduced, and the cost is increased.

Disclosure of Invention

The invention mainly aims to provide an insulin immunogen, a preparation method thereof, an insulin antibody and a kit, and aims to solve the problem of low antibody titer generated by the insulin immunogen in the prior art.

In order to achieve the above object, according to a first aspect of the present invention, there is provided an insulin immunogen, the insulin immunogen is an insulin cross-linked body, the insulin cross-linked body is formed by intramolecular cross-linking and/or intermolecular cross-linking of a plurality of insulin monomers, and the insulin cross-linked body contains one or more cross-linking units represented by formula (1):

wherein ① and ② are each independently selected from intramolecular and/or intermolecular A or B chains,

r1 isR2 and R3 are null; or

R1 and R3 are each independently selected fromor-S-, R2 is selected from the group consisting of C2 to C74 linear or branched alkylene, or the moiety-CH in C2 to C74 linear or branched alkylene₂Optionally substituted by-O-, -S-, -NH-,-COO-、-CO-、arylene, heteroarylene or cycloalkylene; or

R1 and R3 are each independently selected fromor-S-, R2 is selected from arylene or substituted arylene, which means arylene wherein-H on the phenyl ring is substituted with-OH, a halogen element or an alkane.

Further, R1 and R3 are each independently selected fromor-S-, R2 is selected from the group consisting of C12-C62 straight or branched alkylene, preferably C12-C62 straight or branched alkylene, and the moiety-CH₂Optionally substituted by-O-, -S-, -NH-,-COO-, -CO-orAnd (b) substituted, more preferably, R2 is selected from (PEG) n, n is 2-24, and more preferably, n is 4-20.

Furthermore, the average molecular weight of the insulin cross-linked body is 20-150 KD, preferably 50-130 KD.

In order to achieve the above object, according to a second aspect of the present invention, there is provided a method for producing an insulin immunogen, comprising: subjecting a plurality of insulin monomers to intramolecular and/or intermolecular crosslinking by a crosslinking agent to obtain an insulin conjugate, wherein the insulin conjugate comprises one or more crosslinking units represented by the formula (1):

wherein ① and ② are each independently selected from intramolecular and/or intermolecular A or B chains, R1 isR2 and R3 are null; or

Further, the crosslinking agent is a compound obtained by reacting an amino group with a carboxyl groupThe cross-linking agent is a homobifunctional cross-linking agent or a heterobifunctional cross-linking agent containing an R2 group, preferably, the reaction groups of the homobifunctional cross-linking agent or the heterobifunctional cross-linking agent containing an R2 group are independently selected from

Further, in the crosslinking agent, R2 is selected from C12-C62 straight chain or branched chain alkylene, preferably part of-CH in C12-C62 straight chain or branched chain alkylene₂-by-O-, -S-, -NH-,-COO-, -CO-orAnd substituted, more preferably, R2 is selected from (PEG) n, n is 2-24, and more preferably n is 4-20.

Further, the cross-linking agent is selected from EDC, CMC, Bis (NHS) PEG_nAnd n is 2-24, DSS, Sulfo-EMCS, SMCC, Sulfo-SMCC or MBS.

Further, in the crosslinking step, the molar ratio of the crosslinking agent to the insulin monomer molecules is 2: 1-10: 1.

According to a third aspect of the present invention, there is provided an insulin antibody, wherein the insulin antibody is obtained by immunizing an animal with an insulin immunogen, and the insulin immunogen is any one of the insulin immunogens described above or the insulin immunogen prepared by any one of the preparation methods described above.

Further, insulin antibody is obtained by immunizing guinea pigs with insulin immunogen; preferably, the insulin immunogen is subjected to primary immunization and boosting immunization of guinea pigs in sequence to obtain insulin antibodies; more preferably, the amount of the insulin immunogen used in the primary immunization step is 0.5-3 mg, and the amount of the insulin immunogen used in the boosting immunization step is 0.25-1 mg.

According to a fourth aspect of the present invention, there is provided a kit for detecting an insulin antibody, the kit comprising a calibrator or a quality controller for an insulin antibody, the calibrator or the quality controller comprising any one of the insulin antibodies described above.

Furthermore, the kit also comprises an insulin antigen coated solid phase carrier and SPA coupled with a luminescent marker; preferably, the solid phase carrier is a magnetic microsphere and the luminescent marker is ABEI.

By applying the technical scheme of the invention, active groups on two chains of insulin, including two amino groups (Gly and Phe) at the N end, two carboxyl groups (Asn and Thr) at the C end and free amino groups and carboxyl groups on side chains, are utilized to carry out self-crosslinking under the action of a crosslinking agent to obtain a crosslinking body as an immunogen, so that the biological activity of the insulin can be reduced, and the animal can not be subjected to hypoglycemia death under the condition of high-dose immunity, therefore, the crosslinked insulin crosslinking body serving as the immunogen can not only be successfully inoculated, but also can obtain high-titer insulin antibodies. Furthermore, by further controlling the length of the spacer arm in the crosslinking unit contained in the insulin crosslinking body within a proper range and controlling the spacer arm to have hydrophilicity, insulin immunogen with stronger immune response and specificity can be obtained, and further insulin antibody with higher titer can be obtained.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.

Interpretation of terms:

IAA: anti-human insulin antibodies

EMCS: 6- (Maleimido) hexanoic acid succinimidyl ester

EDC: 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride

BSA: bovine serum albumin

MBS: 3-Maleimidobenzoic acid succinimidyl ester

RLU: relative light unit

SPA: staphylococcal protein A

IgG: immunoglobulins

Protein cross-linking agents are a class of small molecule compounds, generally of molecular weight between 200-600, having two or more reactive termini for a particular group (-NH2, -COOH, -HS), which can be coupled to 2 or more molecules separately, thereby binding the molecules together. In the 70 s, glutaraldehyde was commonly used as a protein cross-linking agent to link antibodies and indicators (such as enzymes) and had the disadvantage that the cross-linking groups were random and tended to form disordered polymers. In the 80 s, specific cross-linking agents, such as NHS (for-COOH) and maleimide (for-HS), were more widely used in life science research. By 2010, over a hundred protein cross-linking agents are commonly available on the market. In the research of life science, the skillful application of the cross-linking agent can make a lot of work break through.

Protein cross-linking agents fall into three broad categories: homologous cross-linking agent, heterologous cross-linking agent and photoreactive cross-linking agent.

Homologous cross-linking agent: having two identical activated reactive groups, which applies to: one-step reaction, intramolecular cross-linking, poly-polymerization or stabilization of quaternary structure.

Heterologous cross-linking agent: having two different activation reactive groups. It is applied to: the two-step reaction crosslinking can reduce aggregation and self-crosslinking.

The use method of the cross-linking agent comprises the following steps: firstly, the protein A to be crosslinked and a crosslinking agent react under certain conditions to obtain a protein A-crosslinking agent intermediate, then the redundant crosslinking agent is removed, and the protein B to be crosslinked is added. Polymers may be formed in the product, and this by-product can be controlled by adjusting the reaction conditions. The purpose of the two-step reaction is to reduce the formation of protein multimers by the intramolecular cross-linking agent (heterologous cross-linking agents may also be selected to reduce multimer production).

The carrier protein is linked to the hapten, which is only reactogenic and not immunogenic. And a section of polypeptide sequence (hapten) of the virus envelope protein is connected with carrier protein to prepare vaccine to stimulate immune response. In addition, monoclonal antibodies can also be prepared by the method, and the monoclonal antibodies can be used for finding antigen binding sites on macromolecular proteins. In the process of linking the two, a lower hapten-carrier protein molar ratio results in higher antibody affinity. Furthermore, the hapten is linked to an amino acid in the middle of the carrier protein, which results in a higher antibody titer than when linked to the end.

Various factors are considered in selecting the cross-linking agent: reaction direction, spacer length, water solubility, membrane permeability, the availability of cleavage, the availability of iodination, and the like. By adjusting the length of the spacer arm, the freedom of movement of each monomer of the cross-linked body can be effectively improved, and the steric hindrance effect is further improved, so that the antigen activity of the cross-linked body is improved.

As is known in the background of the present application, insulin consists of an A chain (21 peptide) and a B chain (30 peptide) and has a molecular weight of about 5800 Da. Because the molecular weight of the insulin is small and the antigenicity is poor, the insulin can not be directly used as immunogen to stimulate animal bodies to produce antibodies, and simultaneously the insulin can cause hypoglycemia death of the animals. In order to improve the immunogenicity of insulin, the inventors of the present application have conducted extensive studies on the preparation of insulin immunogen and antibodies thereof, and found that self-crosslinking by using several active groups on both chains of insulin, including two amino groups (Gly and Phe) at the N-terminus, two carboxyl groups (Asn and Thr) at the C-terminus, and free amino and carboxyl groups on the side chains, can reduce the biological activity of insulin by self-crosslinking, and does not cause hypoglycemic death in animals even in the case of high-dose immunization, so that not only successful vaccination but also high-titer insulin antibodies can be obtained using these crosslinked insulin conjugates as immunogen.

Based on the above research results, the applicant proposed the technical solution of the present application. In an exemplary embodiment of the present application, an insulin immunogen is provided, the insulin immunogen being an insulin cross-linked body formed by intramolecular and/or intermolecular cross-linking of a plurality of insulin monomers, the insulin cross-linked body comprising one or more cross-linking units represented by formula (1):

wherein ① and ② are each independently selected from intramolecular and/or intermolecular A or B chains, R1 isR2 and R3 are null; or R1 and R3 are each independently selected fromor-S-, R2 is selected from the group consisting of C2-C74 linear or branched alkylene, or the moiety-CH in C2-C74 linear or branched alkylene₂Optionally substituted by-O-, -S-, -NH-,-COO-、-CO-、arylene, heteroarylene or cycloalkylene; or R1 and R3 are each independently selected fromor-S-, R2 is selected from arylene or substituted arylene, which means arylene wherein-H on the phenyl ring is substituted with-OH, a halogen element or an alkane.

The crosslinked insulin conjugate of the present invention comprising the crosslinking unit represented by the above formula (1) may or may not contain a spacer. When R1 is an amide group and R2 and R3 are empty, the insulin conjugate does not contain a spacer. When R1 and R3 are each independently selected from an amide group or a thioether group, and R2 is the above-mentioned hydrophobic or hydrophilic spacer, an insulin conjugate containing the above-mentioned crosslinking unit as an immunogen not only enables successful vaccination but also enables high titer of insulin antibodies to be obtained. Particularly, when the insulin cross-linked body with the cross-linked structure shown in the formula (1) is provided with the spacer arms, and the spacer arms contain hydrophilic groups, the distance between insulin monomers can be increased, so that the space structure of the insulin cross-linked body can be changed, and compared with the cross-linked insulin monomers, the added spacer arms are weaker than the immunogen of an animal body, so that each cross-linked insulin monomer can more effectively and specifically initiate an immune reaction, and thus, antibodies with more specificity and higher titer are generated.

According to the research result of the application, when the insulin cross-linked body contains the spacer arm and the spacer arm contains the chain structure, the distance between the cross-linked insulin monomers can be increased, and further the space structure of the cross-linked body is changed, so that the insulin cross-linked body has stronger immunogenicity, an immune reaction is effectively caused, and high-titer antibodies are generated. However, as the spacer is longer, the solubility is lower and crosslinking is less likely to occur.

Thus, in a preferred embodiment herein, R2 is selected from the group consisting of C12 to C62 straight or branched chain alkylene, preferably C12 to C62 straight or branched chain alkylene wherein the moiety-CH 2-is replaced by-O-, -S-, -NH-,-COO-, -CO-orMore preferably, R2 is selected from (PEG) n, n is 2-24, and n is 4-20.

R2 as a spacer in the crosslinking unit, when it is a linear or branched alkylene group, can increase the distance between the crosslinking monomers,the relative spatial position and structure between the monomers are changed, so that the insulin cross-linked body has stronger immunoreaction and generates high-titer antibodies. And when the moiety-CH in the linear or branched alkylene of R2₂-by-O-, -S-, -NH-,-COO-, -CO-orWhen the spacer is substituted, the spacer has stronger hydrophilicity, relatively stronger affinity with an animal body and weaker immunogenicity when the spacer is used as an immunogen to immunize an animal, and immune reaction is not easily caused in a cross-linked body, so that the immunogenicity and the immunospecificity of each cross-linked insulin monomer are relatively stronger, and a special strong antibody is more easily generated, and a high-titer insulin antibody is more easily obtained. By controlling the length of the spacer to be within the above-mentioned carbon number range, it is possible to obtain an insulin conjugate which is highly immunogenic and produces a high titer of antibodies.

The antibody titer obtained by immunizing animals with insulin immunogen comprising hydrophilic spacer chains is higher for the same spacer chain length. Among the various hydrophilic spacer arms, the PEG spacer arm has better hydrophilicity, namely the immunogenicity between the spacer arm and an animal body is weaker, so that the immunogenicity of the insulin cross-linked body is relatively enhanced, the immunospecificity of the insulin cross-linked body is higher, and the titer of the generated antibody is higher.

In the insulin crosslinked material of the present application, the spacer may further contain a cyclic structure between the chain structure and the insulin monomer depending on the kind of the crosslinking agent used in the crosslinking. In a preferred embodiment of the present application, the spacer arm further comprises a ring structure, the ring structure being located between the chain structure and the insulin monomer. For example, when the crosslinking agent is Sulfo-EMCS or EMCS, the insulin crosslinked body contains, in addition to a linear alkylene group having 5 carbon atoms, a maleimide residue linked via a thioether bond between the linear alkylene group and an insulin monomer.

The crosslinked insulin having the above-mentioned crosslinking structure has an effect of efficiently inducing an immune reaction to produce a high-titer antibody when used as an immunogen, as compared with conventional insulin. In order to further improve the immunogenicity of the insulin conjugate of the present application and obtain a higher titer of antibodies, in a preferred embodiment of the present application, the average molecular weight of the insulin conjugate is 20 to 150KD, preferably 50 to 130 KDa. The average molecular weight of the insulin cross-linked body is controlled within the range, so that the method has the advantages of stronger and more specific immune response and higher titer of the generated antibody.

In a second exemplary embodiment of the present application, there is provided a method for preparing an insulin immunogen, the method comprising: subjecting a plurality of insulin monomers to intramolecular and/or intermolecular crosslinking by means of a crosslinking agent to obtain an insulin conjugate, the insulin conjugate comprising one or more crosslinking units represented by the formula (1):

wherein ① and ② are each independently selected from intramolecular and/or intermolecular A or B chains, R1 isR2 and R3 are null, or R1 and R3 are each independently selected fromor-S-, R2 is selected from the group consisting of C2-C74 linear or branched alkylene, or the moiety-CH in C2-C74 linear or branched alkylene₂Optionally substituted by-O-, -S-, -NH-,-COO-、-CO-、arylene heteroarylene or cycloalkylene; or R1 and R3 are each independently selected fromor-S-, R2 is selected from arylene or substituted arylene, which means arylene wherein-H on the phenyl ring is substituted with-OH, a halogen element or an alkane.

According to the preparation method, free primary amine groups and free carboxyl groups in a plurality of insulin monomer molecules or among the molecules are mutually crosslinked under the action of the crosslinking agent, and the insulin crosslinked bodies containing the spacing arms with different crosslinking lengths are generated according to different types of the crosslinking agent, so that the obtained insulin immunogen can generate stronger immune reaction in an animal body, and an antibody with relatively higher titer is generated.

In the preparation method, the specific type of the cross-linking agent can be reasonably selected from the existing polypeptide/protein cross-linking agents according to the requirements of cross-linked products. In the present application, the kind of the crosslinking agent is not particularly limited, and any crosslinking agent capable of forming a crosslinked polymer containing the above crosslinking unit is suitable for the production method of the present application.

In a preferred embodiment of the present invention, the crosslinking agent is a homobifunctional crosslinking agent or a heterobifunctional crosslinking agent containing the R2 group, which is a crosslinking agent that crosslinks an amino group and a carboxyl group to form an amide group.

In the above method for preparing insulin immunogen, when the cross-linking agent is a cross-linking agent capable of coupling primary amine group and carboxyl group to form amide bond, the cross-linking agent itself is a bifunctional cross-linking agent with zero length, such as 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride (EDC or EDAC), and the length of the cross-linking body is not increased while forming amide bond. The cross-linked insulin is formed by self-crosslinking of insulin monomers, and the molecular structure of the cross-linked insulin is more complex and the molecular weight is relatively higher than that of monomer molecules, so that the cross-linked insulin does not cause hypoglycemia and death of animals when the animals are immunized at high dose, and can stimulate the animal bodies to generate antibodies better than the monomers.

In the preferred embodiment, when the crosslinking agent is a homo-or hetero-bifunctional crosslinking agent containing the above-mentioned R2 group, the same free groups (e.g., amino group and amino group, or carboxyl group and carboxyl group) within or between the molecules of the insulin monomer can be crosslinked to form a crosslinked body, or different free groups (e.g., amino group and carboxyl group) within or between the molecules of the insulin monomer can be crosslinked to form a crosslinked body, and the R2 group serving as a spacer in the crosslinking agent is carried in the unit forming the crosslinked body. The cross-linking agent is a bifunctional cross-linking agent with the length of an R2 spacer arm, the cross-linked insulin cross-linked body also has the length of the R2 spacer arm, the cross-linked insulin monomers are endowed with larger intervals, the flexibility of each insulin monomer is enhanced, the spatial structure of the cross-linked body is more complex, so that the immune reaction can be generated more effectively, and the high-titer antibody can be obtained.

In the homobifunctional crosslinking agent or heterobifunctional crosslinking agent containing the R2 group, the reactive groups of the crosslinking agents are different according to the specific type. In order to increase the degree of cross-linking reaction between the free amino and carboxyl groups in both chains of the insulin monomer of the present application, in a preferred embodiment of the present application, the reactive groups of the above homobifunctional or heterobifunctional cross-linking agent comprising a group R2 are each independently selected from(N-hydroxysuccinimide ester group) or(maleimido group).

Above mentioned containsHomobifunctional cross-linking agents of reactive groupsCan pass throughThe groups and amino groups form the same amido bonds at two ends of a cross-linking unit, and a partition wall of R2 is added in the middle. Above mentioned containsHomobifunctional crosslinkers of reactive groups, capable of reactingThe group and the amino group after the sulfhydrylation (namely, the amino group is firstly converted into the sulfhydryl, and the sulfhydryl is reacted with the maleimide group) are subjected to the formation of the same thioether bond at two ends of the crosslinking unit, and meanwhile, the partition wall of R2 is added in the middle. One end of the heterobifunctional cross-linking agent contains N-hydroxysuccinimide ester group, the other end of the heterobifunctional cross-linking agent contains maleimide group, one end of the heterobifunctional cross-linking agent reacts with free amino in an insulin monomer to form amido bond, the other end of the heterobifunctional cross-linking agent forms thioether bond with amino after sulfhydrylation, and meanwhile, a spacer arm of R2 is added in the middle.

In order to further improve the convenience of crosslinking based on the above preparation method, in a preferred embodiment of the present application, R2 is selected from C12-C62 linear or branched alkylene, preferably C12-C62 linear or branched alkylene, and is partially-CH₂By O-, -S-, -NH-,-COO-, -CO-orMore preferably, R2 is selected from (PEG) n, n is 2-24, and n is 4-20.

In the preferred embodiment, the length of R2 in the cross-linking agent is selected to be between C12 and C62, so that the solubility of cross-linking can be improved, and the cross-linking efficiency can be improved.

In a preferred embodiment of the present application, the crosslinking agent is selected fromEDC、CMC、Bis(NHS)PEG_nAnd n is 4-20, DSS, Sulfo-EMCS, SMCC, Sulfo-SMCC or MBS. Wherein, the CMC and the EDC have the same crosslinking principle, are based on carbodiimide groups and can be linked with carboxyl (-COOH) and amino (-NH)₂) The cross-linking forms amide bonds, and thus the effect of the cross-linked product is similar. The effect of Sulfo-EMCS, EMCS and DSS is similar, and the carbon chain structure contains 5-6 straight carbon chains; SMCC has similar effect with Sulfo-SMCC, and the carbon chain structure is consistent. The specific structure and action principle of each crosslinking agent are described in detail below.

EDC: 1-ethyl-3- (3-methylenepropyl) carbodiimide hydrochloride, 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide, having the structural formula (2) below:

EDC has water solubility, and can be used for cross-linking for rapidly preparing peptide fragments. Is a zero-length cross-linking agent, and can be cross-linked by available carboxyl (-COOH) and amino (-NH) groups₂) The groups form a cross-linker and the formation of amide linkages provides a neutral linkage.

CMC: 1-cyclohexenyl-3- (2-morpholinoethyl) -carbodiimide-p-tolyenesulfonate, 1-cyclohexyl-2-morpholinoethylcarbodiimide p-toluenesulfonate, having the structural formula (3) below:

CMC is water soluble and is a zero-length cross-linking agent that can be crosslinked by the available carboxyl (-COOH) and amino (-NH) groups₂) The groups form a cross-linker and the formation of amide linkages provides a neutral linkage.

Bis(NHS)PEG_n: is a polyethylene glycol cross-linking agent, both ends of which are N-hydroxysuccinimide ester, and the succinimide ester can react with amino to generate amido bond. By passingFlexible PEG spacer arms irreversibly cross-link proteins or peptides; the polyethylene glycol spacer helps to maintain conjugate solubility; pure compounds with defined structure and molecular weight, ensuring reproducible protein modification effects; PEG spacer arms offer unique advantages, including increased stability, reduced aggregation tendency and reduced immunogenicity. Such as bis (NHS) PEG₅: Bis-N-succinimidyl- (pentaethylene glycol) ester, the structural formula of which is shown as the following formula (4):

further examples are bis (NHS) PEG₉: Bis-N-succinimidyl- (nonaethylene glycol ester) having the structural formula shown in the following formula (5):

DSS: disuccinimidyl suberate, CAS:68528-80-3, having the formula (6) below:

both ends of the N-hydroxysuccinimide ester are N-hydroxysuccinimide ester, the middle part of the N-hydroxysuccinimide ester is a 6-methylene straight chain, and the succinimide ester can react with amino to generate amido bond.

EMCS: 6- (Maleimido) hexanoic acid succinimidyl ester, CAS:55750-63-5, the reaction is directed to amino and sulfhydryl groups. The structural formula is shown as the following formula (7):

Sulfo-EMCS: ee-maleimidocaproic acid sulfosuccinimidyl ester, CAS:215312-86-0, is a water-soluble EMCS.

SMCC: succinimidyl 4- [ N-maleimidomethyl ] cyclohexane-1-carboxylate, succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, CAS: 64987-85-5, the reaction is directed to amino and mercapto groups. The SMCC is a heterobifunctional cross-linking agent, and the structural formula of the SMCC is shown as the following formula (8):

in the formula (8), the reaction groups are N-hydroxysuccinimide ester and maleimide, the N-hydroxysuccinimide ester can react with amino to generate amido bonds, the maleimide reacts with sulfydryl to generate thioether bonds, a part of insulin amino is firstly modified into sulfydryl, and then crosslinking is carried out through SMCC to prepare the insulin self-crosslinking immunogen.

Sulfo-SMCC: sulfosuccinimide 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, sulfosuccinimide 4- (N-maleimidomethyl) cyclohexane-1-carboxylate sodium salt, CAS: 92921-24-9. Is water soluble SMCC. The structural formula is shown as the following formula (9):

in the formula (9), the reactive groups are sulfo-NHS ester and maleimide, the reactivity between amino and sulfydryl (compared with SMCC) is water-soluble, and the crosslinking can be realized in a physiological solution.

The structural formula of MBS is shown in the following formula (10):

the reaction groups are respectively N-hydroxysuccinimide ester and maleimide, the N-hydroxysuccinimide ester can react with amino to generate amido bonds, the maleimide reacts with sulfydryl to generate thioether bonds, a part of insulin amino is firstly transformed into sulfydryl, then the sulfydryl is crosslinked with the insulin amino through MBS to prepare the insulin self-crosslinking immunogen, and the middle spacer arm contains 1 benzene ring.

In the preparation method, compared with insulin monomers, the insulin cross-linked body obtained by cross-linking has the advantages of increased molecular weight and complex spatial structure, so that the animal body is easier to stimulate to produce antibodies. In order to make the crosslinked insulin immunogen more effectively induce the immune response of animals and obtain antibodies with higher titer, in a preferred embodiment of the present application, in the crosslinking step, the molar ratio of the crosslinking agent to the insulin monomer molecules is 2: 1-10: 1, and the dosage ratio of the crosslinking agent to the insulin monomer molecules is controlled within the above range, so that not only can the crosslinking of the insulin monomer be realized by using different crosslinking agents, but also the molecular weight of the obtained insulin crosslinked body is relatively within a proper range (20-150 KD), and the insulin immunogen with the size can generate antibodies with higher titer.

In a third exemplary embodiment of the present application, an insulin antibody is provided, wherein the insulin antibody is obtained by immunizing an animal with an insulin immunogen, and the insulin immunogen is any one of the insulin immunogens described above or prepared by any one of the preparation methods described above. The insulin antibodies obtained by immunizing animals with insulin conjugate as an immunogen according to the present application have the advantage of high potency and, in preferred embodiments, greater antibody specificity.

In the process of immunizing animals to obtain insulin antibodies, the step of immunizing animals is only to adopt the existing immunization method. In a preferred embodiment of the present application, the insulin antibody is obtained by immunizing guinea pigs with an insulin immunogen; preferably, the insulin immunogen is subjected to primary immunization and boosting immunization of guinea pigs in sequence to obtain insulin antibodies; more preferably, the amount of the insulin immunogen used in the primary immunization step is 0.5-3 mg, and the amount of the insulin immunogen used in the boosting immunization step is 0.25-1 mg. Controlling the amount of the insulin immunogen of the present application in the above-mentioned ranges in the primary and booster immunization steps, respectively, has the advantage of being able to elicit an immune response without causing hypoglycemia and death of the animal.

The insulin antibody provided by the application can be used for detecting the titer performance by adopting the existing detection method. For example, an insulin antibody can be detected by a Maglumi 2000 full-automatic chemiluminescence detector according to the kit for detecting an insulin-resistant antibody manufactured under the manufacturing lot number 044160906, and the relative luminescence intensity of the insulin antibody of the present application when diluted 100 times is 30 to 100 ten thousand. It should be noted that manufacturers of the kit for detecting an insulin-resistant antibody produced in 044160906 batch and the full-automatic Maglumi 2000 chemiluminescence determinator are all the limited companies of biomedical engineering of the new industry in Shenzhen.

In a fourth exemplary embodiment of the present application, an insulin antibody detection kit is provided, which comprises an insulin antibody calibrator or quality control, wherein the insulin antibody calibrator or quality control comprises any one of the insulin antibodies.

Compared with the insulin antibody in the prior art, the insulin antibody provided by the application has the advantage of high titer, and can be used as a calibration product or a quality control product in a kit for detecting the insulin antibody.

According to different detection principles of the kit, the kit also comprises an insulin antigen coated solid phase carrier and SPA coupled with a luminescent marker. Wherein, preferably, the solid phase carrier is a magnetic microsphere, and the luminescent marker is ABEI. Of course, the solid phase carrier and the luminescent label can be other common carriers and luminescent labels in the prior art, and are not limited to the above types. Likewise, the kit is suitable for various insulin antibody detection kits, particularly for detection of the concentration of anti-insulin antibody (IAA) by using a chemiluminescent competitive immunoassay sandwich method.

The advantageous effects of the present application will be further described with reference to specific examples.

Example 1: preparation and detection of insulin (EDC method self-crosslinking) polyclonal antibody

1. Principle of immunogen preparation

EDC is used as a carboxyl activating reagent in amide synthesis, and after two C-terminal carboxyl groups (Asn and Thr) are activated in the self-crosslinking process of insulin, EDC can be randomly coupled with amino groups (N-terminal Gly/Phe and Lys) on another insulin molecule to form an insulin crosslinking body. The reaction process is shown in the following formula (11):

2. preparation of immunogen: weighing three parts of 20mg insulin (sigma, cat # I3536) and dissolving in 2ml MES (pH5.0), adding 0.5mg, 1.32mg, 5mg EDC and 12mg EDC (sigma, cat # 03449), stirring at room temperature for 2h, SDS-PAGE result shows that the insulin cross-linked body is dispersed, the sizes of the insulin cross-linked body are respectively concentrated near 20kDa, 50kDa, 130kDa and 150kDa, putting the reaction product into a dialysis bag with cut-off amount of 1.5kDa (the main purpose of selecting the dialysis bag with the cut-off amount is to remove cross-linking agent, and the cut-off amount can effectively remove small molecules such as cross-linking agent) and dialyzing 10L water for more than 24h at 4 ℃, freezing and vacuum drying to obtain solid powder, accurately weighing, and diluting to 2mg/ml concentration by 0.01M pH7.4PBS.

3. Antibody preparation

1) The key of antibody preparation lies in whether the antigen can sufficiently cause the immune animal to generate immune reaction, the species difference between the antigen and the immune animal is better, through comparison, a guinea pig is selected as an immune animal for preparing multiple antibodies (the homology with human insulin is 64%), and the experimental steps refer to the immune step of polyclonal antibodies in chapter three of modern antibody immunity technology and application thereof. The specific immunization steps are carried out in two times:

primary immunization: taking the three insulin immunogens with different molecular weights; mixing with Freund's complete adjuvant at a ratio of 1:1(v/v), adding a stirrer, stirring at 2-8 deg.C and at 2000-2500rpm until complete emulsification (test: adding purified water into a glass beaker, and sucking 10uL of emulsion onto water surface with 10uL pipette, and complete emulsification if no diffusion occurs within 2 min);

for each immunogen of molecular weight, 6 healthy male guinea pigs (about 500 g/pig) were immunized, the guinea pigs were fixed, shaved in an area of 6cmx2cm size in the direction perpendicular to the spinal column on the back, sterilized with alcohol and iodine cotton balls, and injected subcutaneously (or intradermally) with 5 injections of about 0.1mL of emulsion per injection (about 0.5mg of immunogen injected co-).

And (3) boosting immunity: booster immunizations were performed 4 weeks after the primary immunization. Emulsifying the immunogen (same as the primary immunization, except that Freund's complete adjuvant is replaced by Freund's incomplete adjuvant), fixing guinea pigs, selecting an area with the size of 6cmx2cm in the direction perpendicular to the spinal column on the back, shaving, sterilizing with alcohol and iodine cotton balls, injecting subcutaneously (or intradermally) for 5 points, and injecting about 0.1mL of emulsion (about 0.25mg of immunogen per point).

2) Sampling and evaluation of small samples

One week after the boosting, a small amount of blood is collected from leg veins of guinea pigs, serum is separated, and titer detection is performed by using an IAA kit, and considering that the multi-antibody immunity can be different among different guinea pigs, the subsequent contrast test adopts the guinea pig with the highest titer in each group for subsequent contrast.

4. Antibody titer detection

And (3) performing titer detection, namely diluting guinea pig serum by 10, 50 and 100 times with 0.01M pH7.4PBS respectively, performing comparative investigation experiments on a commercial Maglumi 2000 full-automatic chemiluminescence determinator (manufacturer: Shenzhen New Production biomedical engineering Co., Ltd.), and carrying out kit batch number: 044160906, the relative luminescence intensity (RLU) of the three dilution multiple samples was obtained.

5. The titer detection principle is as follows:

the kit detects the concentration of the anti-insulin antibody (IAA) by using a chemiluminescence immune competition sandwich method: the preparation method comprises the steps of coating carboxyl magnetic microspheres with insulin antigens, marking ABEI with SPA, sequentially adding diluted polyclonal antibodies, the insulin-coated magnetic microspheres and the ABEI-marked SPA into a reaction cup, incubating for 20min at 37 ℃, applying a magnetic field for precipitation, removing supernatant, cleaning a precipitation compound for 3 times with washing liquor, directly entering a sample measuring chamber, automatically pumping luminescent substrates 1 and 2 into an instrument, and automatically monitoring relative light intensity (RLU) emitted within 3 s. IAA concentration is proportional to RLU.

Meanwhile, guinea pig serum with the highest titer is selected to be marked with the human anti-IgG antibody ABEI for comparison, and which of the human anti-IgG antibody and the SPA is higher in detection sensitivity is investigated.

Example 2: insulin (bis (NHS) PEG₂) Preparation and detection of polyclonal antibodies

1. Principle of immunogen preparation

Bis(NHS)PEG₂Is a water-soluble polyethylene glycol (PEG) represented by the formula (12):

the PEG spacer arm increases the solubility of cross-linked protein, the reaction group N-hydroxysuccinimide ester (with the same function) points to the amino group on the surface of the protein, the reaction groups at two ends can respectively form amido bonds with the amino groups on different insulin molecules, and the length of the spacer arm is 2 PEG. A crosslinking reaction product thereof with an insulin monomer, containing a crosslinking unit represented by the following formula (13):

2. preparation of immunogens

Three portions of 20mg insulin (sigma, cat # I3536) are weighed out and dissolved in 2ml of 0.1M NaHCO₃(pH8.3) 13.8mg of bis (NHS) PEG pre-dissolved in 0.2ml DMSO was added₂(Mw 400.5), stirring gently at room temperature for 2h, and SDS-PAGE shows that the insulin cross-linked body is dispersed, the size of the insulin cross-linked body is concentrated near 130kDa, the reaction product is filled into a dialysis bag with a cut-off amount of 1.5kDa, dialyzed for more than 24h against 10L of water at 4 ℃, and freeze-dried in vacuum to obtain solid powder, and the solid powder is diluted to a concentration of 2mg/ml by 0.01M PBS (pH7.4PBS) after accurate weighing.

3. Antibody preparation

The antibody preparation procedure was the same as in example 1

4. Antibody titer detection

Titer assay as in example 1

Example 3: preparation and detection of insulin (EMCS cross-linked) polyclonal antibody

1. Principle of immunogen preparation

EMCS is a heterobifunctional cross-linking agent, and the structural formula of the EMCS is shown as the formula (7). Wherein, the reaction groups are respectively N-hydroxysuccinimide ester and maleimide, the N-hydroxysuccinimide ester can react with amino to generate amido bond, the maleimide reacts with sulfhydryl to generate thioether bond, a part of insulin amino is reformed into sulfhydryl, then crosslinking is carried out through EMCS to prepare insulin self-crosslinking immunogen, and the middle spacer arm contains chain structure with 5 carbons and EMCSA cyclic structure. The crosslinked material obtained by the reaction contains a crosslinking unit represented by the following formula (14):

2. preparation of immunogens

insulin-EMCS activating solution is prepared by weighing 20mg of insulin and dissolving in 2ml of 0.1M NaHCO₃(pH8.3), 10mg of EMCS (sigma, cat # M9794) pre-dissolved in 0.2ml of DMSO was added, and the mixture was gently stirred overnight at 4 ℃ to prepare an insulin EMCS activating solution, the activating solution suspension was packed in a cut-off 1.5kDa dialysis bag at 4 ℃ and dialyzed against 10L of 0.1M phosphate buffer solution (pH7.0) for 24 hours or more to prepare an insulin EMCS activating solution, and the concentration was measured with a micro ultraviolet visible spectrophotometer.

insulin-SH solution (amino group modified into sulfhydryl) is prepared by dissolving 20mg of insulin in 2ml of 0.01M phosphate buffer (pH7.4), adding 10mg of 2-iminothiolane hydrochloride (sigma, cat # I6265), stirring at room temperature for 50 min, loading the activated liquid suspension into a dialysis bag with a cut-off of 1.5kDa, dialyzing 10L of 0.1M phosphate buffer (pH 7.0) for more than 4h to obtain insulin-SH solution, and measuring the concentration with a micro ultraviolet visible spectrophotometer.

Mixing insulin-EMCS and insulin-SH solution according to a mass ratio of 1:4, stirring for 2 hours at room temperature, and loading a reaction product into a cut-off 1.5kDa dialysis bag, dialyzing 10L of water for more than 24 hours at 4 ℃ according to SDS-PAGE results, freezing and vacuum-drying to obtain solid powder, accurately weighing, and diluting with 0.01M pH7.4PBS to a concentration of 2 mg/ml.

3. Antibody preparation

Primary immunization: taking the 130KD insulin immunogen; mixing with Freund's complete adjuvant at a ratio of 1:1(v/v), adding a stirrer, stirring at 2-8 deg.C and at 2000-2500rpm until complete emulsification (testing: adding purified water into a glass beaker, and sucking 10uL of emulsion with 10uL pipette to drop on water surface, and complete emulsification if no diffusion occurs within 2 min);

6 healthy male guinea pigs (about 500 g/pig) were selected for immunization, the guinea pigs were fixed, shaved in an area of 6cmx2cm size in the direction perpendicular to the spinal column on the back, sterilized with alcohol and iodine cotton balls, and injected subcutaneously (or intradermally) with 5 drops of about 0.1mL of emulsion per drop (about 3.0mg of immunogen injected).

And (3) boosting immunity: booster immunizations were performed 4 weeks after the primary immunization. Emulsifying the immunogen (same as the primary immunization, except that Freund's complete adjuvant is replaced by Freund's incomplete adjuvant), fixing guinea pigs, selecting an area with the size of 6cmx2cm in the direction perpendicular to the spinal column on the back, shaving, sterilizing by alcohol and iodine cotton balls, injecting subcutaneously (or intradermally) for 5 points, and injecting about 0.1mL of emulsion (about 1.0mg of immunogen per point).

4. Antibody titer detection

Titer assay as in example 1

Example 4: preparation and detection of insulin (SMCC-crosslinked) polyclonal antibody

1. Immunogenic structures

The cross-linking agent SMCC is a heterobifunctional cross-linking agent, the structure of which is shown in the formula (8), wherein the reaction groups are respectively N-hydroxysuccinimide ester and maleimide, the N-hydroxysuccinimide ester can react with amino to generate amido bond, the maleimide reacts with sulfhydryl to generate thioether bond, a part of insulin amino groups are firstly transformed into sulfhydryl, then cross-linking is carried out through SMCC (sigma, product number M5525), the insulin self-crosslinking immunogen is prepared, and the obtained cross-linked body contains a cross-linking unit shown in the following formula (15):

2. preparation of immunogens

The same as example 3, wherein EMCS is replaced by SMCC.

3. Antibody preparation

The antibody preparation procedure was the same as in example 1

4. Antibody titer detection

Titer assay as in example 1

Example 5: preparation and detection of insulin (MBS cross-linked) polyclonal antibody

1. Principle of immunogen preparation

MBS is a heterobifunctional cross-linking agent, and is shown in the structural formula (10), wherein the reaction groups are N-hydroxysuccinimide ester and maleimide respectively, the N-hydroxysuccinimide ester can react with amino to generate amido bond, the maleimide reacts with sulfhydryl to generate thioether bond, a part of insulin amino is firstly transformed into sulfhydryl, then cross-linked with the insulin amino through EMCS to prepare the insulin self-crosslinking immunogen, and the middle spacer arm contains 1 benzene ring and 1 benzene ringThe cyclic structure of (3). The crosslinked product contains a crosslinking unit represented by the following formula (16):

2. preparation of immunogens

The same as example 3, wherein EMCS is changed to MBS.

3. Antibody preparation

The antibody preparation procedure was the same as in example 1

4. Antibody titer detection

Titer assay as in example 1

Example 6 insulin (bis (NHS) PEG₄) Preparation and detection of polyclonal antibodies

1. Principle of immunogen preparation

Bis(NHS)PEG₄Is a water-soluble polyethylene glycol (PEG) represented by the formula (17):

the PEG spacer arm increases the solubility of cross-linked protein, the reaction group N-hydroxysuccinimide ester (with the same function) points to the amino group on the surface of the protein, the reaction groups at two ends can respectively form amido bonds with the amino groups on different insulin molecules, and the length of the spacer arm is 4 PEG. A crosslinking reaction product thereof with an insulin monomer, containing a crosslinking unit represented by the following formula (18):

2. preparation of immunogens

Three 20mg portions of insulin (sigma, cat # I3536) were weighed out and dissolved in 2ml of 0.1M NaHCO3(pH8.3), and 16.8mg of bis (NHS) PEG pre-dissolved in 0.2ml of DMSO was added₄And (3) stirring the mixture for 2 hours at room temperature, wherein SDS-PAGE results show that the insulin crosslinking bodies are dispersed and respectively concentrated in the sizes of about 130kDa, putting reaction products into a dialysis bag with a cut-off amount of 1.5kDa, dialyzing 10L of water at 4 ℃ for more than 24 hours, freezing and drying the mixture in vacuum to obtain solid powder, accurately weighing the solid powder, and diluting the solid powder to a concentration of 2mg/ml by using 0.01M PBS (phosphate buffered saline) pH7.4PBS.

3. Antibody preparation

The antibody preparation procedure was the same as in example 2

4. Antibody titer detection

Titer assay as in example 1

Example 7 insulin (bis (NHS) PEG₂₀) Preparation and detection of polyclonal antibodies

1. Principle of immunogen preparation

Bis(NHS)PEG₂₀Is a water-soluble polyethylene glycol (PEG) represented by formula (19):

the PEG spacer arm increases the solubility of cross-linked protein, the reaction group N-hydroxysuccinimide ester (with the same function) points to the amino group on the surface of the protein, the reaction groups at two ends can respectively form amido bonds with the amino groups on different insulin molecules, and the length of the spacer arm is 20 PEG. A crosslinking reaction product thereof with an insulin monomer, containing a crosslinking unit represented by the following formula (20):

2. preparation of immunogens

Three 20mg portions of insulin (sigma, cat # I3536) were weighed out and dissolved in 2ml of 0.1M NaHCO3(pH8.3), 41mg of bis (NHS) PEG pre-dissolved in 0.2ml DMSO were added₂₀And (3) stirring the mixture for 2 hours at room temperature, wherein SDS-PAGE results show that the insulin crosslinking bodies are dispersed and respectively concentrated in the sizes of about 130kDa, the reaction products are put into a dialysis bag with the cut-off amount of 1.5kDa, dialyzed for more than 24 hours against 10L of water at 4 ℃, freeze-dried in vacuum to obtain solid powder, accurately weighed, and diluted to the concentration of 2mg/ml by 0.01 MpH7.4PBS.

3. Antibody preparation

The antibody preparation procedure was the same as in example 2

4. Antibody titer detection

Titer assay as in example 1

Example 8 insulin (bis (NHS) PEG₂₄) Preparation and detection of polyclonal antibodies

1. Principle of immunogen preparation

Bis(NHS)PEG₂₄Is a water-soluble polyethylene glycol (PEG) represented by the formula (21):

the PEG spacer arm increases the solubility of cross-linked protein, the reaction group N-hydroxysuccinimide ester (with the same function) points to the amino group on the surface of the protein, the reaction groups at two ends can respectively form amido bonds with the amino groups on different insulin molecules, and the length of the spacer arm is 24 PEG. A crosslinking reaction product thereof with an insulin monomer, containing a crosslinking unit represented by the following formula (22):

2. preparation of immunogens

Three 20mg portions of insulin (sigma, cat # I3536) were weighed out and dissolved in 2ml of 0.1M NaHCO3(pH8.3), and 47mg of bis (NHS) PEG pre-dissolved in 0.2ml of DMSO were added₂₄And (3) stirring the mixture for 2 hours at room temperature, wherein SDS-PAGE results show that the insulin crosslinking bodies are dispersed and respectively concentrated in the sizes of about 130kDa, the reaction products are put into a dialysis bag with the cut-off amount of 1.5kDa, dialyzed for more than 24 hours against 10L of water at 4 ℃, freeze-dried in vacuum to obtain solid powder, accurately weighed, and diluted to the concentration of 2mg/ml by 0.01 MpH7.4PBS.

3. Antibody preparation

The antibody preparation procedure was the same as in example 2.

4. Antibody titer detection

The titer was measured as in example 1.

Comparative example 1: preparation and detection of insulin (non-cross-linked) polyclonal antibody

1. Antibody preparation

The antibody preparation process was the same as in example 1, wherein the immunogen was pure insulin.

2. Antibody titer detection

Titer assay as in example 1

Comparative example 2: preparation and detection of insulin (coupled BSA) polyclonal antibody

1. Immunogenic structures

Same as example 5

2. Preparation of immunogens

BSA-MBS activating solution 20mg BSA was dissolved in 2ml 0.1M NaHCO₃(pH8.3), 10mg of Suflo-MBS (sigma, cat # 803227) was added, and the mixture was gently stirred overnight at 4 ℃ to prepare a BSA-MBS activating solution, and the activating solution suspension was packed in a cut-off 1.5kDa dialysis bag at 4 ℃ and dialyzed against 10L of 0.1M phosphate buffer solution (pH7.0) for 24 hours or more to prepare a purified BSA-MBS activating solution, and the concentration was measured with a micro ultraviolet-visible spectrophotometer.

insulin-SH solution (amino modified sulfhydryl) is prepared by dissolving 20mg of insulin in 2ml of 0.01M phosphate buffer (pH7.4), adding 10mg of 2-iminothiolane hydrochloride, stirring at room temperature for 50 minutes, placing the activated solution suspension in a 1.5kDa cut-off dialysis bag, dialyzing 10L of 0.1M phosphate buffer (pH 7.0) for more than 4 hours to obtain insulin-SH solution, and measuring the concentration with a micro ultraviolet visible spectrophotometer.

Mixing BSA-MBS and insulin-SH solutions according to a molar ratio of 1:20, stirring the mixture at 4 ℃ for overnight under mild condition to prepare insulin immunogen to prepare immunogen suspension, filling the immunogen suspension into a dialysis bag with a cut-off amount of 1.5kDa, dialyzing 10L of water at 4 ℃ for more than 24 hours, freezing and drying the mixture in vacuum to obtain solid powder, cutting bands of different cross-linked polymers by adopting a gel cutting mode after SDS-PAGE gel electrophoresis, grinding the gel, adding 0.01M PBS (pH7.4PBS) for dialysis, filtering the gel by a needle filter to obtain insulin solution, measuring the concentration by a micro ultraviolet visible light spectrophotometer (thermo, Nanodorp oneC), and diluting the insulin solution to a concentration of 2mg/ml by 0.01M PBS (pH7.4PBS).

3. Antibody preparation

The antibody preparation procedure was the same as in example 1.

4. Antibody titer detection

The titer was measured as in example 1.

Results and analysis: the results of the titer test of the antibodies prepared in examples 1 to 8 and comparative examples 1 and 2 were compared and analyzed for the influence of the degree of crosslinking, the kind of the secondary antibody and the type of the crosslinking agent on the titer of the antibodies, and the results are shown in tables 1 to 3.

Table 1: immunogenic potency assay for insulin conjugates of varying degrees of crosslinking in example 1

Table 2: example 1 detection of immunogenic potency of insulin Cross-Linked body with human anti-IgG antibody in place of SPA

Table 3: potency assay for insulin polyclonal antibodies prepared with different crosslinkers and crosslinking methods (crosslinking degree n. 130kDa in examples 1-8)

From the above results, it can be seen that:

1) as can be seen from table 1, the basic trends are: the insulin has higher immune effect (especially between 20KD and 130 KD) along with the increase of the crosslinking degree (namely the molecular weight), which shows that the crosslinking body with large insulin crosslinking degree can provide more immune determinants, and the increase of the molecular weight is also beneficial to the identification of the guinea pig immune system, thereby increasing the immune effect.

2) As can be seen from Table 2, when SPA was changed to human anti-IgG antibody, the detection effect was slightly lower than that of SPA, indicating that SPA binds to guinea pig IgG more strongly than to human anti-IgG antibody, although human anti-IgG antibody has a great similarity to guinea pig IgG.

3) As can be seen from the comparison of the examples and comparative examples in Table 3, insulin (comparative example 1) itself is very weak in immunogenicity, and although insulin and guinea pig insulin are not very similar (about 64%), it still fails to induce an effective immune response in guinea pigs, while insulin coupled with BSA increases immunogenicity (comparative example 2), but the immune effect is not very good, and the intensity is only about 2.1 ten thousand after 100-fold dilution, and thus it cannot be used as a calibrator.

4) A comparison of examples 1-3 in Table 3 shows that: the immune effect of the insulin immunogen containing the spacer arms (examples 2 and 3) is obviously higher than that of the immunogen without the spacer arms (example 1), which shows that the immunogenicity can be increased by adding the spacer arms, and simultaneously, the change of the spatial structure can effectively trigger the immune response to generate high-titer antibodies;

5) table 3 the polyclonal antibody titer produced by example 2 was higher, with over 71 million of light intensity diluted 100 times, significantly higher than the antibody titer produced by example 3. This may be due to the bis (NHS) PEG relative to the hydrophobic spacer arm of EMCS₂The spacer arm has better hydrophilicity, namely the immunogenicity of the spacer arm is weaker, so that the insulin cross-linked body is immunizedThe immunogenicity is relatively enhanced, so that the immunospecificity of the insulin cross-linked body is higher, and the titer of the generated antibody is higher.

6) Table 3 the EMCS crosslinker of example 3 is linear and the SMCC of example 4 is hexamethylene, the light intensity of example 4 is 8%, 48% and 61% higher than example 3 at 10, 50 and 100 dilution, respectively. This may be because the linear structure of the EMCS crosslinker is more flexible than the cyclic structure of SMCC, reducing the mutual interference between insulin monomers.

7) In table 3, the antibody titer of example 4 is significantly higher than that of MBS-immunized antibody of example 5, which may be related to benzene rings contained in MBS cross-linked structure, and the benzene rings have poorer affinity with animal body, and are easy to stimulate non-idiotypic immune reaction or affect generation of idiotypic antibody. Similarly, if the cross-linker is substituted MBS, the benzene ring structure it contains will also reduce the potency of the antibody produced.

8) Table 3 shows that the same spacer arm has different lengths and different immune effects from the results of examples 6 to 8; (PEG)_nAnd when the value of n is 4-20, the effect is better than 2 or 24.

From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: by using active groups on two chains of insulin, including two amino groups (Gly and Phe) at the N terminal, two carboxyl groups (Asn and Thr) at the C terminal and free amino groups and carboxyl groups on side chains, a cross-linked body obtained by self-crosslinking under the action of a cross-linking agent is used as an immunogen, so that the biological activity of the insulin can be reduced, and the animal can not be subjected to hypoglycemia death under the condition of high-dose immunization, and therefore, the cross-linked insulin can be used as the immunogen to achieve not only successful inoculation but also high-titer insulin antibodies.

Furthermore, by further controlling the length of the spacer arm in the crosslinking unit contained in the insulin conjugate to be within a proper range and controlling the spacer arm to have hydrophilicity, insulin immunogen with stronger immune response and specificity can be obtained, and further insulin antibody with higher titer can be obtained.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An insulin immunogen, which is characterized in that the insulin immunogen is an insulin cross-linked body, the insulin cross-linked body is formed by intramolecular cross-linking and/or intermolecular cross-linking of a plurality of insulin monomers, and the insulin cross-linked body contains one or more cross-linking units shown as a formula (1):

r1 isSaid R2 and said R3 are null; or,

each of R1 and R3 is independently selected fromor-S-, the R2 is selected from a linear or branched alkylene of C2-C74, or a part-CH in the linear or branched alkylene of C2-C74₂Optionally substituted by-O-, -S-, -NH-,-COO-、-CO-、arylene, heteroarylene or cycloalkylene; or,

each of R1 and R3 is independently selected fromor-S-, the R2 is selected from arylene or substituted arylene, the substituted arylene refers to arylene in which-H on the benzene ring is substituted with-OH, a halogen element or an alkane.

2. The insulin immunogen of claim 1, wherein R1 and R3 are each independently selected fromor-S-, the R2 is selected from C12-C62 straight chain or branched chain alkylene, preferably the part of the C12-C62 straight chain or branched chain alkylene is-CH₂Optionally substituted by-O-, -S-, -NH-,-COO-, -CO-orAnd (b) substituted, more preferably, R2 is selected from (PEG) n, n is 2-24, and further preferably, n is 4-20.

3. The insulin immunogen according to claim 1 or 2, wherein the average molecular weight of the insulin cross-linked body is 20-150 KD, preferably 50-130 KD.

4. A method for producing an insulin immunogen, comprising:

subjecting a plurality of insulin monomers to intramolecular and/or intermolecular crosslinking by a crosslinking agent to obtain an insulin conjugate, wherein the insulin conjugate comprises one or more crosslinking units represented by the formula (1):

wherein ① and ② are independently selected from an intramolecular and/or intermolecular A chain or B chain, and R1 isSaid R2 and said R3 are null; or,

5. The method according to claim 4, wherein the crosslinking agent is a crosslinking agent which crosslinks an amino group and a carboxyl group to form an amide group, or the crosslinking agent is a homobifunctional crosslinking agent or a heterobifunctional crosslinking agent containing the R2 group,

preferably, the reactive groups of the homo-or hetero-bifunctional cross-linker containing said R2 group are each independently selected from

6. The method according to claim 4 or 5, wherein in the crosslinking agent, R2 is selected from C12-C62 linear or branched chain alkylene, preferably part of-CH in the C12-C62 linear or branched chain alkylene₂-by-O-, -S-, -NH-,-COO-, -CO-orAnd (b) substituted, more preferably, R2 is selected from (PEG) n, n is 2-24, and more preferably n is 4-20.

7. The method of claim 4, wherein the crosslinking agent is selected from EDC, CMC, Bis (C: (C) (C))NHS)PEG_nAnd n is 2-24, DSS, Sulfo-EMCS, SMCC, Sulfo-SMCC or MBS.

8. The method according to claim 4, wherein the molar ratio of the crosslinking agent to the insulin monomer molecules in the crosslinking step is 2:1 to 10: 1.

9. An insulin antibody obtained by immunizing an animal with an insulin immunogen, wherein the insulin immunogen is the insulin immunogen of any one of claims 1 to 3 or the insulin immunogen prepared by the preparation method of any one of claims 4 to 8.

10. The insulin antibody of claim 9, wherein the insulin antibody is obtained by immunizing a guinea pig with the insulin immunogen;

preferably, the insulin immunogen is subjected to primary immunization and boosting immunization of the guinea pig in sequence to obtain the insulin antibody;

more preferably, in the primary immunization step, the dosage of the insulin immunogen is 0.5-3 mg, and in the boosting step, the dosage of the insulin immunogen is 0.25-1 mg.

11. A kit for detecting an insulin antibody, the kit comprising a calibrator or a quality control for the insulin antibody, wherein the calibrator or quality control comprises the insulin antibody according to any one of claims 9 or 10.

12. The kit of claim 11, further comprising an insulin antigen coated solid support and a luminescent marker conjugated SPA; preferably, the solid phase carrier is a magnetic microsphere, and the luminescent marker is ABEI.