WO2018006269A1 - 吖啶标记结合物及其制备方法、化学发光免疫检测试剂盒 - Google Patents

吖啶标记结合物及其制备方法、化学发光免疫检测试剂盒 Download PDF

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WO2018006269A1
WO2018006269A1 PCT/CN2016/088685 CN2016088685W WO2018006269A1 WO 2018006269 A1 WO2018006269 A1 WO 2018006269A1 CN 2016088685 W CN2016088685 W CN 2016088685W WO 2018006269 A1 WO2018006269 A1 WO 2018006269A1
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acridine
crosslinking agent
group
intermediate product
labeled
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PCT/CN2016/088685
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English (en)
French (fr)
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钱纯亘
刘陶旭
肖成勇
祝亮
夏福臻
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深圳市亚辉龙生物科技股份有限公司
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Priority to KR1020197002865A priority Critical patent/KR102200905B1/ko
Priority to PCT/CN2016/088685 priority patent/WO2018006269A1/zh
Publication of WO2018006269A1 publication Critical patent/WO2018006269A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/08—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the invention relates to the field of in vitro detection, in particular to an acridine labeling conjugate, a preparation method thereof and a chemiluminescence immunoassay kit.
  • Chemiluminescent Labeling Immunoassay also known as chemiluminescence immunoassay (CLIA) is an immunoassay for direct labeling of antigens, haptens, antibodies or carbohydrates with chemiluminescent agents. Commonly used in the labeling of chemiluminescent substances are acridine compounds (AE). It emits light by the action of activating luminescent reagent (NaOH, H 2 O 2 ), and intense direct illumination is completed in one second, which is a fast blinking luminescence.
  • AE acridine compounds
  • the acridine compound is used for immunoassay.
  • the chemical reaction is simple, rapid, and does not require a catalyst.
  • the small molecule antigen is detected by a competition method, the macromolecule antigen or the antibody is sandwiched, the non-specific binding is small, and the background is low.
  • the combination of macromolecules does not reduce the amount of light produced, thereby increasing sensitivity.
  • the acridine compound can be labeled with an antigen, an antibody or the like to form an acridine labeling conjugate.
  • the quality of acridine-labeled conjugates is directly related to the success of chemiluminescent immunoassays and is therefore a key reagent in the kit.
  • Acridine-labeled conjugates are typically formed by linking an acridine compound to a specific antibody by a suitable method.
  • the acridine compound and antibody will directly affect the luminescence yield, background interference, and affinity of the acridine-labeled conjugate.
  • the currently used acridine labeling conjugate is prepared by a carbodiimide crosslinking method, which is bridged with a carbodiimide bifunctional crosslinking agent to bind the acridine substituent to the label to be labeled.
  • acridine labeling conjugate prepared by the conventional method since the acridine substituent and the label to be labeled need to be directly bound by the carbodiimide, the acridine substituent tends to interfere with the active site on the labeling protein.
  • the marker can selectively bind to less active sites, resulting in decreased activity of the acridine-labeled conjugate, which is sensitive to immunoassays. degree.
  • An acridine labeling conjugate comprising an acridine substituent, a linking carrier, a bifunctional crosslinking agent and a label to be labeled;
  • the bifunctional crosslinking agent is a crosslinking agent containing a maleimide group and a hydrazide group
  • the substance to be labeled is a protein containing a thiol group, a carbonyl group or a carboxyl group, a modified protein, a polypeptide, a modified polypeptide or a carbohydrate;
  • the linking carrier comprises an amino group and a carboxyl group, and the acridine substituent reacts with an amino group on the linking carrier to form a -CO-NH- structure to link the acridine substituent to the linking carrier, the linking vector
  • the upper carboxyl group reacts with a hydrazide group on the bifunctional crosslinking agent to form a -CO-NH-NH- structure to link the linking carrier to the bifunctional crosslinking agent, the bifunctional crosslinking agent
  • the maleimide group reacts with the thiol group on the label to be formed The structure thereby connects the bifunctional crosslinking agent to the label to be labeled.
  • a method for preparing an acridine labeling conjugate comprising the steps of:
  • the intermediate product B is obtained by reacting the activated acridine substituent with a linking carrier, wherein the intermediate product B is an acridine substituent-linking carrier conjugate;
  • the intermediate product B, the bifunctional crosslinking agent and the label to be labeled are crosslinked to obtain an acridine labeling conjugate, wherein the acridine substituent reacts with an amino group on the linking carrier to form -CO-NH- Structure to thereby attach the acridine substituent to the linking carrier, the carboxyl group on the linking carrier reacting with a hydrazide group on the bifunctional crosslinking agent to form a -CO-NH-NH- structure such that a linking carrier coupled to the bifunctional crosslinking agent, the maleimide group on the bifunctional crosslinking agent reacting with a thiol group on the label to be labeled
  • the structure thereby connects the bifunctional crosslinking agent to the label to be labeled.
  • a chemiluminescent immunoassay kit comprising the acridine labeling conjugate of any of the above.
  • Another acridine labeling conjugate comprising an acridine substituent, a linking carrier, a bifunctional crosslinking agent and a label to be labeled;
  • the bifunctional crosslinking agent is a crosslinking agent containing a maleimide group and a hydrazide group
  • the substance to be labeled is a protein containing a thiol group, a carbonyl group or a carboxyl group, a modified protein, a polypeptide, a modified polypeptide or a carbohydrate;
  • the linking carrier comprises an amino group and a disulfide bond
  • the acridine substituent reacts with an amino group on the linking carrier to form a -CO-NH- structure to link the acridine substituent to the linking carrier
  • the disulfide bond on the linking carrier reacts with the maleimide group on the bifunctional crosslinking agent to form Structure to thereby link the linking carrier to the bifunctional crosslinking agent
  • the hydrazide group on the bifunctional crosslinking agent reacts with a carbonyl group or a carboxyl group on the label to form a -NH-NH-CO-configuration
  • the bifunctional crosslinking agent is linked to the label to be labeled.
  • a method for preparing an acridine labeling conjugate comprising the steps of:
  • the intermediate product B is obtained by reacting the activated acridine substituent with a linking carrier, wherein the intermediate product B is an acridine substituent-linking carrier conjugate;
  • the intermediate product B, the bifunctional cross-linking agent and the label to be labeled are cross-linked to obtain an acridine labeling conjugate, wherein the acridine labeling conjugate comprises an acridine substituent, a linking vector, and a bifunctional function.
  • the linking carrier comprising an amino group and a disulfide bond, the acridine substituent reacting with an amino group on the linking carrier to form a -CO-NH- structure such that the acridine substituent is
  • the linking carrier is linked, and a disulfide bond on the linking carrier reacts with a maleimide group on the bifunctional crosslinking agent to form Structure to thereby link the linking carrier to the bifunctional crosslinking agent, the hydrazide group on the bifunctional crosslinking agent reacts with a carbonyl group or a carboxyl group on the label to form a -NH-NH-CO-configuration Thereby the bifunctional crosslinking agent is linked to the label to be labeled.
  • a chemiluminescent immunoassay kit comprising the acridine labeling conjugate of any of the above.
  • the acridine labeling conjugate described above includes acridine substituents, a linking carrier, a bifunctional crosslinking agent, and a label to be labeled which are sequentially linked.
  • the acridine substituent reacts with the amino group on the linking support to form a -CO-NH- structure to attach the acridine substituent to the linking carrier.
  • a linking carrier and a bifunctional crosslinking agent containing a maleimide group and a hydrazide group the acridine substituent and the label to be labeled are not directly bonded by a chemical bond, thereby avoiding the active site of the acridine substituent on the label.
  • the maleimide group and the hydrazide group in the bifunctional crosslinking agent can specifically bind to the label to be labeled containing a mercapto group, a carbonyl group or a carboxyl group, thereby avoiding the inability of the acridine substituent to be combined with a mercapto group, a carbonyl group or a carboxyl group.
  • the defect of binding of the label to be labeled expands the selection range of the label to be labeled, so that the specificity of the acridine labeling conjugate is high, which increases the sensitivity of the use of the acridine labeling conjugate.
  • FIG. 1 is a flow chart showing a method of preparing an acridine labeling conjugate according to an embodiment
  • FIG. 2 is a flow chart showing the preparation of an acridine-labeled conjugate obtained by crosslinking the intermediate product B, the bifunctional crosslinking agent, and the label to be labeled according to an embodiment
  • 3 is a flow chart showing the preparation of an acridine-labeled conjugate obtained by crosslinking the intermediate product B, the bifunctional crosslinking agent, and the label to be labeled according to another embodiment
  • An acridine-labeled conjugate comprising an acridine substituent, a linking carrier, a bifunctional crosslinking agent, and a label to be labeled, which are sequentially linked.
  • the bifunctional crosslinking agent is a crosslinking agent containing a maleimide group and a hydrazide group.
  • the label to be labeled is a protein containing a thiol group, a carbonyl group or a carboxyl group, a modified protein, a polypeptide, a modified polypeptide or a carbohydrate.
  • the linking carrier contains an amino group and a carboxyl group
  • the acridine substituent reacts with the amino group on the linking carrier to form a -CO-NH- structure to link the acridine substituent to the linking carrier.
  • the carboxyl group on the linking carrier reacts with the hydrazide group on the bifunctional crosslinking agent to form a -CO-NH-NH- structure to link the linking carrier to the bifunctional crosslinking agent, and the maleimide on the bifunctional crosslinking agent Forming a reaction with a thiol group on the object to be labeled
  • the structure thereby connects the bifunctional crosslinking agent to the label to be labeled.
  • the linking carrier contains an amino group and a disulfide bond
  • the acridine substituent reacts with the amino group on the linking carrier to form a -CO-NH- structure to link the acridine substituent to the linking carrier.
  • the disulfide bond on the linking carrier reacts with the maleimide group on the bifunctional crosslinking agent to form The structure thereby linking the linking carrier to the bifunctional crosslinking agent, and the hydrazide group on the bifunctional crosslinking agent reacts with the carbonyl or carboxyl group on the label to form a -NH-NH-CO-configuration to thereby bond the bifunctional crosslinking agent with The markers are connected.
  • the acridine substituent is an acridinium ester, an acridine acid, an acridine amide or an acridine sulfonamide.
  • acridinium ester specifically AE-NHS (10-methyl-acridine-9-N-succinimidyl ester carboxylate), DMAE-NHS (2',6'-dimethyl-4) '-(N-succinimide oxycarbonyl)phenyl-acridine-9-formate), ME-DMAE-NHS (2',6'-dimethylcarbonylphenyl-10-methyl-9 - acridine formate-4'-N-succinimidyl ester-trifluoromethanesulfonate) and the like.
  • the linking carrier may be hemocyanin (KLH) containing an amino group, a carboxyl group, and a disulfide bond.
  • KLH hemocyanin
  • other carriers containing an amino group, a carboxyl group, and a disulfide bond may also serve as a linking carrier in the present acridine labeling conjugate.
  • the linking carrier contains three functional groups, an amino group, a carboxyl group and a disulfide bond, and can be combined with a bifunctional crosslinking agent or a label to be labeled.
  • the bifunctional crosslinking agent is a crosslinking agent containing a maleimide group and a hydrazide group.
  • it may be EMCH (N-[ ⁇ -maleimidoacetate] hydrazide ⁇ trifluoroacetic acid), KMUH (N-[-maleimide undecanoic acid] hydrazide ⁇ trifluoroacetic acid) or MPBH. (4-[-N-maleimidophenyl]butanoic acid hydrazide ⁇ hydrochloric acid), etc.
  • the label to be labeled may be a protein, a modified protein, a polypeptide, a modified polypeptide or a carbohydrate having a thiol group, a carbonyl group or a carboxyl group, or a protein or a modified protein which is introduced into a thiol group, a carbonyl group or a carboxyl group by modification.
  • a polypeptide, a modified polypeptide or a carbohydrate may be a protein, a modified protein, a polypeptide, a modified polypeptide or a carbohydrate having a thiol group, a carbonyl group or a carboxyl group, or a protein or a modified protein which is introduced into a thiol group, a carbonyl group or a carboxyl group by modification.
  • the label to be labeled is an antigen, a hapten or an antibody.
  • the acridine substituent is an acridinium ester
  • the linking carrier is a hemocyanin (KLH) containing an amino group, a carboxyl group and a disulfide bond
  • the bifunctional crosslinking agent is a sample containing a maleimide group, and the acridine labeling conjugate is used.
  • the acridine labeling conjugate described above includes acridine substituents, a linking carrier, a bifunctional crosslinking agent, and a label to be labeled which are sequentially linked.
  • a linking carrier containing an amino group, a carboxyl group and a disulfide bond, and a bifunctional crosslinking agent containing a maleimide group and a hydrazide group the acridine substituent and the label to be labeled may be directly bonded without passing through the carbodiimide, thereby avoiding
  • the acridine substitution interferes with the active site on the labeled protein, making the activity of the acridine-labeled conjugate relatively high, increasing the sensitivity of the immunoassay.
  • the maleimide group and the hydrazide group in the bifunctional crosslinking agent can specifically bind to the label to be labeled containing a mercapto group, a carbonyl group or a carboxyl group, thereby avoiding the inability of the acridine substituent to be combined with a mercapto group, a carbonyl group or a carboxyl group.
  • the defect of binding of the label to be labeled expands the selection range of the label to be labeled, so that the specificity of the acridine labeling conjugate is high, which increases the sensitivity of the use of the acridine labeling conjugate.
  • the object to be labeled corresponding to the detected object can be connected correspondingly, because the selection range of the object to be labeled is wide, and even the object to be labeled containing a thiol group, a carbonyl group or a carboxyl group can pass through the bifunctional crosslinking agent and the connection.
  • the carrier binds to the acridine substituent to form an acridine labeling conjugate, so that the acridine labeling conjugate can specifically bind to the analyte at the time of detection, and the sensitivity is high and the labeling rate is high.
  • the acridine-labeled conjugate, the acridine substituent reacts with the amino group on the linking carrier to form a -CO-NH- structure to link the acridine substituent to the linking carrier, and the acridine substituent does not directly occur between the acridine substituent and the label to be labeled.
  • Chemical bond reaction. The traditional method generally uses a carbodiimide bifunctional cross-linker as a bridge to directly bind the acridine substitute to the label to be labeled, so that the labeling rate is low, the activity is not good, and the labeling group has little selectivity, and cannot be directly It is combined with a carbonyl group or a thiol group to be combined.
  • the acridine labeling conjugate of the invention Compared with the traditional acridine labeling conjugate, the acridine labeling conjugate of the invention has high labeling efficiency, can be directly used for detection and quantitative analysis of chemiluminescence immunoassay, and can solve the traditional carbodiimide cross-linking method, etc.
  • the acridine labeling conjugate prepared by the method has the disadvantages of inactivation, low labeling efficiency and less selectivity of the labeling group.
  • the method for preparing the acridine-labeled conjugate shown in FIG. 1 includes the following steps S110 to S120.
  • the intermediate product B is an acridine substitution-linking carrier conjugate
  • the linking carrier contains an amino group and a carboxyl group
  • the linking carrier contains an amino group and a disulfide bond
  • the linking carrier may be a linkage containing an amino group, a carboxyl group and a disulfide bond.
  • the carrier, the acridine substituent reacts with the amino group on the linking carrier to form a -CO-NH- structure to link the acridine substituent to the linking carrier.
  • the acridine substituent is an acridinium ester, an acridine acid, an acridine amide or an acridine sulfonamide.
  • it may be AE-NHS (10-methyl-acridine-9-N-succinimidyl ester carboxylate), DMAE-NHS (2',6'-dimethyl-4'-(N-amber) Imidyloxycarbonyl)phenyl-acridine-9-carboxylate), ME-DMAE-NHS (2',6'-dimethylcarbonylphenyl-10-methyl-9-azidinecarboxylate -4'-N-succinimidyl ester-trifluoromethanesulfonate) and the like.
  • the linking carrier may be hemocyanin (KLH) containing an amino group, a carboxyl group, and a disulfide bond.
  • KLH hemocyanin
  • other carriers containing an amino group, a carboxyl group, and a disulfide bond may also serve as a linking carrier in the present acridine labeling conjugate.
  • the molar ratio of the activated acridine substituent to the linking carrier is from 1 to 5000:1.
  • the molar ratio of the activated acridine substituent to the linking carrier is from 50 to 400:1.
  • the acridine substituent can be obtained by activating an acridine compound by a hydroxysuccinimide, a succinimide or a carboxylic acid.
  • the acridine substituent is a hydroxysuccinimide-activated acridine compound
  • the linking carrier may be a hemocyanin (KLH) containing an amino group, a carboxyl group and a disulfide bond, and the activated acridine substituent is reacted with the linking carrier.
  • KLH hemocyanin
  • the specific reaction process of the above reaction formula is: dissolving KLH in PBS buffer (pH 7.4), adding an acridinium ester solution dissolved in a DMSO solvent, and reacting at 20 ° C to 30 ° C for 1 h. 5 mL of 7KD molecular weight deionization column (Thermofish) was used as a buffer exchange buffer with 150 mM PBS (pH 7.4) as a buffer exchange solution, and the free acridine ester and reaction by-products were removed to obtain an intermediate product B ( ⁇ ).
  • the pyridine substitution-linker carrier conjugate, in this example the intermediate product B is specifically acridinium ester-KLH.
  • the acridine labeling conjugate comprises an acridine substituent, a linking carrier, a bifunctional crosslinking agent and a label to be labeled
  • the linking carrier contains an amino group and a carboxyl group
  • the acridine substituent reacts with an amino group on the linking carrier to form a -CO -NH- structure thereby linking the acridine substituent to the linking carrier
  • the carboxyl group attached to the carrier reacts with the hydrazide group on the bifunctional crosslinking agent to form a -CO-NH-NH- structure to thereby link the carrier to the dual function Crosslinking agent
  • the maleimide group on the bifunctional crosslinking agent reacts with the sulfhydryl group on the label to be labeled
  • the structure thereby connects the bifunctional crosslinking agent to the label to be labeled.
  • the intermediate product B obtained by the step contains a functional group amino group, a carboxyl group, a disulfide bond or the like attached to the carrier.
  • the intermediate product B may be first combined with the bifunctional crosslinking agent and then combined with the label to be labeled by the bifunctional crosslinking agent, or the bifunctional crosslinking agent may be first combined with the label to be labeled to form a bifunctional crosslinking agent - to be labeled The substance is then combined with the intermediate product B.
  • the intermediate product B is reacted with a hydrazide group on the bifunctional crosslinking agent to form a -CO-NH-NH- structure to link the linking carrier to the bifunctional crosslinking agent and then cross-link through the bifunctional crosslinking.
  • the maleimide group on the agent reacts with the sulfhydryl group on the label to form The structure thereby connects the bifunctional crosslinker to the label to be labeled to give an acridine labelled conjugate.
  • S120 includes the following steps S120A and S120B.
  • the intermediate product D is an acridine substitution-linking carrier-bifunctional crosslinking agent conjugate
  • the bifunctional crosslinking agent is a crosslinking agent containing a maleimide group and a hydrazide group
  • the linkage in the intermediate product B The carboxyl group on the support reacts with the hydrazide group on the bifunctional crosslinker to form a -CO-NH-NH- structure to link the intermediate product B to the bifunctional crosslinker.
  • the bifunctional crosslinking agent is a crosslinking agent containing a maleimide group and a hydrazide group.
  • it may be EMCH (N-[ ⁇ -maleimidoacetic acid]-hydrazide ⁇ trifluoroacetic acid), KMUH (N-[K-maleimide undecanoic acid]-hydrazide ⁇ trifluoroacetic acid Or MPBH (4-[4-N-maleimidophenyl]-butyric acid hydrazide ⁇ hydrochloric acid).
  • the molar ratio of the bifunctional crosslinking agent to the intermediate product B is from 1 to 10000:1.
  • the molar ratio of the bifunctional crosslinking agent to the intermediate product B is from 5 to 5000:1.
  • the reaction of the intermediate product B with the bifunctional crosslinking agent to obtain the intermediate product D is as follows:
  • the intermediate product B is reacted with a bifunctional crosslinking agent (EMCH) in a carbodiimide solvent (EDC) solution
  • the carbodiimide solvent may be selected from dicyclohexylcarbodiimide or 1-(3). - dimethylaminopropyl)-3-ethylcarbodiimide and N,N-diisopropylcarbodiimide.
  • EDC is selected from dicyclohexylcarbodiimide or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.
  • the intermediate product B is reacted with a bifunctional crosslinking agent (EMCH) by a carbodiimide solvent to increase the product rate.
  • EMCH bifunctional crosslinking agent
  • the molar ratio of the bifunctional crosslinking agent to the carbodiimide solution may be from 10:1 to 1:10. Further molar ratio of bifunctional crosslinker to carbodiimide is from 5:1 to 1:5.
  • the specific reaction process of the above reaction formula is: adding EDC and EMCH to the intermediate product B (azetidine ester-KLH).
  • the EDC and EMCH moles are from 10:1 to 1:10.
  • the column was passed 3 times to remove free EDC and EMCH and by-products to obtain intermediate D (acridine).
  • the intermediate product D is specifically acridinium ester-KLH-EMCH.
  • the intermediate product D is combined with the label to be labeled to obtain an acridine labeling conjugate.
  • the label to be labeled is a protein containing a thiol group, a carbonyl group or a carboxyl group, a modified protein, a polypeptide, a modified polypeptide or a carbohydrate, and the maleimide group and the label to be labeled on the bifunctional crosslinking agent in the intermediate product D Thiol-based reaction
  • the structure thus connects the intermediate product D to the object to be labeled.
  • the label to be labeled may be a protein, a modified protein, a polypeptide, a modified polypeptide or a carbohydrate having a thiol group, a carbonyl group or a carboxyl group, or a protein or a modified protein which is introduced into a thiol group, a carbonyl group or a carboxyl group by modification.
  • a polypeptide, a modified polypeptide or a carbohydrate may be a protein, a modified protein, a polypeptide, a modified polypeptide or a carbohydrate having a thiol group, a carbonyl group or a carboxyl group, or a protein or a modified protein which is introduced into a thiol group, a carbonyl group or a carboxyl group by modification.
  • the label to be labeled is an antigen, a hapten or an antibody.
  • the molar ratio of the intermediate product D to the label to be labeled is from 0.01 to 100:1. Further, the molar ratio of the intermediate product D to the label to be labeled is 0.25 to 5:1.
  • the intermediate product D is acridinium ester-KLH-EMCH, and the label to be labeled is a thiol-containing label to be labeled.
  • the reaction formula of the intermediate product D combined with the label to be labeled to obtain an acridine labeling conjugate is as follows:
  • the specific reaction process of the above reaction formula is as follows: the intermediate product D (azetidine ester-KLH-EMCH) is added to the purified solution containing thiol group (-SH), and reacted for 1 to 3 hours to obtain an acridine labeling conjugate.
  • the intermediate product D azetidine ester-KLH-EMCH
  • -SH thiol group
  • the linking carrier comprises an amino group and a disulfide bond
  • the acridine substituent reacts with an amino group on the linking carrier to form a -CO-NH- structure to link the acridine substituent to the linking carrier.
  • the disulfide bond on the linking carrier reacts with the maleimide group on the bifunctional crosslinking agent to form The structure thereby linking the linking carrier to the bifunctional crosslinking agent, and the hydrazide group on the bifunctional crosslinking agent reacts with the carbonyl or carboxyl group on the label to form a -NH-NH-CO-configuration to thereby bond the bifunctional crosslinking agent with The markers are connected.
  • the intermediate product B is formed by reacting a disulfide bond with a maleimide group on the bifunctional crosslinking agent.
  • the structure thereby connects the linking carrier to the bifunctional crosslinking agent.
  • the hydrazide group on the bifunctional crosslinker reacts with the carbonyl or carboxyl group on the label to form a -NH-NH-CO-configuration to link the bifunctional crosslinker to the label to be labeled.
  • S120 includes the following steps S120a, S120b, and S120c.
  • the intermediate product B is subjected to an activation treatment to activate a disulfide bond on the linking carrier in the intermediate product B to form a mercapto group, to obtain an intermediate product F.
  • intermediate product F is an acridine substituent-linking carrier conjugate containing a thiol group.
  • a thiol group can be formed by activation of a disulfide bond on a linking carrier in the intermediate product B by a thiol-containing solution, thereby obtaining an intermediate product F.
  • the thiol-containing solution may be DTT (dithiothreitol) or the like.
  • the reaction product of the intermediate product B is activated to obtain the intermediate product F as follows:
  • the specific reaction process of the above reaction formula is as follows: the purified intermediate product B (azetidine ester-KLH) is added to DTT at a final concentration of 1 mM to 10 mM, and mixed, and reacted at 20 ° C to 30 ° C for 10 min to 60 min. 5 mL of 7KD molecular weight deionization column (Thermofish) was used as a buffer exchange buffer with 150 mM PBS (pH 7.4) as a buffer exchange solution, and the free DTT and reaction by-products were removed to obtain an intermediate product F.
  • the intermediate product F is specifically acridinium ester-KLH-SH.
  • the intermediate product I is a label-bifunctional cross-linker conjugate
  • the label is a protein containing a thiol group, a carbonyl group or a carboxyl group, a modified protein, a polypeptide, a modified polypeptide or a carbohydrate
  • the bifunctional crosslinking agent is a crosslinking agent containing a maleimide group and a hydrazide group, and a hydrazide group on the bifunctional crosslinking agent reacts with a carbonyl group or a carboxyl group on the label to form a -NH-NH-CO-configuration to thereby bond
  • the linker is linked to the label to be labeled.
  • the label to be labeled may be a protein having a thiol group, a carbonyl group or a carboxyl group, and a modified egg.
  • the white, polypeptide, modified polypeptide or carbohydrate may also be a protein, a modified protein, a polypeptide, a modified polypeptide or a carbohydrate which is introduced into the thiol group, the carbonyl group or the carboxyl group by modification.
  • the label to be labeled is an antigen, a hapten or an antibody.
  • the bifunctional crosslinking agent is a crosslinking agent containing a maleimide group and a hydrazide group.
  • it may be EMCH (N-[ ⁇ -maleimidoacetic acid]-hydrazide ⁇ trifluoroacetic acid), KMUH (N-[K-maleimide undecanoic acid]-hydrazide ⁇ trifluoroacetic acid Or MPBH (4-[4-N-maleimidophenyl]-butyric acid hydrazide ⁇ hydrochloric acid).
  • the molar ratio of the bifunctional crosslinking agent to the label to be labeled is from 1 to 10000:1. Further, the molar ratio of the bifunctional crosslinking agent to the label to be labeled is 50 to 1000:1.
  • the reaction formula of the label and the bifunctional cross-linking agent to form the intermediate product I is as follows:
  • -R is -H, -OH or an aromatic compound.
  • the label to be labeled is reacted with a bifunctional crosslinking agent (EMCH) in a carbodiimide solvent (EDC) solution
  • the carbodiimide solvent may be selected from dicyclohexylcarbodiimide or 1-(3). - dimethylaminopropyl)-3-ethylcarbodiimide and N,N-diisopropylcarbodiimide.
  • EDC is selected from dicyclohexylcarbodiimide or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.
  • the product to be labeled is reacted with a bifunctional crosslinking agent (EMCH) by a carbodiimide solvent to increase the product rate.
  • EMCH bifunctional crosslinking agent
  • the molar ratio of the bifunctional crosslinking agent to the carbodiimide solution may be from 10:1 to 1:10. Further the molar ratio of the one-step bifunctional crosslinking agent to the carbodiimide solution is from 5:1 to 1:5.
  • the specific reaction process of the above reaction formula is: adding EDC and EMCH to the solution to be labeled.
  • the EDC and EMCH moles are from 10:1 to 1:10.
  • 5 mL of a 7 KD molecular weight deionization column (Thermofish Co., Ltd.) was used as a buffer exchange buffer with 150 mM PBS (pH 7.4) as a buffer exchange solution, and the free EDC and EMCH and by-products were removed to obtain an intermediate product I.
  • the intermediate product I is specifically the label-EMCH to be labeled.
  • the intermediate product I is combined with the intermediate product F to obtain an acridine labelled conjugate.
  • the bifunctional crosslinker in the intermediate product I reacts with a mercapto group on the intermediate product F to form a maleimide group
  • the structure thus connects the intermediate product I to the intermediate product F.
  • the molar ratio of the intermediate product I to the intermediate product F is from 0.01 to 100:1. Further, the molar ratio of the intermediate product I to the intermediate product F is from 0.2 to 20:1.
  • the reaction formula of the intermediate product I combined with the intermediate product F to obtain the acridine labeling conjugate is as follows:
  • the specific reaction process of the above reaction formula is: adding the object to be labeled, -EMCH, to the acridinium ester-KLH-SH solution, and reacting for 1 to 5 hours to obtain an acridine-labeled conjugate.
  • the acridine labeling conjugate is specifically an acridinium ester-KLH-EMCH-tag to be labeled.
  • the preparation method of the above acridine labeling conjugate is simple in process, and the acridine substituent and the label to be labeled may not pass through the carbodiimide by a linking carrier and a bifunctional crosslinking agent containing a maleimide group and a hydrazide group. Direct binding prevents the acridine substitution from interfering with the active site on the labeled protein, making the activity of the acridine-labeled conjugate relatively high, increasing the sensitivity of the immunoassay.
  • the maleimide group and the hydrazide group in the bifunctional crosslinking agent can specifically bind to the label-containing substance containing a mercapto group, a carbonyl group or a carboxyl group, thereby avoiding the fact that the acridine substituent cannot be combined with a mercapto group, a carbonyl group or a carboxyl group.
  • the defect of label binding increases the selection range of the label to be labeled, so that the specificity of the acridine labeling conjugate is high, increasing the sensitivity of the use of the acridine labeling conjugate.
  • the object to be labeled corresponding to the detected object can be connected correspondingly, because the selection range of the object to be labeled is wide, and even the object to be labeled containing a thiol group, a carbonyl group or a carboxyl group can pass through the bifunctional crosslinking agent and the connection.
  • the carrier binds to the acridine substituent to form an acridine labeling conjugate, so that upon detection, the acridine labeling conjugate can specifically bind to the analyte, increasing sensitivity and labeling rate.
  • the invention also discloses a chemiluminescent immunoassay kit comprising the above acridine labeling conjugate.
  • the chemiluminescent kit further comprises at least one of a centrifugal desalting column and a centrifugal ultrafiltration tube.
  • the chemiluminescent kit can be connected to the corresponding label to be detected in the acridine labeling conjugate according to the needs of the detection, because the selection range of the label to be labeled is wide, even if it contains a thiol group, a carbonyl group or a carboxyl group.
  • the label to be labeled can also form an acridine labeling conjugate by a bifunctional cross-linking agent and a linking carrier to form an acridine labeling conjugate, so that the acridine labeling conjugate can specifically bind to the analyte during detection, thereby improving sensitivity and Marking rate.
  • the reagents used were purchased from Sigma-aldrich, Inc., unless otherwise stated.
  • the acridinium esters present in the examples are all N-hydroxysuccinimide activated acridinium esters.
  • KLH represents a hemocyanin containing an amino group, a carboxyl group, and a disulfide bond.
  • EDC represents dicyclohexylcarbodiimide or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.
  • DTT represents dithiothreitol.
  • KLH was dissolved in 1 mL of 150 mM PBS buffer (pH 7.4) to a final concentration of 20 nmol/L. 10 ⁇ L of 10 mmol/L acridinium ester dissolved in DMSO solvent was added. The reaction was carried out at 25 ° C for 1 h, and 5 mL of a 7 KD molecular weight dehydration column (Thermofish Co., Ltd.) was used as a buffering buffer in 150 mM PBS (pH 7.4) buffer, and the column was passed 3 times to remove free acridinium ester and reaction by-products. Acridine ester-KLH solution.
  • the purified acridinium ester-KLH solution was added to DTT at a final concentration of 5 mM, mixed, and reacted at 25 ° C for 0.5 h, using a 5 mL 7KD molecular weight cut off desalination column (Thermofish) in 150 mM PBS (pH 7.4) buffer. As a liquid exchange buffer, the column was passed 3 times, and free DTT and reaction by-products were removed to obtain acridinium ester-KLH-SH.
  • the activated thyroglobulin-EMCH was added to the purified acridinium ester-KLH-SH solution and reacted for 2 hours to obtain an acridine-labeled conjugate (azetidine-KLH-EMCH-thyroglobulin) solution.
  • KLH was dissolved in 1 mL of 150 mM PBS buffer (pH 7.4) to a final concentration of 20 nmol/L, and 10 ⁇ L of 10 mmol/L acridinium ester dissolved in DMSO solvent was added. Reaction at 25 ° C for 1 h, with 5 mL A 7KD molecular weight deionization column (Thermofish Co., Ltd.) was used as a buffer exchange buffer with 50 mM MES (pH 4.5) buffer, and the free acridine ester and reaction by-products were removed by a three-pass column to obtain an acridinium ester-KLH solution.
  • EDC final concentration of 5 mmol/L
  • EMCH final concentration of 10 mmol/L
  • 150 mM PBS pH 7.4 buffer as a buffering buffer, 3 times through the column, remove free EDC and EMCH and by-products, to obtain activated acridinium ester-KLH-EMCH;
  • anti-procalcitonin mouse monoclonal antibody antibody (manufacturer: Hytest, Cat. No.: 4PC47-6F10, 6.67 nmol) was dissolved in 150 mM PBS (pH 7.4) buffer solution and treated with DTT at a final concentration of 5 mM, 25 ° C. The reaction was carried out for 0.5 h, and 5 mL of a 7 KD molecular weight dehydration column (Thermofish) was used as a buffer exchange buffer in 150 mM PBS (pH 7.4), and the column was passed 3 times to remove free DTT and reaction by-products to obtain a thiolated antibiotic.
  • Procalcitonin antibody (anti-procalcitonin antibody-SH).
  • the activated acridinium ester-KLH-EMCH was added to the purified anti-procalcitonin antibody-SH solution and reacted for 2 hours to obtain an acridine labeled conjugate (acridine ester-KLH-EMCH-anti-calcitonin small Mouse monoclonal antibody) solution.
  • KLH was dissolved in 1 mL of 150 mM PBS buffer (pH 7.4) to a final concentration of 20 nmol/L.
  • 10 ⁇ L of 10 mmol/L acridinium ester dissolved in DMSO solvent was added, and reacted at 25 ° C for 1 h, using 5 mL of 7KD molecular weight dehydration column (Thermo fish The company) used 150 mM PBS (pH 7.4) buffer as a buffering solution, and passed the column 3 times to remove the free acridinium ester and reaction by-products to obtain an acridinium ester-KLH solution.
  • the final concentration of 10 mmol/L of EMCH was added, and the reaction was carried out for 1 hour.
  • the final concentration was 20 mmol/L of lysine for 30 min, and 5 mL of 7 KD molecular weight dehydration column was used.
  • Thermo Fisher Company 150 mM PBS (pH 7.4) buffer was used as a buffering buffer, and the reaction was carried out 3 times to remove the free reaction by-products to obtain activated ⁇ 2 glycoprotein-EMCH.
  • the purified acridinium ester-KLH solution was added to a final concentration of 5 mM DTT, mixed, and reacted at 25 ° C for 0.5 h, using 5 mL of a 7 KD molecular weight cut off desalination column (Thermofish) in 150 mM PBS (pH 7.4) buffer.
  • the transfusion buffer was passed through the column for 3 times to remove free DTT and reaction by-products to obtain an acridinium ester-KLH-SH solution.
  • the activated ⁇ 2 glycoprotein-EMCH was added to the purified acridinium ester-KLH-SH solution and reacted for 2 hours to obtain a conjugate acridinium ester-KLH-EMCH-glycoprotein solution.
  • This example is a scheme in which the activated acridine ester is labeled with thyroglobulin under the same conditions under the same conditions, and 1 mg of thyroglobulin (manufacturer: biospacific, article number: J19400, 1.5 nmol) is dissolved in 1 mL of 150 mM PBS buffer (pH 7.4). Into, 10 ⁇ L of 10 mmol/L acridinium ester dissolved in DMSO solvent was added and reacted at 25 ° C for 1 h.
  • This example is a protocol in which an activated acridine ester is labeled with a mouse monoclonal antibody against procalcitonin under the same conditions, and 1 mg of anti-procalcitonin mouse monoclonal antibody (manufacturer: Hytest, The product number: 4PC47-6F10, 6.67nmol) was dissolved in 1mL 150mM PBS buffer (pH 7.4), 10ul of 10mmol/L acridinium ester dissolved in DMSO solvent was added, and reacted at 25 ° C for 1h, using 5mL 7KD molecular weight deionization column ( Thermo fish) 150 mM PBS (pH 7.4) buffer as a buffering buffer, 3 times to remove free acridinium ester and reaction by-products to obtain acridinium ester-anti-procalcitonin mouse monoclonal Antibody solution.
  • This example is a scheme in which the activated acridine ester is labeled with ⁇ 2 microglobulin under the same conditions under the same conditions, and 1 mg of ⁇ 2 glycoprotein (manufacturer: abgree, article number: G7018, 16.7 nmol) is dissolved in 1 mL of 150 mM PBS buffer (pH 7.4). Into, 10 ul of 10 mmol/L acridinium ester dissolved in DMSO solvent was added and reacted at 25 ° C for 1 h.
  • Example 1 and Comparative Example 1 were each used for chemiluminescence immunoassay detection of thyroglobulin antibody (Anti-Tg).
  • Anti-Tg thyroglobulin antibody
  • To a 500 IU/mL thyroglobulin antibody sample 40 ⁇ g of thyroglobulin-coated magnetic beads were added, and 4 nmol of acridine ester-labeled thyroglobulin prepared in Example 1 and Comparative Example 1 were separately added, and the reaction was washed and then washed.
  • Example 2 and Comparative Example 2 were each used for chemiluminescence immunoassay detection of procalcitonin (PCT).
  • PCT procalcitonin
  • Example 3 and Comparative Example 3 were used for chemiluminescence immunoassay detection of anti- ⁇ 2 glycoprotein I antibodies, respectively.
  • acridinium ester-labeled ⁇ 2 glycoprotein prepared in Example 3 and Comparative Example 3 were separately added, and the reaction was washed and washed.
  • 100 ⁇ L of HNO 3 -H 2 O 2 solution and 100 ⁇ L of sodium hydroxide solution were added successively, and the luminescence value was measured by an iFlash3000 chemiluminescence immunoassay analyzer, and three measurements were performed in parallel. The average value is shown in Table 3. The greater the luminescence value of the results obtained by immunoassay, the better the activity of the acridinium ester label.
  • the acridinium ester-KLH-EMCH-thyroglobulin solution obtained in Example 1 was used for chemiluminescence immunoassay detection of a series of concentrations of thyroglobulin antibody (Anti-Tg).
  • Anti-Tg thyroglobulin antibody
  • the luminescence value was measured by an iFlash3000 type chemiluminescence immunoassay analyzer using an O 2 solution and 100 ⁇ L of a sodium hydroxide solution. The results are shown in Table 4.
  • the thyroglobulin antibody concentration was plotted on the X-axis and the relative luminescence value was plotted on the
  • Example 1 From the results of Table 4 and Figure 4, the preparation of acridinium ester-KLH-EMCH-thyroglobulin of Example 1 can be applied to chemiluminescence immunoassay with good results.
  • the acridinium ester-KLH-EMCH-label preparation method prepared in Example 1 has good applicability.

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Abstract

公开了一种吖啶标记结合物及其制备方法、化学发光免疫检测试剂盒。吖啶标记结合物包括依次连接的吖啶取代物、连接载体、双功能交联剂和待标记物。吖啶取代物与连接载体上的氨基反应形成-CO-NH-结构从而将吖啶取代物与连接载体连接。

Description

吖啶标记结合物及其制备方法、化学发光免疫检测试剂盒 技术领域：
本发明涉及体外检测领域，尤其涉及一种吖啶标记结合物及其制备方法、化学发光免疫检测试剂盒。
背景技术：
化学发光标记免疫分析又称化学发光免疫分析(CLIA)，是用化学发光剂直接标记抗原、半抗原、抗体或碳水化合物的免疫分析方法。常用于标记的化学发光物质有吖啶类化合物(acridinium ester，AE)。其通过起动发光试剂(NaOH、H2O2)作用而发光，强烈的直接发光在一秒钟内完成，为快速的闪烁发光。
吖啶类化合物作为标记物用于免疫分析，其化学反应简单、快速、无须催化剂，检测小分子抗原采用竞争法，大分子抗原或者抗体则采用夹心法，非特异性结合少，本底低，与大分子的结合不会减小所产生的光量，从而增加灵敏度。吖啶类化合物可标记抗原抗体等形成吖啶标记结合物。吖啶标记结合物质量的好坏直接关系到化学发光免疫技术的成功与否，因此是试剂盒中一个很关键的试剂。吖啶标记结合物一般通过吖啶类化合物与特异性抗体经适当方法连接而成，吖啶类化合物和抗体将直接影响吖啶标记结合物的发光产率、背景干扰及亲和力等能力。
目前常用的吖啶标记结合物的制备方法是碳二亚胺交联法，以碳二亚胺双功能交联剂为桥，使吖啶取代物与待标记物结合。然而，传统方法制得的吖啶标记结合物中，由于吖啶取代物与待标记物需要通过碳二亚胺直接结合，吖啶取代物往往会对待标记蛋白上的活性位点形成干扰，待标记物上可选择结合的活性位点少，造成吖啶标记结合物的活性降低，影响免疫分析的灵敏 度。
发明内容：
基于此，有必要提供一种活性相对较高的吖啶标记结合物及其制备方法、化学发光免疫检测试剂盒。
一种吖啶标记结合物，包括依次连接的吖啶取代物、连接载体、双功能交联剂和待标记物；
所述双功能交联剂为含有马来酰亚胺基及酰肼基的交联剂；
所述待标记物为含有巯基、羰基或羧基的蛋白、改性蛋白、多肽、改性多肽或碳水化合物；
所述连接载体含有氨基和羧基，所述吖啶取代物与所述连接载体上的氨基反应形成-CO-NH-结构从而将所述吖啶取代物与所述连接载体连接，所述连接载体上的羧基与所述双功能交联剂上的酰肼基反应形成-CO-NH-NH-结构从而将所述连接载体与所述双功能交联剂连接，所述双功能交联剂上的马来酰亚胺基与所述待标记物上的巯基反应形成
Figure PCTCN2016088685-appb-000001
结构从而将所述双功能交联剂与所述待标记物连接。
一种吖啶标记结合物的制备方法，包括如下步骤：
用活化后的吖啶取代物与连接载体反应得到中间产物B，其中，所述中间产物B为吖啶取代物-连接载体结合物；以及
将所述中间产物B、双功能交联剂以及待标记物交联反应后得到吖啶标记结合物，其中，所述吖啶取代物与所述连接载体上的氨基反应形成-CO-NH-结构从而将所述吖啶取代物与所述连接载体连接，所述连接载体上的羧基与所述双功能交联剂上的酰肼基反应形成-CO-NH-NH-结构从而将所述连接载 体与所述双功能交联剂连接，所述双功能交联剂上的马来酰亚胺基与所述待标记物上的巯基反应形成
Figure PCTCN2016088685-appb-000002
结构从而将所述双功能交联剂与所述待标记物连接。
一种化学发光免疫检测试剂盒，包括上述任一项所述的吖啶标记结合物。
另一种吖啶标记结合物，包括依次连接的吖啶取代物、连接载体、双功能交联剂和待标记物；
所述双功能交联剂为含有马来酰亚胺基及酰肼基的交联剂；
所述待标记物为含有巯基、羰基或羧基的蛋白、改性蛋白、多肽、改性多肽或碳水化合物；
所述连接载体含有氨基及二硫键，所述吖啶取代物与所述连接载体上的氨基反应形成-CO-NH-结构从而将所述吖啶取代物与所述连接载体连接，所述连接载体上的二硫键与所述双功能交联剂上的马来酰亚胺基反应形成
Figure PCTCN2016088685-appb-000003
结构从而将所述连接载体与所述双功能交联剂连接，所述双功能交联剂上的酰肼基与所述待标记物上的羰基或羧基反应形成-NH-NH-CO-构从而将所述双功能交联剂与所述待标记物连接。
一种吖啶标记结合物的制备方法，包括如下步骤：
用活化后的吖啶取代物与连接载体反应得到中间产物B，其中，所述中间产物B为吖啶取代物-连接载体结合物；以及
将所述中间产物B、双功能交联剂以及待标记物交联反应后得到吖啶标记结合物，其中，所述吖啶标记结合物包括依次连接的吖啶取代物、连接载 体、双功能交联剂和待标记物，所述连接载体含有氨基及二硫键，所述吖啶取代物与所述连接载体上的氨基反应形成-CO-NH-结构从而将所述吖啶取代物与所述连接载体连接，所述连接载体上的二硫键与所述双功能交联剂上的马来酰亚胺基反应形成
Figure PCTCN2016088685-appb-000004
结构从而将所述连接载体与所述双功能交联剂连接，所述双功能交联剂上的酰肼基与所述待标记物上的羰基或羧基反应形成-NH-NH-CO-构从而将所述双功能交联剂与所述待标记物连接。
一种化学发光免疫检测试剂盒，包括上述任一项所述的吖啶标记结合物。
上述吖啶标记结合物包括依次连接的吖啶取代物、连接载体、双功能交联剂和待标记物。吖啶取代物与连接载体上的氨基反应形成-CO-NH-结构从而将吖啶取代物与连接载体连接。通过含有连接载体以及含有马来酰亚胺基及酰肼基的双功能交联剂，吖啶取代物与待标记物没有直接通过化学键结合，避免了吖啶取代物对待标记物上的活性位点形成干扰，使得吖啶标记结合物的活性相对较高，提高免疫分析的灵敏度。此外，双功能交联剂中的马来酰亚胺基及酰肼基可与含有巯基、羰基或羧基的待标记物特异性结合，避免了以往吖啶取代物不能与含有巯基、羰基或羧基的待标记物结合的缺陷，扩大待标记物的选择范围，使得吖啶标记结合物的特异性高，增加了吖啶标记结合物的使用灵敏度。
附图说明
图1为一实施方式的吖啶标记结合物的制备方法的流程图；
图2为一实施方式的由中间产物B、双功能交联剂以及待标记物交联反应后得到吖啶标记结合物的制备流程图；
图3为另一实施方式的由中间产物B、双功能交联剂以及待标记物交联反应后得到吖啶标记结合物的制备流程图；
图4为测试例4中得到的系列浓度甲状腺球蛋白抗体样本检验结果散点图。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合附图和具体实施例对本发明的具体实施方式做详细的说明。在下面的描述中阐述了很多具体细节以便于充分理解本发明。但是本发明能够以很多不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似改进，因此本发明不受下面公开的具体实施的限制。
一种吖啶标记结合物，包括依次连接的吖啶取代物、连接载体、双功能交联剂和待标记物。双功能交联剂为含有马来酰亚胺基及酰肼基的交联剂。待标记物为含有巯基、羰基或羧基的蛋白、改性蛋白、多肽、改性多肽或碳水化合物。
一实施方式中，连接载体含有氨基和羧基，吖啶取代物与连接载体上的氨基反应形成-CO-NH-结构从而将吖啶取代物与连接载体连接。连接载体上的羧基与双功能交联剂上的酰肼基反应形成-CO-NH-NH-结构从而将连接载体与双功能交联剂连接，双功能交联剂上的马来酰亚胺基与待标记物上的巯基反应形成
Figure PCTCN2016088685-appb-000005
结构从而将双功能交联剂与待标记物连接。
另一实施方式中，连接载体含有氨基及二硫键，吖啶取代物与连接载体上的氨基反应形成-CO-NH-结构从而将吖啶取代物与连接载体连接。连接载体上的二硫键与双功能交联剂上的马来酰亚胺基反应形成
Figure PCTCN2016088685-appb-000006
结构从而将连接载体与双功能交联剂连接，双功能交联剂上的酰肼基与待标记物上的羰基或羧基反应形成-NH-NH-CO-构从而将双功能交联剂与待标记物连接。
具体的，吖啶取代物为吖啶酯、吖啶酸、吖啶酰胺或吖啶磺酰胺。标记用吖啶酯，具体可以为AE-NHS(10-甲基-吖啶-9-N-琥珀酰亚胺酯甲酸酯)、DMAE-NHS(2’，6’-二甲基-4’-(N-琥珀酰亚胺氧羰基)苯基-吖啶-9-甲酸酯)、ME-DMAE-NHS(2’，6’-二甲基羰基苯基-10-甲基-9-吖啶甲酸酯-4’-N-琥珀酰亚胺酯-三氟甲基磺酸盐)等。
具体的，连接载体可以为含有氨基、羧基及二硫键的血蓝蛋白(KLH)。当然，在其它同时含有氨基、羧基及二硫键的载体也可以作为本吖啶标记结合物中的连接载体。连接载体同时含有氨基、羧基及二硫键这三种功能性基团，可以与双功能交联剂或待标记物发生多种结合方式。
具体的，双功能交联剂为含有马来酰亚胺基及酰肼基的交联剂。具体可以为EMCH(N-[ε-马来酰亚胺乙酸]酰肼·三氟乙酸)、KMUH(N-[-马来酰亚胺十一烷酸]酰肼·三氟乙酸)或MPBH(4-[-N-马来酰亚胺苯基]丁酸酰肼·盐酸)等
具体的，待标记物可以为本身就具有巯基、羰基或羧基的蛋白、改性蛋白、多肽、改性多肽或碳水化合物，也可以为通过修饰后引入巯基、羰基或羧基的蛋白、改性蛋白、多肽、改性多肽或碳水化合物。
具体的，待标记物为抗原、半抗原或抗体。
以吖啶取代物为吖啶酯，连接载体为含有氨基、羧基及二硫键的血蓝蛋白(KLH)、双功能交联剂为含有马来酰亚胺基为例，吖啶标记结合物具有如下结构式：
Figure PCTCN2016088685-appb-000007
其中，
Figure PCTCN2016088685-appb-000008
表示吖啶类化合物，
Figure PCTCN2016088685-appb-000009
表示血蓝蛋白，
Figure PCTCN2016088685-appb-000010
为待标记物。
另一实施方式的吖啶标记结合物具有如下结构式：
Figure PCTCN2016088685-appb-000011
其中，
Figure PCTCN2016088685-appb-000012
表示吖啶类化合物，
Figure PCTCN2016088685-appb-000013
表示血蓝蛋白，
Figure PCTCN2016088685-appb-000014
为待标记物。
上述吖啶标记结合物包括依次连接的吖啶取代物、连接载体、双功能交联剂和待标记物。通过含有氨基、羧基及二硫键的连接载体以及含有马来酰亚胺基及酰肼基的双功能交联剂，吖啶取代物与待标记物可以不通过碳二亚胺直接结合，避免了吖啶取代物对待标记蛋白上的活性位点形成干扰，使得吖啶标记结合物的活性相对较高，提高免疫分析的灵敏度。
此外，双功能交联剂中的马来酰亚胺基及酰肼基可与含有巯基、羰基或羧基的待标记物特异性结合，避免了以往吖啶取代物不能与含有巯基、羰基或羧基的待标记物结合的缺陷，扩大待标记物的选择范围，使得吖啶标记结合物的特异性高，增加了吖啶标记结合物的使用灵敏度。可以根据检测的需要，相应的连接该被检测物对应的待标记物，因为待标记物的选择范围宽，即使是含有巯基、羰基或羧基的待标记物也能通过双功能交联剂以及连接载体与吖啶取代物结合形成吖啶标记结合物，因此在检测时，吖啶标记结合物可与被检测物特异性结合，灵敏度高，标记率高。
这种吖啶标记结合物，吖啶取代物与连接载体上的氨基反应形成-CO-NH-结构从而将吖啶取代物与连接载体连接，吖啶取代物与待标记物之间没有直接发生化学键反应。而传统的方法一般以碳二亚胺双功能交联剂为桥，使吖啶取代物与待标记物直接结合，这样标记率较低，活性不好，且标记基团选择性少，不能直接跟含羰基、巯基的待结合物结合。与传统吖啶标记结合物相比，本发明的吖啶标记结合物标记效率较高，可以直接用于化学发光免疫分析的检测和定量分析，并且能解决传统的碳二亚胺交联法等方法制备的吖啶标记结合物原料失活、标记效率低、标记基团选择性少等缺点。
如图1所示的上述的吖啶标记结合物的制备方法，包括如下步骤S110～S120。
S110、用活化后的吖啶取代物与连接载体反应得到中间产物B。
其中，中间产物B为吖啶取代物-连接载体结合物，连接载体含有氨基和羧基或者连接载体含有氨基及二硫键，更进一步的，连接载体可以为含有氨基、羧基及二硫键的连接载体，吖啶取代物与连接载体上的氨基反应形成-CO-NH-结构从而将吖啶取代物与连接载体连接。
具体的，吖啶取代物为吖啶酯、吖啶酸、吖啶酰胺或吖啶磺酰胺。具体可以为AE-NHS(10-甲基-吖啶-9-N-琥珀酰亚胺酯甲酸酯)、DMAE-NHS(2’，6’-二甲基-4’-(N-琥珀酰亚胺氧羰基)苯基-吖啶-9-甲酸酯)、ME-DMAE-NHS(2’，6’-二甲基羰基苯基-10-甲基-9-吖啶甲酸酯-4’-N-琥珀酰亚胺酯-三氟甲基磺酸盐)等。
具体的，连接载体可以为含有氨基、羧基及二硫键的血蓝蛋白(KLH)。当然，在其它同时含有氨基、羧基及二硫键的载体也可以作为本吖啶标记结合物中的连接载体。
用活化后的吖啶取代物与连接载体反应得到中间产物B的操作中，活化后的吖啶取代物与连接载体的摩尔比为1～5000∶1。
更进一步的，活化后的吖啶取代物与连接载体的摩尔比为50～400∶1。
吖啶取代物可通过羟基琥珀酰亚胺、琥珀酰亚胺或羧酸等活化吖啶类化合物后得到。
吖啶取代物为羟基琥珀酰亚胺活化的吖啶化合物、连接载体可以为含有氨基、羧基及二硫键的血蓝蛋白(KLH)为例，用活化后的吖啶取代物与连接载体反应得到中间产物B的反应式如下：
Figure PCTCN2016088685-appb-000015
其中，
Figure PCTCN2016088685-appb-000016
表示吖啶类化合物，
Figure PCTCN2016088685-appb-000017
表示血蓝蛋白。
上述反应式的具体反应过程为：将KLH溶解于PBS缓冲液(pH 7.4)中，加入用DMSO溶剂溶解的吖啶酯溶液，于20℃～30℃反应1h。用5mL 7KD截留分子量的脱盐柱(Thermo fish公司)以150mM PBS(pH 7.4)缓冲液作为换液缓冲液，过柱3遍，除去游离的吖啶酯及反应副产物，得到中间产物B(吖啶取代物-连接载体结合物)，本实施例中中间产物B具体为吖啶酯-KLH。
S120、将中间产物B、双功能交联剂以及待标记物交联反应后得到吖啶标记结合物。
其中，吖啶标记结合物包括依次连接的吖啶取代物、连接载体、双功能交联剂和待标记物，连接载体含有氨基和羧基，吖啶取代物与连接载体上的氨基反应形成-CO-NH-结构从而将吖啶取代物与连接载体连接，连接载体上的羧基与双功能交联剂上的酰肼基反应形成-CO-NH-NH-结构从而将连接载体与所述双功能交联剂连接，双功能交联剂上的马来酰亚胺基与待标记物上的巯基反应形成
Figure PCTCN2016088685-appb-000018
结构从而将双功能交联剂与待标记物连接。
通过步骤得到的中间产物B，含有连接载体上的功能性基团氨基、羧基及二硫键等。中间产物B可以先与双功能交联剂结合再通过双功能交联剂与待标记物结合，也可以是先将双功能交联剂与待标记物结合，形成双功能交联剂-待标记物，然后再与中间产物B结合。
在一个实施方式中，中间产物B通过羧基与双功能交联剂上的酰肼基反应形成-CO-NH-NH-结构从而将连接载体与双功能交联剂连接，再通过双功能交联剂上的马来酰亚胺基与待标记物上的巯基反应形成
Figure PCTCN2016088685-appb-000019
结构从而将双功能交联剂与待标记物连接得到吖啶标记结合物。具体如图2所示，S120 包括以下步骤S120A和S120B。
S120A、用中间产物B与双功能交联剂反应生成中间产物D。
其中，中间产物D为吖啶取代物-连接载体-双功能交联剂结合物，双功能交联剂为含有马来酰亚胺基及酰肼基的交联剂，中间产物B中的连接载体上的羧基与双功能交联剂上的酰肼基反应形成-CO-NH-NH-结构从而将中间产物B与双功能交联剂连接。
具体的，双功能交联剂为含有马来酰亚胺基及酰肼基的交联剂。具体可以为EMCH(N-[ε-马来酰亚胺乙酸]-酰肼·三氟乙酸)、KMUH(N-[K-马来酰亚胺十一烷酸]-酰肼·三氟乙酸)或MPBH(4-[4-N-马来酰亚胺苯基]-丁酸酰肼·盐酸)等。
用中间产物B与双功能交联剂反应生成中间产物D的操作中，双功能交联剂与中间产物B的摩尔比为1～10000∶1。
更进一步的，双功能交联剂与中间产物B的摩尔比为5～5000∶1。
以中间产物B为吖啶酯-KLH、双功能交联剂为EMCH为例，中间产物B与双功能交联剂反应得到中间产物D的反应式如下：
Figure PCTCN2016088685-appb-000020
其中，
Figure PCTCN2016088685-appb-000021
表示吖啶类化合物，
Figure PCTCN2016088685-appb-000022
表示血蓝蛋白。
本实施方式中，中间产物B与双功能交联剂(EMCH)在碳二亚胺溶剂(EDC)溶液中反应，碳二亚胺溶剂可以选自二环己基碳二亚胺或1-(3-二甲氨基丙基)-3-乙基碳二亚胺和N，N-二异丙基碳二亚胺。本实施方式中，EDC选用二环己基碳二亚胺或1-(3-二甲氨基丙基)-3-乙基碳二亚胺。通过碳二亚胺溶剂促进中间产物B与双功能交联剂(EMCH)反应，提高产物率。
具体的，双功能交联剂与碳二亚胺溶的摩尔比例可以为10∶1～1∶10。更进一步的双功能交联剂与碳二亚胺溶的摩尔比例为5∶1～1∶5。
上述反应式的具体反应过程为：将EDC和EMCH加入到中间产物B(吖啶酯-KLH)中。其中，EDC与EMCH摩尔为10∶1～1∶10。在20℃～25℃反应10min～60min后。用5mL 7KD截留分子量的脱盐柱(Thermo fish公司)以150mM PBS(pH 7.4)缓冲液作为换液缓冲液，过柱3遍，除去游离的EDC和EMCH及副产物，得到中间产物D(吖啶取代物-连接载体-双功能交联剂结合物)，本实施方式中，中间产物D具体为吖啶酯-KLH-EMCH。
S120B、将中间产物D与待标记物结合得到吖啶标记结合物。
其中，待标记物为含有巯基、羰基或羧基的蛋白、改性蛋白、多肽、改性多肽或碳水化合物，中间产物D中的双功能交联剂上的马来酰亚胺基与待标记物上的巯基反应形成
Figure PCTCN2016088685-appb-000023
结构从而将中间产物D与待标记物连接。
具体的，待标记物可以为本身就具有巯基、羰基或羧基的蛋白、改性蛋白、多肽、改性多肽或碳水化合物，也可以为通过修饰后引入巯基、羰基或羧基的蛋白、改性蛋白、多肽、改性多肽或碳水化合物。
本实施方式中，待标记物为抗原、半抗原或抗体。
将中间产物D与待标记物结合得到吖啶标记结合物的操作中，中间产物D与待标记物的摩尔比为0.01～100∶1。进一步的，中间产物D与待标记物的摩尔比为0.25～5∶1。
以中间产物D为吖啶酯-KLH-EMCH，待标记物为含有巯基的待标记物 为例，中间产物D与待标记物结合得到吖啶标记结合物的反应式如下：
Figure PCTCN2016088685-appb-000024
其中，
Figure PCTCN2016088685-appb-000025
表示吖啶类化合物，
Figure PCTCN2016088685-appb-000026
表示血蓝蛋白，
Figure PCTCN2016088685-appb-000027
为待标记物。
上述反应式的具体反应过程为：将中间产物D(吖啶酯-KLH-EMCH)加入纯化好含有巯基(-SH)的待标记物溶液中，反应1h～3h，得到吖啶标记结合物。
在另一个吖啶标记结合物的制备方法中，连接载体含有氨基及二硫键，吖啶取代物与连接载体上的氨基反应形成-CO-NH-结构从而将吖啶取代物与连接载体连接，连接载体上的二硫键与双功能交联剂上的马来酰亚胺基反应形成
Figure PCTCN2016088685-appb-000028
结构从而将连接载体与双功能交联剂连接，双功能交联剂上的酰肼基与待标记物上的羰基或羧基反应形成-NH-NH-CO-构从而将双功能交联剂与待标记物连接。中间产物B通过二硫键与双功能交联剂上的马来酰亚胺基反应形成
Figure PCTCN2016088685-appb-000029
结构从而将连接载体与双功能交联剂连接。双功能交联剂上的酰肼基与待标记物上的羰基或羧基反应形成-NH-NH-CO-构从而将双功能交联剂与待标记物连接。具体如图3所示，在本实施方式中，S120包括 以下步骤S120a、S120b以及S120c。
S120a、将中间产物B进行活化处理，使得中间产物B中的连接载体上的二硫键活化形成巯基，得到中间产物F。
其中，中间产物F为含有巯基的吖啶取代物-连接载体结合物。
具体的，可以通过含巯基的溶液对中间产物B中的连接载体上的二硫键活化形成巯基，从而得到中间产物F。含巯基的溶液可以为DTT(二硫苏糖醇)等。
以中间产物B为吖啶酯-KLH、活化处理剂为DTT溶液为例，将中间产物B进行活化处理得到中间产物F的反应式如下：
Figure PCTCN2016088685-appb-000030
其中，
Figure PCTCN2016088685-appb-000031
表示吖啶类化合物，
Figure PCTCN2016088685-appb-000032
表示血蓝蛋白。
上述反应式的具体反应过程为：将纯化后的中间产物B(吖啶酯-KLH)，加入终浓度为1mM～10mM的DTT，混匀，20℃～30℃反应10min～60min。用5mL 7KD截留分子量的脱盐柱(Thermofish公司)以150mM PBS(pH 7.4)缓冲液作为换液缓冲液，过柱3遍，除去游离的DTT及反应副产物，得到中间产物F。本实施方式中，中间产物F具体为吖啶酯-KLH-SH。
S120b、将待标记物与双功能交联剂反应生成中间产物I。
其中，中间产物I为待标记物-双功能交联剂结合物，待标记物为含有巯基、羰基或羧基的蛋白、改性蛋白、多肽、改性多肽或碳水化合物，双功能交联剂为含有马来酰亚胺基及酰肼基的交联剂，双功能交联剂上的酰肼基与待标记物上的羰基或羧基反应形成-NH-NH-CO-构从而将双功能交联剂与待标记物连接。
具体的，待标记物可以为本身就具有巯基、羰基或羧基的蛋白、改性蛋 白、多肽、改性多肽或碳水化合物，也可以为通过修饰后引入巯基、羰基或羧基的蛋白、改性蛋白、多肽、改性多肽或碳水化合物。
本实施方式中，待标记物为抗原、半抗原或抗体。
具体的，双功能交联剂为含有马来酰亚胺基及酰肼基的交联剂。具体可以为EMCH(N-[ε-马来酰亚胺乙酸]-酰肼·三氟乙酸)、KMUH(N-[K-马来酰亚胺十一烷酸]-酰肼·三氟乙酸)或MPBH(4-[4-N-马来酰亚胺苯基]-丁酸酰肼·盐酸)等。
将待标记物与双功能交联剂反应生成中间产物I的操作中，双功能交联剂与待标记物的摩尔比为1～10000∶1。更进一步的，双功能交联剂与待标记物的摩尔比为50～1000∶1
以待标记物为含有羰基或羧基的待标记物、双功能交联剂为EMCH为例，待标记物与双功能交联剂反应生成中间产物I的反应式如下：
Figure PCTCN2016088685-appb-000033
其中，
Figure PCTCN2016088685-appb-000034
为待标记物，-R为-H、-OH或芳香族化合物。
本实施方式中，待标记物与双功能交联剂(EMCH)在碳二亚胺溶剂(EDC)溶液中反应，碳二亚胺溶剂可以选自二环己基碳二亚胺或1-(3-二甲氨基丙基)-3-乙基碳二亚胺和N，N-二异丙基碳二亚胺。本实施方式中，EDC选用二环己基碳二亚胺或1-(3-二甲氨基丙基)-3-乙基碳二亚胺。通过碳二亚胺溶剂促进待标记物与双功能交联剂(EMCH)反应，提高产物率。
具体的，双功能交联剂与碳二亚胺溶的摩尔比例可以为10∶1～1∶10。更进 一步的双功能交联剂与碳二亚胺溶的摩尔比例为5∶1～1∶5。
上述反应式的具体反应过程为：将EDC和EMCH加入到待标记物溶液中。其中，EDC与EMCH摩尔为10∶1～1∶10。在20℃～25℃反应10min～60min后。用5mL 7KD截留分子量的脱盐柱(Thermo fish公司)以150mM PBS(pH 7.4)缓冲液作为换液缓冲液，过柱3遍，除去游离的EDC和EMCH及副产物，得到中间产物I。本实施方式中，中间产物I具体为待标记物-EMCH。
S120c、将中间产物I与中间产物F结合得到吖啶标记结合物。
其中，中间产物I中的双功能交联剂上马来酰亚胺基与中间产物F上的巯基反应形成
Figure PCTCN2016088685-appb-000035
结构从而将中间产物I与所述中间产物F连接。
具体的，将中间产物I与中间产物F结合得到吖啶标记结合物的操作中，中间产物I与中间产物F的摩尔比为0.01～100∶1。更进一步的，中间产物I与中间产物F的摩尔比为0.2～20∶1。
以中间产物I为待标记物-EMCH、中间产物F为吖啶酯-KLH-SH为例，中间产物I与中间产物F结合得到吖啶标记结合物的反应式如下：
Figure PCTCN2016088685-appb-000036
其中，
Figure PCTCN2016088685-appb-000037
表示吖啶类化合物，
Figure PCTCN2016088685-appb-000038
表示血蓝蛋白，
Figure PCTCN2016088685-appb-000039
为待标记 物。
上述反应式的具体反应过程为：将待标记物-EMCH加入吖啶酯-KLH-SH溶液中，反应1h～5h，得到吖啶标记结合物。本实施方式中，吖啶标记结合物具体为吖啶酯-KLH-EMCH-待标记物。
上述吖啶标记结合物的制备方法，工艺简单，通过连接载体以及含有马来酰亚胺基及酰肼基的双功能交联剂，吖啶取代物与待标记物可以不通过碳二亚胺直接结合，避免了吖啶取代物对待标记蛋白上的活性位点形成干扰，使得吖啶标记结合物的活性相对较高，提高免疫分析的灵敏度。双功能交联剂中的马来酰亚胺基及酰肼基可与含有巯基、羰基或羧基的待标记物特异性结合，避免了以往吖啶取代物不能与含有巯基、羰基或羧基的待标记物结合的缺陷，扩大待标记物的选择范围，使得吖啶标记结合物的特异性高，增加了吖啶标记结合物的使用灵敏度。可以根据检测的需要，相应的连接该被检测物对应的待标记物，因为待标记物的选择范围宽，即使是含有巯基、羰基或羧基的待标记物也能通过双功能交联剂以及连接载体与吖啶取代物结合形成吖啶标记结合物，因此在检测时，吖啶标记结合物可与被检测物特异性结合，提高灵敏度高和标记率。
本发明还公开了一种化学发光免疫检测试剂盒，该化学发光免疫检测试剂盒包括上述吖啶标记结合物。
在一个实施方式中，这种化学发光试剂盒还包括离心脱盐柱和离心超滤管中的至少一种。
这种化学发光试剂盒可以根据检测的需要，在吖啶标记结合物中相应的连接该被检测物对应的待标记物，因为待标记物的选择范围宽，即使是含有巯基、羰基或羧基的待标记物也能通过双功能交联剂以及连接载体与吖啶取代物结合形成吖啶标记结合物，因此在检测时，吖啶标记结合物可与被检测物特异性结合，提高灵敏度高和标记率。
以下为具体实施例。
实施例中，除非特殊说明，否则所使用的试剂购自Sigma-aldrich公司，为分析纯。实施例中出现的吖啶酯均为N-羟基琥珀酰亚胺活化的吖啶酯。KLH表示含有氨基、羧基及二硫键的血蓝蛋白。EDC表示二环己基碳二亚胺或1-(3-二甲氨基丙基)-3-乙基碳二亚胺。DTT表示二硫苏糖醇。
实施例1
将KLH溶解于1mL150mM PBS缓冲液(pH 7.4)中，终浓度为20nmol/L。加入10μL溶解于DMSO溶剂的10mmol/L吖啶酯。于25℃反应1h，用5mL7KD截留分子量的脱盐柱(Thermo fish公司)以150mM PBS(pH 7.4)缓冲液作为换液缓冲液，过柱3遍，除去游离的吖啶酯及反应副产物，得到吖啶酯-KLH溶液。
将1mg甲状腺球蛋白(厂家：biospacific，货号：J19400，1.5nmol)溶解于1mL 50mM MES缓冲液(pH 4.5)中，加入EDC(终浓度为5mmol/L)和EMCH(终浓度为10mmol/L)，25℃条件下反应30分钟后，用5mL 7KD截留分子量的脱盐柱(Thermo fish公司)以150mM PBS(pH 7.4)缓冲液作为换液缓冲液，过柱3遍，除去游离的EDC和EMCH及副产物，得到活化后的甲状腺球蛋白-EMCH。
将纯化后的吖啶酯-KLH溶液，加入终浓度为5mM的DTT处理，混匀，25℃反应0.5h，用5mL 7KD截留分子量的脱盐柱(Thermofish公司)以150mM PBS(pH 7.4)缓冲液作为换液缓冲液，过柱3遍，除去游离的DTT及反应副产物，得到吖啶酯-KLH-SH。
将活化后的甲状腺球蛋白-EMCH加入纯化好的吖啶酯-KLH-SH溶液中，反应2h，得到吖啶标记结合物(吖啶酯-KLH-EMCH-甲状腺球蛋白)溶液。
实施例2
将KLH溶解于1mL150mM PBS缓冲液(pH 7.4)中，终浓度为20nmol/L，加入10μL溶解于DMSO溶剂的10mmol/L吖啶酯。于25℃反应1h，用5mL 7KD截留分子量的脱盐柱(Thermo fish公司)以50mM MES(pH 4.5)缓冲液作为换液缓冲液，过柱3遍，除去游离的吖啶酯及反应副产物，得到吖啶酯-KLH溶液。
将EDC(终浓度为5mmol/L)和EMCH(终浓度为10mmol/L)加入到吖啶酯-KLH中，25℃反应30分钟后，用5mL 7KD截留分子量的脱盐柱(Thermo fish公司)以150mM PBS(pH 7.4)缓冲液作为换液缓冲液，过柱3遍，除去游离的EDC和EMCH及副产物，得到活化后的吖啶酯-KLH-EMCH；
将1mg抗降钙素原的小鼠单克隆抗体抗体(厂家：Hytest，货号：4PC47-6F10，6.67nmol)溶解于150mM PBS(pH 7.4)缓冲液溶液，用终浓度5mM的DTT处理，25℃反应0.5h，用5mL 7KD截留分子量的脱盐柱(Thermofish公司)以150mM PBS(pH 7.4)缓冲液作为换液缓冲液，过柱3遍，除去游离的DTT及反应副产物，得到巯基化的抗降钙素原抗体(抗降钙素原抗体-SH)。
将活化后的吖啶酯-KLH-EMCH加入纯化好的抗降钙素原抗体-SH溶液中，反应2h，得到吖啶标记结合物(吖啶酯-KLH-EMCH-抗降钙素原小鼠单克隆抗体)溶液。
实施例3
将KLH溶解于1mL150mM PBS缓冲液(pH 7.4)中，终浓度20nmol/L，加入10μL溶解于DMSO溶剂的10mmol/L吖啶酯，于25℃反应1h，用5mL7KD截留分子量的脱盐柱(Thermo fish公司)以150mM PBS(pH 7.4)缓冲液作为换液缓冲液，过柱3遍，除去游离的吖啶酯及反应副产物，得到吖啶酯-KLH溶液。
将1mg的β2糖蛋白(厂家：abgree，货号：G7018，16.7nmol)溶解于1mL 50mM MES缓冲液(pH 4.5)中，加入偏高碘酸钠(终浓度为10mmol/L)，25℃反应30分钟后，加20ul的乙二醇淬灭反应30min，用5mL 7KD截留分子量的脱盐柱(Thermo fish公司)以150mM PBS(pH 7.4)缓冲液作为换液 缓冲液，过柱3遍，除去游离反应副产物，加入终浓度为10mmol/L的EMCH，反应1h，加入终浓度为20mmol/L的赖氨酸反应30min，用5mL 7KD截留分子量的脱盐柱(Thermo fish公司)以150mM PBS(pH 7.4)缓冲液作为换液缓冲液，过柱3遍，除去游离反应副产物，得到活化后的β2糖蛋白-EMCH。
将纯化后的吖啶酯-KLH溶液，加入终浓度5mM的DTT处理，混匀，25℃反应0.5h，用5mL 7KD截留分子量的脱盐柱(Thermofish公司)以150mM PBS(pH 7.4)缓冲液作为换液缓冲液，过柱3遍，除去游离的DTT及反应副产物，得到吖啶酯-KLH-SH溶液。
将活化后的β2糖蛋白-EMCH加入纯化好的吖啶酯-KLH-SH溶液中，反应2h，得到结合物吖啶酯-KLH-EMCH-糖蛋白溶液。
对比例1
此实施例为活化后的吖啶酯在同等条件下用常规方法标记甲状腺球蛋白的方案，将1mg甲状腺球蛋白(厂家：biospacific，货号：J19400，1.5nmol)溶解于1mL150mM PBS缓冲液(pH 7.4)中，加入10μL溶解于DMSO溶剂的10mmol/L吖啶酯，于25℃反应1h。用5mL 7KD截留分子量的脱盐柱(Thermo fish公司)以150mM PBS(pH 7.4)缓冲液作为换液缓冲液，过柱3遍，除去游离的吖啶酯及反应副产物，得到吖啶酯-甲状腺球蛋白溶液。
对比例2
此实施例为活化后的吖啶酯在同等条件下用常规方法标记抗降钙素原的小鼠单克隆抗体的方案，将1mg抗降钙素原的小鼠单克隆抗体(厂家：Hytest，货号：4PC47-6F10，6.67nmol)溶解于1mL150mM PBS缓冲液(pH 7.4)中，加入10ul溶解于DMSO溶剂的10mmol/L吖啶酯，于25℃反应1h，用5mL 7KD截留分子量的脱盐柱(Thermo fish公司)以150mM PBS(pH 7.4)缓冲液作为换液缓冲液，过柱3遍，除去游离的吖啶酯及反应副产物，得到吖啶酯-抗降钙素原的小鼠单克隆抗体溶液。
对比例3
此实施例为活化后的吖啶酯在同等条件下用常规方法标记β2微球蛋白的方案，将1mgβ2糖蛋白(厂家：abgree，货号：G7018，16.7nmol)溶解于1mL150mM PBS缓冲液(pH 7.4)中，加入10ul溶解于DMSO溶剂的10mmol/L吖啶酯，于25℃反应1h。用5mL 7KD截留分子量的脱盐柱(Thermo fish公司)以150mM PBS(pH 7.4)缓冲液作为换液缓冲液，过柱3遍，除去游离的吖啶酯及反应副产物，得到吖啶酯-β2糖蛋白溶液。
测试例1
将实施例1和对比例1中得到的结合物分别用于甲状腺球蛋白抗体(Anti-Tg)的化学发光免疫分析检测。向500IU/mL的甲状腺球蛋白抗体样本中，加入40μg甲状腺球蛋白包被的磁珠，再分别加入实施例1和对比例1制备的4nmoL吖啶酯标记甲状腺球蛋白，孵育反应后清洗，先后加入100μLHNO3-H2O2溶液和100μL的氢氧化钠溶液，用iFlash3000型化学发光免疫分析仪测量发光值，平行进行3份测量，取平均值。结果见表1。免疫分析所得结果的发光值越大，说明吖啶酯标记物的活性越好。
表1：实施例1和对比例1中得到的结合物检测Anti-Tg对比结果
吖啶酯标记物 平均发光值(RLU)
实施例1 2015672
对比例1 431557
由表1结果可知，采用实施例1制备得到的吖啶标记结合物的活性显著优于对比例1制备的结合物。
测试例2
将实施例2和对比例2中得到的结合物分别用于降钙素原(PCT)的化学发光免疫分析检测。向30ng/ml的降钙素原样本中，加入40μg抗降钙素原的小鼠单克隆抗体包被的磁珠，再分别加入实施例2和对比例2制备的10nmol 吖啶酯标记甲状腺球蛋白，孵育反应后清洗，先后加入100μLHNO3-H2O2溶液和100μL的氢氧化钠溶液，用iFlash3000型化学发光免疫分析仪测量发光值，平行进行3份测量，取平均值，结果见表2。免疫分析所得结果的发光值越大，说明吖啶酯标记物的活性越好。
表2：实施例2和对比例2中得到的结合物检测PCT对比结果
吖啶酯标记物 平均发光值(RLU)
实施例2 6358457
对比例2 865473
由表2结果可知，采用实施例2制备得到的吖啶标记结合物的活性显著优于对比例2制备的结合物。
测试例3
将实施例3和对比例3中得到的结合物分别用于抗β2糖蛋白I抗体的化学发光免疫分析检测。向50AU/mL的抗β2糖蛋白I抗体样本中，加入40μgβ2糖蛋白包被的磁珠，再分别加入实施例3和对比例3制备的1nmol吖啶酯标记β2糖蛋白，孵育反应后清洗，先后加入100μL HNO3-H2O2溶液和100μL的氢氧化钠溶液，用iFlash3000型化学发光免疫分析仪测量发光值，平行进行3份测量，取平均值结果见表3。免疫分析所得结果的发光值越大，说明吖啶酯标记物的活性越好。
表3：实施例3和对比例3中得到的结合物检测抗β2糖蛋白I抗体对比结果
吖啶酯标记物 平均发光值(RLU)
实施例3 1843162
对比例3 546317
由表3的结果可知，采用实施例3制备得到的吖啶标记结合物的活性显著优于对比例3制备的结合物。
测试例4
将实施例1中得到的吖啶酯-KLH-EMCH-甲状腺球蛋白溶液用于系列浓度的甲状腺球蛋白抗体(Anti-Tg)的化学发光免疫分析检测。向系列浓度的Anti-Tg样本溶液中分别加入40μg甲状腺球蛋白包被的磁珠试剂和4nmoL吖啶酯-KLH-EMCH-甲状腺球蛋白试剂，孵育反应后清洗，先后加入100μL HNO3-H2O2溶液和100μL的氢氧化钠溶液，用iFlash3000型化学发光免疫分析仪测量发光值，结果见表4。以甲状腺球蛋白抗体浓度为X轴，以相对发光值为Y轴，由表4数据作图，得图4。
表4：系列浓度甲状腺球蛋白抗体样本的免疫检测结果
Figure PCTCN2016088685-appb-000040
由表4和图4结果可知，实施例1制备吖啶酯-KLH-EMCH-甲状腺球蛋白可应用于化学发光免疫分析且效果良好。实施例1制备的吖啶酯-KLH-EMCH-标记物制备方法具有良好的适用性。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (17)

  1. 一种吖啶标记结合物，其特征在于，包括依次连接的吖啶取代物、连接载体、双功能交联剂和待标记物；
    所述双功能交联剂为含有马来酰亚胺基及酰肼基的交联剂；
    所述待标记物为含有巯基、羰基或羧基的蛋白、改性蛋白、多肽、改性多肽或碳水化合物；
    所述连接载体含有氨基和羧基，所述吖啶取代物与所述连接载体上的氨基反应形成-CO-NH-结构从而将所述吖啶取代物与所述连接载体连接，所述连接载体上的羧基与所述双功能交联剂上的酰肼基反应形成-CO-NH-NH-结构从而将所述连接载体与所述双功能交联剂连接，所述双功能交联剂上的马来酰亚胺基与所述待标记物上的巯基反应形成
    Figure PCTCN2016088685-appb-100001
    结构从而将所述双功能交联剂与所述待标记物连接。
  2. 根据权利要求1所述的吖啶标记结合物，其特征在于，所述吖啶取代物为吖啶酯、吖啶酸、吖啶酰胺或吖啶磺酰胺。
  3. 根据权利要求1所述的吖啶标记结合物，其特征在于，所述连接载体为含有氨基、羧基及二硫键的血蓝蛋白。
  4. 根据权利要求1所述的吖啶标记结合物，其特征在于，所述待标记物为抗原、半抗原或抗体。
  5. 一种吖啶标记结合物的制备方法，包括如下步骤0
    用活化后的吖啶取代物与连接载体反应得到中间产物B，其中，所述中间产物B为吖啶取代物-连接载体结合物；以及
    将所述中间产物B、双功能交联剂以及待标记物交联反应后得到吖啶标记结合物，其中，所述吖啶标记结合物包括依次连接的吖啶取代物、连接载 体、双功能交联剂和待标记物，所述连接载体含有氨基和羧基，所述吖啶取代物与所述连接载体上的氨基反应形成-CO-NH-结构从而将所述吖啶取代物与所述连接载体连接，所述连接载体上的羧基与所述双功能交联剂上的酰肼基反应形成-CO-NH-NH-结构从而将所述连接载体与所述双功能交联剂连接，所述双功能交联剂上的马来酰亚胺基与所述待标记物上的巯基反应形成
    Figure PCTCN2016088685-appb-100002
    结构从而将所述双功能交联剂与所述待标记物连接。
  6. 根据权利要求5所述的方法，其特征在于，将所述中间产物B、双功能交联剂以及待标记物交联反应后得到吖啶标记结合物的操作具体包括以下步骤：
    用中间产物B与双功能交联剂反应生成中间产物D，其中，所述中间产物D为吖啶取代物-连接载体-双功能交联剂结合物，所述双功能交联剂为含有马来酰亚胺基及酰肼基的交联剂，所述中间产物B中的连接载体上的羧基与所述双功能交联剂上的酰肼基反应形成-CO-NH-NH-结构从而将所述中间产物B与所述双功能交联剂连接；以及
    将所述中间产物D与待标记物结合得到吖啶标记结合物，其中，所述待标记物为含有巯基、羰基或羧基的蛋白、改性蛋白、多肽、改性多肽或碳水化合物，所述中间产物D中的双功能交联剂上的马来酰亚胺基与所述待标记物上的巯基反应形成
    Figure PCTCN2016088685-appb-100003
    结构从而将所述中间产物D与所述待标记物连接。
  7. 根据权利要求5所述的方法，其特征在于，用活化后的吖啶取代物与连接载体反应得到中间产物B的操作中，所述活化后的吖啶取代物与所述连接载体的摩尔比为1～5000∶1。
  8. 根据权利要求6所述的方法，其特征在于，用中间产物B与双功能交联剂反应生成中间产物D的操作中，所述双功能交联剂与所述中间产物B的摩尔比为1～10000∶1。
  9. 一种化学发光免疫检测试剂盒，其特征在于，包括权利要求1～4中任一项所述的吖啶标记结合物。
  10. 一种吖啶标记结合物，其特征在于，包括依次连接的吖啶取代物、连接载体、双功能交联剂和待标记物；
    所述双功能交联剂为含有马来酰亚胺基及酰肼基的交联剂；
    所述待标记物为含有巯基、羰基或羧基的蛋白、改性蛋白、多肽、改性多肽或碳水化合物；
    所述连接载体含有氨基及二硫键，所述吖啶取代物与所述连接载体上的氨基反应形成-CO-NH-结构从而将所述吖啶取代物与所述连接载体连接，所述连接载体上的二硫键与所述双功能交联剂上的马来酰亚胺基反应形成
    Figure PCTCN2016088685-appb-100004
    结构从而将所述连接载体与所述双功能交联剂连接，所述双功能交联剂上的酰肼基与所述待标记物上的羰基或羧基反应形成-NH-NH-CO-构从而将所述双功能交联剂与所述待标记物连接。
  11. 根据权利要求10所述的吖啶标记结合物，其特征在于，所述吖啶取代物为吖啶酯、吖啶酸、吖啶酰胺或吖啶磺酰胺。
  12. 根据权利要求10所述的吖啶标记结合物，其特征在于，所述连接载体为含有氨基、羧基及二硫键的血蓝蛋白。
  13. 根据权利要求10所述的吖啶标记结合物，其特征在于，所述待标记物为抗原、半抗原或抗体。
  14. 一种吖啶标记结合物的制备方法，包括如下步骤：
    用活化后的吖啶取代物与连接载体反应得到中间产物B，其中，所述中间产物B为吖啶取代物-连接载体结合物；以及
    将所述中间产物B、双功能交联剂以及待标记物交联反应后得到吖啶标记结合物，其中，所述吖啶标记结合物包括依次连接的吖啶取代物、连接载体、双功能交联剂和待标记物，所述连接载体含有氨基及二硫键，所述吖啶取代物与所述连接载体上的氨基反应形成-CO-NH-结构从而将所述吖啶取代物与所述连接载体连接，所述连接载体上的二硫键与所述双功能交联剂上的马来酰亚胺基反应形成
    Figure PCTCN2016088685-appb-100005
    结构从而将所述连接载体与所述双功能交联剂连接，所述双功能交联剂上的酰肼基与所述待标记物上的羰基或羧基反应形成-NH-NH-CO-构从而将所述双功能交联剂与所述待标记物连接。
  15. 根据权利要求14所述的方法，其特征在于，将所述中间产物B、双功能交联剂以及待标记物交联反应后得到吖啶标记结合物的操作具体包括以下步骤：
    将中间产物B进行活化处理，使得所述中间产物B中的连接载体上的二硫键活化形成巯基，得到中间产物F，其中，所述中间产物F为含有巯基的吖啶取代物-连接载体结合物；
    将待标记物与双功能交联剂反应生成中间产物I，其中，所述中间产物I为待标记物-双功能交联剂结合物，所述待标记物为含有巯基、羰基或羧基的蛋白、改性蛋白、多肽、改性多肽或碳水化合物，所述双功能交联剂为含有马来酰亚胺基及酰肼基的交联剂，所述双功能交联剂上的酰肼基与所述待标 记物上的羰基或羧基反应形成-NH-NH-CO-构从而将所述双功能交联剂与所述待标记物连接；以及
    将所述中间产物I与所述中间产物F结合得到吖啶标记结合物，其中，所述中间产物I中的双功能交联剂上马来酰亚胺基与所述中间产物F上的巯基反应形成
    Figure PCTCN2016088685-appb-100006
    结构从而将所述中间产物I与所述中间产物F连接。
  16. 根据权利要求14所述的方法，其特征在于，用活化后的吖啶取代物与连接载体反应得到中间产物B的操作中，所述活化后的吖啶取代物与所述连接载体的摩尔比为1～5000∶1。
  17. 一种化学发光免疫检测试剂盒，其特征在于，包括权利要求10～13中任一项所述的吖啶标记结合物。
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