WO2018006268A1 - 吖啶标记结合物及其制备方法、化学发光试剂盒 - Google Patents

吖啶标记结合物及其制备方法、化学发光试剂盒 Download PDF

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WO2018006268A1
WO2018006268A1 PCT/CN2016/088625 CN2016088625W WO2018006268A1 WO 2018006268 A1 WO2018006268 A1 WO 2018006268A1 CN 2016088625 W CN2016088625 W CN 2016088625W WO 2018006268 A1 WO2018006268 A1 WO 2018006268A1
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acridine
polyamino acid
labeled
protein
conjugate
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French (fr)
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钱纯亘
肖成勇
祝亮
刘陶旭
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 PCT/CN2016/088625 priority Critical patent/WO2018006268A1/zh
Priority to EP16907773.2A priority patent/EP3483172B1/en
Publication of WO2018006268A1 publication Critical patent/WO2018006268A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B15/00—Acridine dyes
    • 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
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B69/00—Dyes not provided for by a single group of this subclass
    • C09B69/10—Polymeric dyes; Reaction products of dyes with monomers or with macromolecular compounds
    • C09B69/109—Polymeric dyes; Reaction products of dyes with monomers or with macromolecular compounds containing other specific dyes
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • 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 kit.
  • Chemiluminescent Labeling Immunoassay also known as chemiluminescence immunoassay (CLIA) is an immunoassay for direct labeling of antigens, haptens or antibodies with chemiluminescent agents.
  • the chemiluminescent substances used for labeling include acridine substitutes, and depending on the substituents, acridine substituents are classified into two types: acridinium ester (AE) and acridinesulfonamide, both of which are effective luminescence.
  • AE acridinium ester
  • acridinesulfonamide both of which are effective luminescence.
  • the marker emits light by the action of the luminescent reagent (NaOH, H 2 O 2 ), and the intense direct illumination is completed in one second, which is a fast scintillation luminescence.
  • acridine substitution is used for immunoassay.
  • the chemical reaction is simple, rapid, and free of catalyst.
  • the small molecule antigen is detected by competition method, the macromolecular antigen 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.
  • Such compounds are characterized by the mechanism of luminescence: 1.
  • the non-luminescent substituent moiety attached to the acridine ring is detached from the acridine ring before the formation of the electronically excited intermediate in the luminescent reaction, ie,
  • the light-emitting portion is separated from the light-emitting portion, and thus its luminous efficiency is substantially unaffected by the substituent structure.
  • the acridine ester or acridine sulfonamide compound does not require a catalyst for chemiluminescence, and emits light in a dilute alkaline solution having H 2 O 2 . Therefore, it has many advantages in the application of chemiluminescence detection.
  • the main advantages are: 1 low background luminescence and high signal-to-noise ratio; 2 less luminescence reaction interference factors; 3 fast light concentration, high luminous efficiency and high luminous intensity; 4 easy to protein The photon yield does not decrease after bonding and bonding; 5 the marker is stable (can be stored for several months at 2-8 ° C). Acridine substitutes are therefore a very effective and very good chemiluminescent label.
  • the acridine labeling conjugate is a complex obtained by combining an acridine substituent with a label (antibody, antigen, etc.).
  • the quality of acridine-labeled conjugates is directly related to the success of chemiluminescent immunoassay techniques and is therefore referred to as a key reagent.
  • the currently used acridine labeling conjugate is prepared by a carbodiimide crosslinking method, which is bridged with a carbodiimide crosslinking agent to bind the acridine substituent to the protein to be labeled.
  • acridine labeling conjugate prepared by the conventional method the acridine substitution is combined with the protein to be labeled by carbodiimide, and the acridine substitution tends to interfere with the active site on the labeled protein, resulting in acridine labeling.
  • the reduced activity of the conjugate affects the sensitivity of the immunoassay.
  • An acridine labeling conjugate comprising an acridine substitution, a polyamino acid, and a protein to be labeled;
  • the polyamino acid is a polyamino acid containing a carboxyl group and an amino group, and the polyamino acid forms a chemical bond by reacting an amino group on the polyamino acid with the acridine substituent;
  • the protein to be labeled is an amino group-containing protein, a modified protein, a polypeptide or a modified polypeptide, and an amino group on the protein to be labeled reacts with a carboxyl group on the polyamino acid to form a -NH-CO- structure, thereby A polyamino acid and the protein to be labeled are linked together.
  • a method for preparing the above acridine labeling conjugate comprising the steps of:
  • the acridine-substituent-polyamino acid conjugate after activation with a carboxyl group is cross-linked with the protein to be labeled, and fully reacted to obtain an acridine-labeled conjugate, wherein the acridine-labeled conjugate comprises acridine-substituted in turn.
  • a protein, a polyamino acid, and a protein to be labeled wherein the protein to be labeled is an amino group-containing protein, a modified protein, a polypeptide or a modified polypeptide, and an amino group on the protein to be labeled reacts with a carboxyl group on the polyamino acid to form The -NH-CO- structure thereby linking the polyamino acid and the protein to be labeled together.
  • the polyamino acid is a polyamino acid containing a carboxyl group and an amino group, and the polyamino acid can form a chemical bond by reacting an amino group on the polyamino acid with the acridine substituent;
  • the protein to be labeled is an amino group-containing protein, a modified protein, a polypeptide or a modified polypeptide, and an amino group on the protein to be labeled can react with a carboxyl group on the polyamino acid to form a -NH-CO- structure, thereby The polyamino acid and the protein to be labeled are linked together.
  • the acridine labeling conjugate comprises an acridine substitution, a polyamino acid and a protein to be labeled which are sequentially linked, and the polyamino acid forms a chemical bond by reacting an amino group on the polyamino acid with an acridine substituent, and an amino group on the protein to be labeled Reacts with a carboxyl group on a polyamino acid to form a -NH-CO- structure to link the polyamino acid and the protein to be labeled, and the binding site is relatively determined, thereby preventing the acridine substitution from interfering with the active site on the labeled protein.
  • the activity of this acridine-labeled conjugate is relatively high.
  • polyamino acids increase the steric hindrance of the acridine-labeled conjugate, thereby increasing the sensitivity of the acridine-labeled conjugate.
  • FIG. 1 is a flow chart showing a method of preparing an acridine labeling conjugate according to an embodiment
  • Test Example 2 is a scattergram of the test results of the series of concentration TSH antigen samples obtained in Test Example 2.
  • An acridine-labeled conjugate comprising an acridine substitution, a polyamino acid, and a protein to be labeled, which are sequentially linked.
  • the polyamino acid is a polyamino acid containing a carboxyl group and an amino group, and the polyamino acid forms a chemical bond by reacting an amino group on the polyamino acid with an acridine substituent.
  • the polyamino acid may be a polylysine having a degree of polymerization of more than 25.
  • the polyamino acid is polylysine having a degree of polymerization of from 100 to 200.
  • the protein to be labeled is an amino group-containing protein, a modified protein, a polypeptide or a modified polypeptide, and the amino group on the labeled protein reacts with a carboxyl group on the polyamino acid to form a -NH-CO- structure to thereby link the polyamino acid to the protein to be labeled.
  • the protein to be labeled may be a protein or a polypeptide having an amino group by itself, or may be a modified protein or a modified polypeptide which is introduced into the amino group by modification.
  • the protein to be labeled is an antigen, a hapten or an antibody.
  • the acridine substituent can be an acridinium ester (DMAE-NHS, AE-NHS), acridinic acid (9-acridinecarboxylic acid), acridine amide or acridinesulfonamide (NSP-SA-NHS).
  • acridinium ester DMAE-NHS, AE-NHS
  • acridinic acid 9-acridinecarboxylic acid
  • NSP-SA-NHS acridinesulfonamide
  • the amino group on the polyamino acid reacts with a different group (carboxyl, succinimide ester, etc.) on the acridine substituent to form a chemical bond.
  • the acridinium ester can be AE-NHS (10-methyl-acridine-9-N-succinimidyl ester carboxylate), DMAE-NHS (2',6'-dimethyl-4' -(N-succinimideoxycarbonyl)phenyl-acridine-9-carboxylate),
  • the protein to be labeled carries an amino residue, and n is an integer greater than 25, preferably an integer of 100 to 200.
  • the acridine labeling conjugate comprises an acridine substitution, a polyamino acid and a protein to be labeled which are sequentially linked, and the polyamino acid forms a chemical bond by reacting an amino group on the polyamino acid with an acridine substituent, and an amino group on the protein to be labeled Reacts with a carboxyl group on a polyamino acid to form a -NH-CO- structure to link the polyamino acid and the protein to be labeled, and the binding site is relatively determined, thereby preventing the acridine substitution from interfering with the active site on the labeled protein.
  • the activity of this acridine-labeled conjugate is relatively high.
  • polyamino acids increase the steric hindrance of the acridine-labeled conjugate, thereby increasing the sensitivity of the acridine-labeled conjugate.
  • the active site of the protein to be labeled is effectively protected from the inactivation of the protein to be labeled, and the acridine substitution, polyamino acid and The proteins to be labeled are sequentially connected, and have the characteristics of stability and controllable connection amount.
  • the acridine labeling conjugate can be directly used for detection and quantitative analysis of chemiluminescence immunoassay, and can solve the disadvantages of inactivation and signal difference of acridine labeling conjugate materials prepared by conventional carbodiimide cross-linking methods and the like. .
  • the method for preparing the above acridine labeling conjugate as shown in FIG. 1 comprises the following steps:
  • the molar ratio of the activated acridine substituent to the polyamino acid is from 1 to 10000:1.
  • the molar ratio of the acridine substituent to the polyamino acid is from 10 to 200:1.
  • the activated acridine substituent can be a carboxyl activated acridine substituent.
  • the activated acridine substitution can be activated by carbodiimide and hydroxysuccinimide.
  • the specific activation operation is as follows: acridine substitution is added to the buffer, and after fully dissolved, EDC and NHS are added at 25 ° C. The reaction was carried out for 10 min to give an activated acridine substitution.
  • the buffer is phosphate buffer, carbonate buffer, 2-(N-morpholino)ethanesulfonic acid (MES) buffer or piperazine-N,N' bis(2-ethanesulfonic acid) ( PIPES) buffer, the pH of the reaction process is 4-10.
  • the polyamino acid is a polyamino acid containing a carboxyl group and an amino group, and the polyamino acid forms a chemical bond by reacting an amino group on the polyamino acid with an acridine substituent.
  • the polyamino acid may be a polylysine having a degree of polymerization of more than 25.
  • the polyamino acid is polylysine having a degree of polymerization of from 100 to 200.
  • the acridine substituent can be an acridinium ester (DMAE-NHS, AE-NHS), acridinic acid (9-acridinecarboxylic acid), acridine amide or acridinesulfonamide (NSP-SA-NHS).
  • acridinium ester DMAE-NHS, AE-NHS
  • acridinic acid 9-acridinecarboxylic acid
  • NSP-SA-NHS acridinesulfonamide
  • the amino group on the polyamino acid reacts with a different group (carboxyl, succinimide ester, etc.) on the acridine substituent to form a chemical bond.
  • the acridinium ester can be AE-NHS (10-methyl-acridine-9-N-succinimidyl ester carboxylate), DMAE-NHS (2',6'-dimethyl-4' -(N-succinimideoxycarbonyl)phenyl-acridine-9-carboxylate),
  • the acridine substituent-polyamino acid conjugate is covalently cross-linked to obtain an acridine-substituted polyamino acid conjugate.
  • the specific reaction process of the above reaction formula is: adding polyamino acid to the buffer, fully dissolving, adding excess acridinium ester, reacting at 4 ° C to 37 ° C for 0.5 h to 12 h, and purifying to obtain acridinium ester - Polyamino acid conjugate.
  • the buffer is phosphate buffer, carbonate buffer, 2-(N-morpholino)ethanesulfonic acid (MES) buffer or piperazine-N,N' bis(2-ethanesulfonic acid) ( PIPES) buffer
  • the pH of the reaction process is 4-10.
  • the amino group on the polyamino acid can be regarded as a complete reaction, and does not compete with the amino group on the protein to be labeled in the next step, so that it is not necessary to block the acridinium ester-multiple An amino group on a polyamino acid conjugate.
  • the purification of the acridine-substituent-polyamino acid conjugate can be performed in combination with one or more of several operations of ultrafiltration purification, desalting column purification, and dialysis purification.
  • the crosslinking agent comprises carbodiimide and hydroxysuccinimide, the molar ratio of carbodiimide to acridine-substituted polyamino acid conjugate is 5 to 1000:1, carbodiimide and hydroxysuccinimide The molar ratio is from 10:1 to 1:20.
  • the carbodiimide is selected from the group consisting of dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and N,N'-diisopropylcarbodiimide. At least one of the amines.
  • the molar ratio of carbodiimide to acridine substituted-polyamino acid conjugate is from 20 to 500:1.
  • the hydroxysuccinimide is selected from at least one of N-hydroxysuccinimide and N-hydroxysulfosuccinimide.
  • the molar ratio of carbodiimide to hydroxysuccinimide is from 3:1 to 1:10.
  • n is an integer greater than 25, preferably an integer of 100 to 200.
  • the specific reaction process of the above reaction formula is: adding acridine-substituent-polyamino acid conjugate to the buffer, fully dissolving, adding EDC and NHS, and reacting at 25 ° C for 10 min to obtain acridine-substituted acridine-substituted- Polyamino acid conjugates.
  • the buffer is phosphate buffer, carbonate buffer, 2-(N-morpholino)ethanesulfonic acid (MES) buffer or piperazine-N,N' bis(2-ethanesulfonic acid) ( PIPES) buffer, the pH of the reaction process is 4-10.
  • S30 further comprises adding a mercapto compound (mercaptoethanol, decylthreitol, cysteine, cysteine hydrochloride, etc.) after the carboxyl group on the acridine-substituent-polyamino acid conjugate is activated. Quenching the cross-linking agent activity (or purifying the cross-linking agent by purification) to obtain an acridine-substituted poly-amino acid conjugate after activation of the carboxyl group, and the acridine-substituent-polyamino acid conjugate after activation of the carboxyl group The amino group is blocked.
  • a mercapto compound mercaptoethanol, decylthreitol, cysteine, cysteine hydrochloride, etc.
  • the acridine-substituted poly-amino acid conjugate after activation of the carboxyl group obtained by S30 is cross-linked with the protein to be labeled, and fully reacted to obtain an acridine-labeled conjugate.
  • the resulting acridine-labeled conjugate comprises an acridine substitution, a polyamino acid, and a protein to be labeled, which are sequentially linked.
  • the protein to be labeled is an amino group-containing protein, a modified protein, a polypeptide or a modified polypeptide, and an amino group on the labeled protein reacts with a carboxyl group on the polyamino acid to form a -NH-CO- structure to thereby form a polyamino acid. Connected to the protein to be labeled.
  • the protein to be labeled may be a protein or a polypeptide having an amino group by itself, or may be a modified protein or a modified polypeptide which is introduced into the amino group by modification.
  • the protein to be labeled is an antigen, a hapten or an antibody.
  • the molar ratio of the acridine-substitution-polyamino acid conjugate to the protein to be labeled is 5:1 to 1:5. .
  • the molar ratio of the acridine-substituted polyamino acid conjugate to the protein to be labeled is from 2:1 to 1:2.
  • acridinium ester as AE-NHS and polyamino acid as polylysine as an example
  • the acridine-substituted-polyamino acid conjugate after carboxyl activation is cross-linked with the protein to be labeled to obtain acridine-labeled conjugate reaction.
  • the formula is as follows:
  • the protein to be labeled carries an amino residue, and n is an integer greater than 25, preferably an integer of 100 to 200.
  • the specific reaction process of the above reaction formula is: adding a carboxylate-activated acridine-substituent-polyamino acid conjugate to the buffer, and after fully dissolving, adding the protein to be labeled, and reacting at 4 ° C to 37 ° C for 0.5 h to 12 h, After the reaction, it was purified to obtain an acridine-labeled conjugate.
  • the buffer is phosphate buffer, carbonate buffer, 2-(N-morpholino)ethanesulfonic acid (MES) buffer or piperazine-N,N' bis(2-ethanesulfonic acid) ( PIPES) buffer, the pH of the reaction process is 4-10.
  • the polyamino acid forms a chemical bond by reacting an amino group on the polyamino acid with an acridine substituent, and the amino group on the protein to be labeled and the polyamino acid
  • the carboxyl group reacts to form a -NH-CO- structure to link the polyamino acid and the protein to be labeled, and the binding site is relatively determined, thereby avoiding the interference of the acridine substituent on the active site on the labeled protein, the acridine label
  • the acridine-labeled conjugate prepared by the method of preparation of the conjugate has a relatively high activity.
  • polyamino acids increase the steric hindrance of the acridine-labeled conjugate, thereby increasing the sensitivity of the acridine-labeled conjugate.
  • the invention also discloses a chemiluminescence kit for binding the protein to be labeled to form the acridine labeling conjugate described above.
  • Chemiluminescence kits include: acridine substituted and polyamino acids.
  • the acridine substituent can be an acridinium ester (DMAE-NHS, AE-NHS), acridinic acid (9-acridinecarboxylic acid), acridine amide or acridinesulfonamide (NSP-SA-NHS).
  • acridinium ester DMAE-NHS, AE-NHS
  • acridinic acid 9-acridinecarboxylic acid
  • NSP-SA-NHS acridinesulfonamide
  • the amino group on the polyamino acid reacts with a different group (carboxyl, succinimide ester, etc.) on the acridine substituent to form a chemical bond.
  • the acridinium ester can be AE-NHS (10-methyl-acridine-9-N-succinimidyl ester carboxylate), DMAE-NHS (2',6'-dimethyl-4' -(N-succinimideoxycarbonyl)phenyl-acridine-9-carboxylate),
  • the polyamino acid is a polyamino acid containing a carboxyl group and an amino group, and the polyamino acid forms a chemical bond by reacting an amino group on the polyamino acid with an acridine substituent.
  • the polyamino acid may be a polylysine having a degree of polymerization of more than 25.
  • the polyamino acid is polylysine having a degree of polymerization of from 100 to 200.
  • the polyamino acid may be bovine serum albumin, chicken serum albumin or hemocyanin.
  • the protein to be labeled is an amino group-containing protein, a modified protein, a polypeptide or a modified polypeptide, and the amino group on the labeled protein reacts with a carboxyl group on the polyamino acid to form a -NH-CO- structure to thereby link the polyamino acid to the protein to be labeled.
  • the protein to be labeled may be a protein or a polypeptide having an amino group itself, or may be modified. A modified protein or modified polypeptide of an amino group is introduced.
  • the protein to be labeled is an antigen, a hapten or an antibody.
  • the chemiluminescent kit further comprises at least one of a centrifugal desalting column and a centrifugal ultrafiltration tube.
  • the chemiluminescence kit can form a chemical bond through the acridine substitution, the polyamino acid and the protein to be labeled, and the binding site is relatively determined, thereby avoiding the formation of interference between the acridine substituent and the active site on the labeled protein.
  • the activity of the acridine labelled conjugate is relatively high.
  • polyamino acids increase the steric hindrance of the acridine-labeled conjugate, thereby increasing the sensitivity of the acridine-labeled conjugate.
  • the acridinium esters present in the examples are all carboxyl-activated acridinium esters.
  • the specific activation procedure is as follows: acridine ester is added to a phosphate buffer solution of pH 7, and after fully dissolved, EDC is added in a molar ratio of 1:1. And NHS, wherein the molar ratio of NHS to acridinium ester is 100:1, and reacted at 25 ° C for 10 min to obtain an activated acridine substitute.
  • the polylysine was dissolved in 1 mL of 150 mM PBS buffer (pH 7.4) to a final concentration of 10 nmol/L.
  • 10 ⁇ L of 10 mmol/L acridinium ester dissolved in DMSO solvent was added, and reacted at 25 ° C for 1 h, with 5 mL of 7KD molecular weight cut off.
  • a desalting 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 to obtain an acridine-polylysine solution.
  • EDC final concentration: 5 mmol/L
  • NHS final concentration: 10 mmol/L
  • the polylysine was dissolved in 1 mL of 150 mM PBS buffer (pH 7.4) to a final concentration of 10 nmol/L.
  • 10 ⁇ L of 10 mmol/L acridinium ester dissolved in DMSO solvent was added, and reacted at 25 ° C for 1 h, with 5 mL of 7KD molecular weight cut off.
  • a desalting 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 to obtain an acridine-polylysine solution.
  • EDC final concentration: 5 mmol/L
  • NHS final concentration: 10 mmol/L
  • 5 mL of 7KD molecular weight dehydration column was used as a buffer exchange buffer in 150 mM PBS (pH 7.4), and the column was passed 3 times to remove the free acridine ester and the reaction pair.
  • the product gave an acridine-polylysine solution.
  • EDC final concentration: 5 mmol/L
  • NHS final concentration: 10 mmol/L
  • 1 mg of anti-PTH monoclonal antibody (manufacturer: Abnova, Cat. No.: PAB5103, 6.67 nmol) was dissolved in 1 mL of 150 mM PBS buffer (pH 7.4), 10 ⁇ L of 10 mmol/L acridinium ester dissolved in DMSO solvent was added, and reacted at 25 ° C for 1 h.
  • 150 mM PBS (pH 7.4) buffer as a buffering buffer, and passing the column 3 times to remove free acridinium ester and reaction by-products to obtain acridinium ester- PTH monoclonal antibody solution.
  • the conjugates obtained in Examples 1 to 3 and the comparative examples were used for chemiluminescence immunoassay detection of PTH.
  • To 1000pg/mL of PTH sample 50 ⁇ g of anti-PTH monoclonal antibody-coated magnetic beads were added, and 1pmol of acridinium ester-labeled anti-PTH antibody prepared by each method was added separately. After incubation, the reaction was washed, and 100 ⁇ L of HNO 3 -H was added successively. 2 O 2 solution and 100 ⁇ L of sodium hydroxide solution were measured for luminescence value by iFlash3000 type chemiluminescence immunoassay analyzer, and 3 measurements were taken in parallel, and the average value was obtained; the results are shown in Table 1. The greater the luminescence value of the results obtained by immunoassay, the better the activity of the acridinium ester label.
  • the acridinium ester-polylysine-anti-PTH monoclonal antibody solution obtained in Example 1 was used for chemiluminescence immunoassay detection of a series of concentrations of PTH.
  • the luminescence value was measured with an iFlash3000 type chemiluminescence immunoassay analyzer using a -H 2 O 2 solution and 100 ⁇ L of a sodium hydroxide solution; the results are shown in Table 2.
  • the PTH antigen concentration was plotted on the X-axis and the relative luminescence value was plotted on the Y-axis, and the data in Table 2 was plotted to obtain Figure 2.
  • PTH antigen concentration Luminous value (RLU) 0pg/mL 1528 13.91pg/mL 33615 25.74pg/mL 121932 140.79pg/mL 602764 696.54pg/mL 2848273 3701.6pg/mL 14004950
  • the acridinium ester-polylysine-antibody prepared in Example 1 can be applied to chemiluminescence immunoassay with good results.
  • the acridinium ester-polylysine-label system of Example 1 The preparation method 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、化学发光试剂盒，吖啶标记结合物，包括依次连接的吖啶取代物、多聚氨基酸和待标记蛋白；所述多聚氨基酸为含有羧基和氨基的多聚氨基酸；所述待标记蛋白为含有氨基的蛋白、改性蛋白、多肽或改性多肽。

Description

吖啶标记结合物及其制备方法、化学发光试剂盒 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、半抗原或抗体的免疫分析方法。用于标记的化学发光物质包括吖啶取代物，根据取代基的不同，吖啶取代物分为两类：吖啶酯(acridinium ester，AE)和吖啶磺酰胺，二者均为有效的发光标记物，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。
吖啶取代物作为化学发光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。这类化合物从发光的机理来说特点是：1、发光反应中在形成电子激发态中间体之前，联结于吖啶环上的不发光的取代基部分从吖啶环上脱离开来，即未发光部分与发光部分分离，因而其发光效率基本不受取代基结构的影响。2、吖啶酯或吖啶磺酰胺类化合物化学发光不需要催化剂，在有H2O2的稀碱性溶液中即能发光。因此应用于化学发光检测具有许多优越性，优点主要有：①背景发光低，信噪比高；②发光反应干扰因素少；③光释放快速集中、发光效率高、发光强度大；④易于与蛋白质联结且联结后光子产率不减少；⑤标记物稳定(在2-8℃下可保存数月之久)。因此吖啶取代物是一类非常有效、非常好的化学发光标记物。
吖啶标记结合物为吖啶取代物与待标记物(抗体、抗原，等)结合得到的复合物。吖啶标记结合物质量的好坏直接关系到化学发光免疫分析技术的成功与否，因此被称为关键的试剂。
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，以碳二亚胺交联剂为桥，使吖啶取代物与待标记蛋白结合。然而，传统方法制得的吖啶标记结合物中，吖啶取代物与待标记蛋白通过碳二亚胺结合，吖啶取代物往往会对待标记蛋白上的活性位点形成干扰，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、化学发光试剂盒。
一种吖啶标记结合物，包括依次连接的吖啶取代物、多聚氨基酸和待标记蛋白；
所述多聚氨基酸为含有羧基和氨基的多聚氨基酸，所述多聚氨基酸通过所述多聚氨基酸上的氨基与所述吖啶取代物反应形成化学键连接；
所述待标记蛋白为含有氨基的蛋白、改性蛋白、多肽或改性多肽，所述待标记蛋白上的氨基与所述多聚氨基酸上的羧基反应形成-NH-CO-结构从而将所述多聚氨基酸和所述待标记蛋白连接到一起。
一种上述的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和多聚氨基酸共价交联，充分反应后得到吖啶取代物-多聚氨基酸结合物，其中，所述多聚氨基酸为含有羧基和氨基的多聚氨基酸，所述多聚氨基酸通过所述多聚氨基酸上的氨基与所述吖啶取代物反应形成化学键连接；
对所述吖啶取代物-多聚氨基酸结合物进行纯化；
用交联剂对纯化后的所述吖啶取代物-多聚氨基酸结合物上的羧基进行活化；以及
用羧基活化后的所述吖啶取代物-多聚氨基酸结合物与待标记蛋白交联，充分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、多聚氨基酸和待标记蛋白，所述待标记蛋白为含有氨基的蛋白、改性蛋白、多肽或改性多肽，所述待标记蛋白上的氨基与所述多聚氨基酸上的羧基反应形成-NH-CO-结构从而将所述多聚氨基酸和所述待标记蛋白连接到一起。
一种化学发光试剂盒，用于结合待标记蛋白形成如上述的吖啶标记结合物，所述化学发光试剂盒包括：吖啶取代物和多聚氨基酸；
所述多聚氨基酸为含有羧基和氨基的多聚氨基酸，所述多聚氨基酸可以通过所述多聚氨基酸上的氨基与所述吖啶取代物反应形成化学键连接；
所述待标记蛋白为含有氨基的蛋白、改性蛋白、多肽或改性多肽，所述待标记蛋白上的氨基可以与所述多聚氨基酸上的羧基反应形成-NH-CO-结构从而将所述多聚氨基酸和所述待标记蛋白连接到一起。
这种吖啶标记结合物包括依次连接的吖啶取代物、多聚氨基酸和待标记蛋白，多聚氨基酸通过多聚氨基酸上的氨基与吖啶取代物反应形成化学键连接，待标记蛋白上的氨基与多聚氨基酸上的羧基反应形成-NH-CO-结构从而将多聚氨基酸和待标记蛋白连接到一起，结合位点相对确定，避免了吖啶取代物对待标记蛋白上的活性位点形成干扰，这种吖啶标记结合物的活性相对较高。此外，多聚氨基酸可使吖啶标记结合物的空间位阻增大，从而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为测试例2中得到的系列浓度TSH抗原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、多聚氨基酸和待标记蛋白。
多聚氨基酸为含有羧基和氨基的多聚氨基酸，多聚氨基酸通过多聚氨基酸上的氨基与吖啶取代物反应形成化学键连接。
本实施方式中，多聚氨基酸可以为聚合度大于25的多聚赖氨酸。优选的，多聚氨基酸为聚合度为100～200的多聚赖氨酸。
待标记蛋白为含有氨基的蛋白、改性蛋白、多肽或改性多肽，待标记蛋白上的氨基与多聚氨基酸上的羧基反应形成-NH-CO-结构从而将多聚氨基酸和待标记蛋白连接到一起。
待标记蛋白可以为本身就具有氨基的蛋白或多肽，也可以为通过修饰后引入氨基的改性蛋白或改性多肽。
本实施方式中，待标记蛋白为抗原、半抗原或抗体。
吖啶取代物可以为吖啶酯(DMAE-NHS、AE-NHS)、吖啶酸(9-吖啶甲酸)、吖啶酰胺或吖啶磺酰胺(NSP-SA-NHS)。根据吖啶取代物的具体不同，多聚氨基酸上的氨基与吖啶取代物上的不同基团(羧基、琥珀酰亚胺酯等)反应形成化学键连接。
具体的，吖啶酯可以为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。
以吖啶酯为AE-NHS，多聚氨基酸为多聚赖氨酸为例，此时，吖啶标记 结合物具有如下结构式：
Figure PCTCN2016088625-appb-000001
其中，
Figure PCTCN2016088625-appb-000002
为待标记蛋白，待标记蛋白上带有氨基残基，n为大于25的整数，优选为100～200的整数。
这种吖啶标记结合物包括依次连接的吖啶取代物、多聚氨基酸和待标记蛋白，多聚氨基酸通过多聚氨基酸上的氨基与吖啶取代物反应形成化学键连接，待标记蛋白上的氨基与多聚氨基酸上的羧基反应形成-NH-CO-结构从而将多聚氨基酸和待标记蛋白连接到一起，结合位点相对确定，避免了吖啶取代物对待标记蛋白上的活性位点形成干扰，这种吖啶标记结合物的活性相对较高。
此外，多聚氨基酸可使吖啶标记结合物的空间位阻增大，从而增加了吖啶标记结合物的使用灵敏度。
与传统技术相比，这种含有多聚氨基酸的吖啶标记结合物中，待标记蛋白的活性位点得到有效保护，避免待标记蛋白失活，通过化学键将吖啶取代物、多聚氨基酸和待标记蛋白依次连接，具有稳定、连接量可控等特点。
这种吖啶标记结合物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、信号差等缺点。
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：
S10、用活化后的吖啶取代物和多聚氨基酸共价交联，充分反应后得到吖啶取代物-多聚氨基酸结合物。
用活化后的吖啶取代物和多聚氨基酸共价交联的操作中，活化后的吖啶取代物和多聚氨基酸的摩尔比为1～10000∶1。
优选的，吖啶取代物和多聚氨基酸的摩尔比为10～200∶1。
活化后的吖啶取代物可以为羧基活化的吖啶取代物。活化后的吖啶取代物可以为采用碳二亚胺和羟基琥珀酰亚胺活化，具体的活化操作如下：在缓冲液中加入吖啶取代物，充分溶解后，加入EDC和NHS，于25℃反应10min，得到活化后的吖啶取代物。其中，缓冲液为磷酸盐缓冲液、碳酸盐缓冲液、2-(N-吗啉代)乙磺酸(MES)缓冲液或哌嗪-N，N’双(2-乙磺酸)(PIPES)缓冲液，反应过程的pH为4～10。
多聚氨基酸为含有羧基和氨基的多聚氨基酸，多聚氨基酸通过多聚氨基酸上的氨基与吖啶取代物反应形成化学键连接。
本实施方式中，多聚氨基酸可以为聚合度大于25的多聚赖氨酸。优选的，多聚氨基酸为聚合度为100～200的多聚赖氨酸。
吖啶取代物可以为吖啶酯(DMAE-NHS、AE-NHS)、吖啶酸(9-吖啶甲酸)、吖啶酰胺或吖啶磺酰胺(NSP-SA-NHS)。根据吖啶取代物的具体不同，多聚氨基酸上的氨基与吖啶取代物上的不同基团(羧基、琥珀酰亚胺酯等)反应形成化学键连接。
具体的，吖啶酯可以为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。
以吖啶酯为AE-NHS，多聚氨基酸为多聚赖氨酸为例，吖啶取代物和多聚氨基酸共价交联得到吖啶取代物-多聚氨基酸结合物的反应式如下：
Figure PCTCN2016088625-appb-000003
上述反应式的具体反应过程为：在缓冲液中加入多聚氨基酸，充分溶解后加入过量的吖啶酯，于4℃～37℃反应0.5h～12h，反应后经过纯化得到吖啶酯-多聚氨基酸结合物。其中，缓冲液为磷酸盐缓冲液、碳酸盐缓冲液、2-(N-吗啉代)乙磺酸(MES)缓冲液或哌嗪-N，N’双(2-乙磺酸)(PIPES)缓冲液，反应过程的pH为4～10。
由于吖啶酯相对于多聚氨基酸是过量的，因此多聚氨基酸上的氨基可以视为反应完全，不会与下一步中待标记蛋白上的氨基进行竞争，从而可以不必封闭吖啶酯-多聚氨基酸结合物上的氨基。
S20、对S10得到的吖啶取代物-多聚氨基酸结合物进行纯化。
对吖啶取代物-多聚氨基酸结合物进行纯化的操作可以为超滤纯化、脱盐柱纯化和透析纯化几种操作中的一种或几种联用。
S30、用交联剂对S20得到的纯化后的吖啶取代物-多聚氨基酸结合物上的羧基进行活化。
交联剂包括碳二亚胺和羟基琥珀酰亚胺，碳二亚胺与吖啶取代物-多聚氨基酸结合物的摩尔比为5～1000∶1，碳二亚胺与羟基琥珀酰亚胺的摩尔比为10∶1～1∶20。
优选的，碳二亚胺选自二环己基碳二亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺和N，N′-二异丙基碳二亚胺中的至少一种。
优选的，碳二亚胺与吖啶取代物-多聚氨基酸结合物的摩尔比为20～500∶1。
优选的，羟基琥珀酰亚胺选自N-羟基琥珀酰亚胺和N-羟基磺基琥珀酰亚胺中的至少一种。
优选的，碳二亚胺与羟基琥珀酰亚胺的摩尔比为3∶1～1∶10。
以吖啶酯为AE-NHS，多聚氨基酸为多聚赖氨酸为例，采用碳二亚胺和羟基琥珀酰亚胺对吖啶取代物-多聚氨基酸结合物的羧基进行活化的反应式如下：
Figure PCTCN2016088625-appb-000004
其中，n为大于25的整数，优选为100～200的整数。
上述反应式的具体反应过程为：在缓冲液中加入吖啶取代物-多聚氨基酸结合物，充分溶解后，加入EDC和NHS，于25℃反应10min，得到羧基活化后的吖啶取代物-多聚氨基酸结合物。其中，缓冲液为磷酸盐缓冲液、碳酸盐缓冲液、2-(N-吗啉代)乙磺酸(MES)缓冲液或哌嗪-N，N’双(2-乙磺酸)(PIPES)缓冲液，反应过程的pH为4～10。
优选的，S30还包括在吖啶取代物-多聚氨基酸结合物上的羧基被活化后，加入巯基化合物(巯基乙醇、巯基苏糖醇、半胱氨酸、半胱氨酸盐酸盐，等)淬灭交联剂活性(或者经纯化除去交联剂)，得到羧基活化后的吖啶取代物-多聚氨基酸结合物，并且该羧基活化后的吖啶取代物-多聚氨基酸结合物中的氨基封闭。
S40、用S30得到的羧基活化后的吖啶取代物-多聚氨基酸结合物与待标记蛋白交联，充分反应后得到吖啶标记结合物。
得到的吖啶标记结合物包括依次连接的吖啶取代物、多聚氨基酸和待标记蛋白。
待标记蛋白为含有氨基的蛋白、改性蛋白、多肽或改性多肽，待标记蛋白上的氨基与多聚氨基酸上的羧基反应形成-NH-CO-结构从而将多聚氨基酸 和待标记蛋白连接到一起。
待标记蛋白可以为本身就具有氨基的蛋白或多肽，也可以为通过修饰后引入氨基的改性蛋白或改性多肽。
本实施方式中，待标记蛋白为抗原、半抗原或抗体。
用羧基活化后的吖啶取代物-多聚氨基酸结合物与待标记蛋白交联的操作中，吖啶取代物-多聚氨基酸结合物与待标记蛋白的摩尔比为5∶1～1∶5。
优选的，吖啶取代物-多聚氨基酸结合物与待标记蛋白的摩尔比为2∶1～1∶2。
以吖啶酯为AE-NHS，多聚氨基酸为多聚赖氨酸为例，羧基活化后的吖啶取代物-多聚氨基酸结合物和待标记蛋白交联，得到吖啶标记结合物的反应式如下：
Figure PCTCN2016088625-appb-000005
其中，
Figure PCTCN2016088625-appb-000006
为待标记蛋白，待标记蛋白上带有氨基残基，n为大于25的整数，优选为100～200的整数。
上述反应式的具体反应过程为：在缓冲液中加入羧基活化后的吖啶取代物-多聚氨基酸结合物，充分溶解后，加入待标记蛋白，于4℃～37℃反应0.5h～12h，反应后经过纯化得到吖啶标记结合物。其中，缓冲液为磷酸盐缓冲液、碳酸盐缓冲液、2-(N-吗啉代)乙磺酸(MES)缓冲液或哌嗪-N，N’双(2-乙磺酸)(PIPES)缓冲液，反应过程的pH为4～10。
这种吖啶标记结合物的制备方法，多聚氨基酸通过多聚氨基酸上的氨基与吖啶取代物反应形成化学键连接，待标记蛋白上的氨基与多聚氨基酸上的 羧基反应形成-NH-CO-结构从而将多聚氨基酸和待标记蛋白连接到一起，结合位点相对确定，避免了吖啶取代物对待标记蛋白上的活性位点形成干扰，这种吖啶标记结合物的制备方法制得的吖啶标记结合物的活性相对较高。
此外，多聚氨基酸可使吖啶标记结合物的空间位阻增大，从而增加了吖啶标记结合物的使用灵敏度。
本发明还公开了一种化学发光试剂盒，用于结合待标记蛋白形成上述的吖啶标记结合物。
化学发光试剂盒包括：吖啶取代物和多聚氨基酸。
吖啶取代物可以为吖啶酯(DMAE-NHS、AE-NHS)、吖啶酸(9-吖啶甲酸)、吖啶酰胺或吖啶磺酰胺(NSP-SA-NHS)。根据吖啶取代物的具体不同，多聚氨基酸上的氨基与吖啶取代物上的不同基团(羧基、琥珀酰亚胺酯等)反应形成化学键连接。
具体的，吖啶酯可以为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。
多聚氨基酸为含有羧基和氨基的多聚氨基酸，多聚氨基酸通过多聚氨基酸上的氨基与吖啶取代物反应形成化学键连接。
本实施方式中，多聚氨基酸可以为聚合度大于25的多聚赖氨酸。优选的，多聚氨基酸为聚合度为100～200的多聚赖氨酸。
本实施方式中，多聚氨基酸可以为牛血清白蛋白、鸡血清白蛋白或血蓝蛋白。
待标记蛋白为含有氨基的蛋白、改性蛋白、多肽或改性多肽，待标记蛋白上的氨基与多聚氨基酸上的羧基反应形成-NH-CO-结构从而将多聚氨基酸和待标记蛋白连接到一起。
待标记蛋白可以为本身就具有氨基的蛋白或多肽，也可以为通过修饰后 引入氨基的改性蛋白或改性多肽。
本实施方式中，待标记蛋白为抗原、半抗原或抗体。
优选的，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通过吖啶取代物、多聚氨基酸和待标记蛋白依次形成化学键连接，结合位点相对确定，避免了吖啶取代物对待标记蛋白上的活性位点形成干扰，形成的吖啶标记结合物的活性相对较高。
此外，多聚氨基酸可使吖啶标记结合物的空间位阻增大，从而增加了吖啶标记结合物的使用灵敏度。
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羧基活化的吖啶酯，具体活化操作如下：在pH为7的磷酸盐缓冲液中加入吖啶酯，充分溶解后，加入摩尔比为1∶1的EDC和NHS，其中，NHS与吖啶酯的摩尔比为100∶1，于25℃反应10min，得到活化后的吖啶取代物。
实施例1
将多聚赖氨酸溶解于1mL150mM PBS缓冲液(pH 7.4)中，终浓度10nmol/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-多聚赖氨酸溶液。
向上述纯化过的吖啶-多聚赖氨酸溶液中加入EDC(终浓度为5mmol/L)和NHS(终浓度为10mmol/L)。加入25℃反应30分钟后，加入终浓度为10mM的巯基乙醇，得到活化后的吖啶-多聚赖氨酸；
在加入1mg抗PTH单克隆抗体(厂家：Abnova，货号：PAB5103， 6.67nmol)，混匀，置于25℃反应1h，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NHS及反应副产物，得到结合物吖啶酯-多聚赖氨酸-PTH单克隆抗体溶液。
实施例2
将多聚赖氨酸溶解于1mL150mM PBS缓冲液(pH 7.4)中，终浓度10nmol/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-多聚赖氨酸溶液。
向上述纯化过的吖啶-多聚赖氨酸溶液中加入EDC(终浓度为5mmol/L)和NHS(终浓度为10mmol/L)。加入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，除去游离的吖啶酯及反应副产物，得到吖啶-多聚赖氨酸溶液。
在加入1mg抗PTH单克隆抗体(厂家：Abnova，货号：PAB5103，6.67nmol)，混匀，置于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NHS及反应副产物，得到结合物吖啶酯-多聚赖氨酸-PTH单克隆抗体溶液。
实施例3
将多聚赖氨酸溶解于1mL150mM PBS缓冲液(pH 7.4)中，终浓度10nmol/L，加入10μL溶解于DMSO溶剂的10mmol/L吖啶酯，于25℃反应1h，，用4mL 30KD截留分子量的超滤管(Millipore公司)，以150mM PBS(pH 7.4)缓冲液作为换液缓冲液，超滤3遍，除去游离的吖啶酯及反应 副产物，得到吖啶-多聚赖氨酸溶液。
向上述纯化过的吖啶-多聚赖氨酸溶液中加入EDC(终浓度为5mmol/L)和NHS(终浓度为10mmol/L)。加入25℃反应30分钟后，用4mL 30KD截留分子量的超滤管(Millipore公司)，以150mM PBS(pH 7.4)缓冲液作为换液缓冲液，超滤3遍，得到活化后的吖啶-多聚赖氨酸；
在加入1mg抗PTH单克隆抗体(厂家：Abnova，货号：PAB5103，6.67nmol)，混匀，置于25℃反应1h，用4mL 30KD截留分子量的超滤管(Millipore公司)，以150mM PBS(pH 7.4)缓冲液作为换液缓冲液，超滤3遍，除去游离的NHS及反应副产物，得到结合物吖啶酯-多聚赖氨酸-PTH单克隆抗体溶液。
对比例
将1mg抗PTH单克隆抗体(厂家：Abnova，货号：PAB5103，6.67nmol)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-PTH单克隆抗体溶液。
测试例1
将实施例1～3以及对比例中得到的结合物用于PTH的化学发光免疫分析检测。向1000pg/mL的PTH样本中，加入50μg抗PTH单克隆抗体包被的磁珠，再分别加入各方法制备的1pmol吖啶酯标记抗PTH抗体，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：各方法标记的吖啶酯标物检测PTH对比结果
吖啶酯标记物 平均发光值(RLU)
实施例1 3124571
实施例2 2816943
实施例3 2642157
对比例 752644
由表1结果可知，采用实施例1～3制备的吖啶酯标记试剂，试剂的活性显著优于对比例制备的。
测试例2
将实施例1中得到的吖啶酯-多聚赖氨酸-抗PTH的单克隆抗体溶液用于系列浓度的PTH的化学发光免疫分析检测。向系列浓度的PTH样本溶液中分别加入50μg PTH单克隆抗体包被的磁珠试剂和1pmol吖啶酯-多聚赖氨酸-抗PTH单克隆抗体试剂，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2。以PTH抗原浓度为X轴，以相对发光值为Y轴，由表2数据作图，得图2。
表2：系列浓度TSH抗原样本的免疫检测结果
PTH抗原浓度 发光值(RLU)
0pg/mL 1528
13.91pg/mL 33615
25.74pg/mL 121932
140.79pg/mL 602764
696.54pg/mL 2848273
3701.6pg/mL 14004950
由表2和图2结果可知，实施例1制备的吖啶酯-多聚赖氨酸-抗体可应用于化学发光免疫分析且效果良好。实施例1的吖啶酯-多聚赖氨酸-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一种或几种实施方式，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 (10)

  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、多聚氨基酸和待标记蛋白；
    所述多聚氨基酸为含有羧基和氨基的多聚氨基酸，所述多聚氨基酸通过所述多聚氨基酸上的氨基与所述吖啶取代物反应形成化学键连接；
    所述待标记蛋白为含有氨基的蛋白、改性蛋白、多肽或改性多肽，所述待标记蛋白上的氨基与所述多聚氨基酸上的羧基反应形成-NH-CO-结构从而将所述多聚氨基酸和所述待标记蛋白连接到一起。
  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，所述多聚氨基酸为聚合度大于25的多聚赖氨酸。
  4. 根据权利要求1或3所述的吖啶标记结合物，其特征在于，所述待标记蛋白为抗原、半抗原或抗体。
  5. 一种权利要求1～4中任一项所述的吖啶标记结合物的制备方法，其特征在于，包括如下步骤：
    用活化后的吖啶取代物和多聚氨基酸共价交联，充分反应后得到吖啶取代物-多聚氨基酸结合物，其中，所述多聚氨基酸为含有羧基和氨基的多聚氨基酸，所述多聚氨基酸通过所述多聚氨基酸上的氨基与所述吖啶取代物反应形成化学键连接；
    对所述吖啶取代物-多聚氨基酸结合物进行纯化；
    用交联剂对纯化后的所述吖啶取代物-多聚氨基酸结合物上的羧基进行活化；以及
    用羧基活化后的所述吖啶取代物-多聚氨基酸结合物与待标记蛋白交联，充分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、多聚氨基酸和待标记蛋白，所述待标记蛋白为含有氨基的蛋白、改性蛋白、多肽或改性多肽，所述待标记蛋白上的氨基与所述多聚氨基 酸上的羧基反应形成-NH-CO-结构从而将所述多聚氨基酸和所述待标记蛋白连接到一起。
  6. 根据权利要求5所述的吖啶标记结合物的制备方法，其特征在于，所述用活化后的吖啶取代物和多聚氨基酸共价交联的操作中，所述活化后的吖啶取代物和所述多聚氨基酸的摩尔比为1～10000∶1。
  7. 根据权利要求5所述的吖啶标记结合物的制备方法，其特征在于，所述用羧基活化后的所述吖啶取代物-多聚氨基酸结合物与待标记蛋白交联的操作中，所述吖啶取代物-多聚氨基酸结合物与所述待标记蛋白的摩尔比为5∶1～1∶5。
  8. 根据权利要求5所述的吖啶标记结合物的制备方法，其特征在于，所述用交联剂对纯化后的所述吖啶取代物-多聚氨基酸结合物上的羧基进行活化的操作中，所述交联剂包括碳二亚胺和羟基琥珀酰亚胺。
  9. 根据权利要求8所述的吖啶标记结合物的制备方法，其特征在于，所述碳二亚胺选自二环己基碳二亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺和N，N′-二异丙基碳二亚胺中的至少一种，所述碳二亚胺与所述吖啶取代物-多聚氨基酸结合物的摩尔比为5～1000∶1；
    所述羟基琥珀酰亚胺选自N-羟基琥珀酰亚胺和N-羟基磺基琥珀酰亚胺中的至少一种，所述碳二亚胺与所述羟基琥珀酰亚胺的摩尔比为10∶1～1∶20。
  10. 一种化学发光试剂盒，用于结合待标记蛋白形成如权利要求1～4中任一项所述的吖啶标记结合物，其特征在于，所述化学发光试剂盒包括：吖啶取代物和多聚氨基酸；
    所述多聚氨基酸为含有羧基和氨基的多聚氨基酸，所述多聚氨基酸可以通过所述多聚氨基酸上的氨基与所述吖啶取代物反应形成化学键连接；
    所述待标记蛋白为含有氨基的蛋白、改性蛋白、多肽或改性多肽，所述待标记蛋白上的氨基可以与所述多聚氨基酸上的羧基反应形成-NH-CO-结构从而将所述多聚氨基酸和所述待标记蛋白连接到一起。
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CN112710827A (zh) * 2020-12-15 2021-04-27 宝诺威(苏州)生物技术有限公司 多聚赖氨酸化合物在增强化学发光免疫灵敏度上的应用及方法
CN113419059A (zh) * 2021-06-30 2021-09-21 迈克生物股份有限公司 用于化学发光免疫分析方法的封闭剂
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