Deoxypyridol artificial hapten as well as preparation method and application thereof

Abstract

The invention belongs to the field of immunodetection, and particularly relates to a deoxypyridinol artificial hapten and a preparation method and application thereof. The deoxypyridol artificial hapten provided by the invention maximally retains the characteristic structure of deoxypyridol, and has an active group capable of being coupled with carrier protein, the obtained artificial antigen can be used as an antigenic determinant, a deoxypyridol antibody is obtained by immunizing an experimental animal, and the antigenic activity of the deoxypyridol is improved. The prepared artificial hapten, artificial antigen and antibody are used for preparing a reagent for detecting deoxypyridol to realize detection of deoxypyridol, the prepared reagent for detecting deoxypyridol is high in sensitivity and few in cross, and a convenient, rapid and accurate way is provided for detection of deoxypyridol.

Description

Technical Field

[0001] The invention belongs to the field of immunoassay, and in particular relates to a deoxypyridinol artificial hapten and a preparation method and application thereof. Background Art

[0002] Osteoporosis is a disabling degenerative bone disease that affects the elderly population, especially postmenopausal women, resulting in 1.5 million bone fractures per year. This metabolic disease is caused by a bone turnover process when bone resorption exceeds bone formation. Current methods for the diagnosis of osteoporosis Current methods for the diagnosis of osteoporosis involve histomorphometric studies and density measurements. Efforts to prevent this disease and develop effective anti-resorptive treatments have increased the demand for reliable and non-invasive biochemical markers of bone resorption. Traditional markers of bone resorption, namely urinary calcium and hydroxyproline, lack clinical sensitivity and specificity for the diagnosis of osteoporosis. Deoxypyridinoline (DPD), as a sensitive and specific biochemical indicator reflecting bone metabolism, can not only diagnose osteoporosis early and reduce the incidence of fractures, but also be used to assess the prognosis of osteoporosis, providing a simple, specific and sensitive clinical indicator for monitoring and preventing premature bone loss. In addition, DPD is important in reflecting increased bone resorption during pregnancy and lactation. DPD can play a huge role in the diagnosis of benign bone lesions. Compared with traditional biochemical indicators, it has stronger specificity and higher sensitivity. Therefore, the rapid detection of DPD indicators has important practical significance. Summary of the invention

[0003] In view of the need for rapid detection of deoxypyridinol (DPD) indicators in the prior art, the present invention provides a deoxypyridinol artificial hapten and a preparation method and application thereof. The specific technical scheme is as follows:

[0004] In a first aspect, the present invention provides a deoxypyridinol artificial hapten, the structure of which is shown in Formula IV:

[0005]

[0006] In a second aspect, the present invention provides a method for preparing the above-mentioned deoxypyridinol artificial hapten, comprising the following steps:

[0007] S1: reacting N-Boc-D-glutamic acid-1-tert-butyl ester with isobutyl chloroformate and diazomethane in the presence of triethylamine, and separating and purifying to obtain an intermediate product Ⅰ as shown in formula Ⅰ;

[0008]

[0009] S2: reacting the intermediate product I with hydrobromic acid, separating and purifying to obtain the intermediate product II as shown in formula II;

[0010]

[0011] S3: The intermediate product II reacts with N-Boc-L-lysine methyl ester in the presence of a base and a catalyst, and the intermediate product III is obtained after separation and purification as shown in formula III;

[0012]

[0013] S4: The intermediate product III is hydrolyzed under alkaline conditions, and after separation and purification, the deoxypyridinol artificial hapten shown in formula IV is obtained.

[0014] Furthermore, in S1, the specific steps are:

[0015] Dissolve N-Boc-D-glutamic acid-1-tert-butyl ester in dichloromethane, add triethylamine, and place in an ice-water bath. Add isobutyl chloroformate and react in an ice-water bath. Add diazomethane dichloromethane solution and continue to react in an ice-water bath. Add ethyl acetate and water for liquid separation and extraction. Dry over anhydrous sodium sulfate, filter, and concentrate the organic phase to obtain intermediate product Ⅰ as shown in formula Ⅰ.

[0016] Furthermore, the mass ratio of the N-Boc-D-glutamic acid-1-tert-butyl ester to isobutyl chloroformate is 5:2-3.

[0017] Furthermore, the mass ratio of the N-Boc-D-glutamic acid-1-tert-butyl ester to isobutyl chloroformate is 2:1.

[0018] Furthermore, in S1, before adding ethyl acetate and water for separation and extraction, acetic acid is added to quench the remaining diazomethane.

[0019] Furthermore, in S2, the specific steps are:

[0020] Dissolve the intermediate product I in tetrahydrofuran, add thymol blue as an indicator, stir in an ice-water bath, add hydrobromic acid aqueous solution dropwise until the solution turns purple-red, terminate the reaction, add ethyl acetate and water for liquid separation and extraction, dry over anhydrous sodium sulfate, filter, concentrate the organic phase, add appropriate amount of acetonitrile to dissolve, add silica gel, spin dry, and purify by column chromatography: the eluent is PE / EA4 / 1 to obtain the intermediate product II.

[0021] Furthermore, in S2, before adding ethyl acetate and water for separation and extraction, sodium bicarbonate is added to quench the remaining acid and neutralize the reaction mixture.

[0022] Furthermore, in S3, the base is potassium carbonate, and the catalyst is 1,8-diazabicyclo(5,4,0)-7-undecene (DBU).

[0023] Furthermore, in S3, the specific steps are:

[0024] The intermediate product II was dissolved in acetonitrile, N-Boc-L-lysine methyl ester and potassium carbonate were added, nitrogen protection was applied, stirring was performed at room temperature, the mixture was filtered, the filtrate was dried by rotary evaporation, methanol was added to dissolve, 1,8-diazabicyclo(5,4,0)-7-undecene (DBU) was added, stirring was performed at room temperature, ethyl acetate and water were added, separation and extraction were performed, the mixture was dried over anhydrous sodium sulfate, filtered, the organic phase was concentrated, an appropriate amount of acetonitrile was added to dissolve the mixture, silica gel was added, the mixture was dried by rotary evaporation, and the intermediate product III was obtained by column chromatography: the eluent was PE / EA1 / 4.

[0025] Furthermore, the mass ratio of the intermediate product II to N-Boc-L-lysine methyl ester is 10:4-5.

[0026] Furthermore, the mass ratio of the intermediate product II to N-Boc-L-lysine methyl ester is 9:4.

[0027] Furthermore, in S3, after adding DBU, oxygen is introduced.

[0028] Furthermore, in S4, the specific steps are:

[0029] The intermediate product III was dissolved in ethanol, 10% sodium hydroxide solution was added, the mixture was stirred at room temperature, ethyl acetate and water were added for separation and extraction, the mixture was dried over anhydrous sodium sulfate, filtered, the organic phase was concentrated, and the deoxypyridinol artificial hapten was obtained by spin drying.

[0030] Furthermore, in S4, before adding ethyl acetate and water for separation extraction, the pH value is adjusted to 5 with acetic acid.

[0031] In a third aspect, the present invention provides a deoxypyridinol artificial antigen, which is obtained by coupling the above-mentioned deoxypyridinol artificial hapten with a carrier protein.

[0032] Furthermore, the carrier protein is bovine serum albumin.

[0033] In a fourth aspect, the present invention provides a method for preparing the above-mentioned deoxypyridinol artificial antigen, comprising the following steps:

[0034] S1: reacting the deoxypyridinol artificial hapten as claimed in claim 1 with N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide in a solvent to obtain a reaction solution I;

[0035] S2: dissolving the carrier protein to obtain reaction solution II;

[0036] S3: Mix the reaction solution I and the reaction solution II. After the reaction is completed, filter and centrifuge, and take the supernatant to obtain the deoxypyridinol artificial antigen.

[0037] Furthermore, in S1, the solvent is N,N-dimethylformamide (DMF).

[0038] Furthermore, in S1, the specific steps are:

[0039] Take deoxypyridinol hapten, add N,N-dimethylformamide (DMF), then add N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), stir at room temperature for reaction, centrifuge and take supernatant after the reaction is completed to obtain reaction solution I.

[0040] Furthermore, the mass ratio of the deoxypyridinol hapten to N-hydroxysuccinimide is 1:0.5-1.

[0041] Furthermore, the mass ratio of the deoxypyridinol hapten to N-hydroxysuccinimide is 1:0.8.

[0042] Furthermore, in S2, the carrier protein is bovine serum albumin.

[0043] Furthermore, in S2, the carrier protein is dissolved in PBS buffer to obtain reaction solution II.

[0044] Furthermore, in S3, the specific steps are:

[0045] Under rapid stirring, reaction liquid I was slowly added dropwise to reaction liquid II, the volume ratio of reaction liquid I to reaction liquid II was 1:5, the obtained mixed solution was kept at 4°C, transferred to a dialysis bag, and dialyzed with a hydrochloric acid solution of pH = 2. After completion, it was transferred to a dialysis bag and dialyzed with PBS buffer. After the dialysis was completed, the supernatant was centrifuged to obtain the deoxypyridinol artificial antigen.

[0046] Furthermore, during the mixing process of S3, the mass ratio of the deoxypyridinol artificial hapten in S1 to the carrier protein in S2 is 1:1 to 1.5.

[0047] Furthermore, during the mixing process of S3, the mass ratio of the deoxypyridinol artificial hapten used in S1 to the carrier protein used in S2 was 1:1.25.

[0048] In a fifth aspect, the present invention provides a deoxypyridinol antibody, which is prepared by immunizing experimental animals with the above-mentioned deoxypyridinol artificial antigen.

[0049] In a sixth aspect, the present invention provides a method for preparing the above-mentioned deoxypyridinol antibody, comprising the following steps:

[0050] (1) mixing a 1 mg / mL deoxypyridinol artificial antigen PBS solution with Freund's complete adjuvant to obtain a mixed solution;

[0051] (2) injecting the mixed solution obtained in step (1) into an immunized animal;

[0052] (3) Blood is collected from the immunized animal, immune serum is separated from the blood, and deoxypyridinol antibodies are separated.

[0053] Furthermore, in the above-mentioned method for preparing nicardipine antibodies, in step (1), the volume ratio of the deoxypyridinol artificial antigen PBS solution to Freund's complete adjuvant is 1:1.

[0054] Furthermore, in the above-mentioned method for preparing the deoxypyridinol antibody, in step (2), the mixed solution prepared in step (1) is used to inject the immunized animal, and 21 days later, the deoxypyridinol artificial antigen PBS solution and Freund's complete adjuvant mixed solution are injected into the New Zealand white rabbit again, and then the injection is performed every 14 days, for a total of 5 injections.

[0055] In a seventh aspect, the present invention provides the use of the above-mentioned deoxypyridinol artificial hapten, deoxypyridinol artificial antigen, or deoxypyridinol antibody in the preparation of a reagent for detecting deoxypyridinol.

[0056] Furthermore, the reagent for detecting deoxypyridinol is one or more of a homogeneous enzyme immunoassay reagent, an enzyme-linked immunosorbent assay reagent, a colloidal gold labeling reagent, a chemiluminescent labeling reagent, a radioactive labeling reagent, a fluorescent labeling reagent, and a magnetic particle labeling reagent.

[0057] Furthermore, the reagent for detecting deoxypyridinol is a homogeneous enzyme immunoassay reagent.

[0058] In an eighth aspect, the present invention provides a homogeneous enzyme immunoassay reagent for deoxypyridinol, wherein the homogeneous enzyme immunoassay reagent comprises an anti-deoxypyridinol specific antibody and an enzyme reagent;

[0059] The anti-deoxypyridinol specific antibody is the deoxypyridinol antibody according to claim 6;

[0060] The enzyme reagent consists of a deoxypyridinol enzyme-labeled conjugate and an enzyme substrate. The deoxypyridinol enzyme-labeled conjugate is obtained by coupling the deoxypyridinol artificial hapten described in claim 1 with glucose-6-phosphate dehydrogenase, and the enzyme substrate is glucose-6-phosphate.

[0061] In a ninth aspect, the present invention provides a method for preparing the above-mentioned deoxypyridinol homogeneous enzyme immunoassay reagent, comprising the following steps:

[0062] (1) Preparation of reagent A:

[0063] The deoxypyridinol artificial hapten and glucose-6-phosphate dehydrogenase are coupled to obtain a deoxypyridinol enzyme-labeled conjugate; the prepared deoxypyridinol enzyme-labeled conjugate is dispersed in a Tris buffer;

[0064] (2) Preparation of reagent B:

[0065] Disperse glucose-6-phosphate, nicotinamide adenine dinucleotide, and deoxypyridinol antibody in Tris buffer;

[0066] The reagent A and the reagent B are deoxypyridinol homogeneous enzyme immunoassay reagents.

[0067] Furthermore, in the preparation of reagent A, the Tris buffer has a concentration of 100 mM and a pH of 8.1.

[0068] Furthermore, in the preparation of reagent A, the concentration of deoxypyridinol enzyme-labeled conjugate is 0.01% to 1%.

[0069] Furthermore, the preparation of reagent B is as follows: 5 g, 10 mM nicotinamide adenine dinucleotide and 1.5 g, 10 mM glucose-6-phosphate are dissolved in 1 L 50 mM, pH = 8.0 Tris buffer, and the deoxypyridinol antibody prepared in Example 2 is dispersed therein, and the ratio of the deoxypyridinol antibody to the enzyme substrate is 1:100 to 1:5000.

[0070] Compared with the prior art, the present invention has the following beneficial effects:

[0071] The invention provides an artificial hapten of deoxypyridinol, which retains the characteristic structure of deoxypyridinol to the greatest extent and has an active group that can be coupled with a carrier protein. The artificial antigen obtained by coupling can be used as an antigenic determinant, and deoxypyridinol antibodies are obtained by immunizing experimental animals. The prepared artificial hapten, artificial antigen or antibody is used to detect deoxypyridinol, with high sensitivity and less cross-talk, thereby providing a convenient, rapid and accurate way for the detection of deoxypyridinol. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 The figure is a schematic diagram of the reaction formula for preparing the deoxypyridinol artificial antigen (IV) of the present invention.

[0073] Wherein, THF represents tetrahydrofuran, DCM represents dichloromethane, DBU represents 1,8-diazabicyclo(5,4,0)-7-undecene, EDC represents 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, NHS represents N-hydroxysuccinimide, DMF represents N,N-dimethylformamide, and BSA represents bovine serum albumin.

[0074] Figure 2The mass spectrum of hapten intermediate I in Example 1 of the present invention; wherein Rel. Abundance (%) represents relative abundance, and m / z represents charge-to-mass ratio.

[0075] Figure 3 The mass spectrum of hapten intermediate II in Example 1 of the present invention; wherein Rel. Abundance (%) represents relative abundance, and m / z represents charge-to-mass ratio.

[0076] Figure 4 The mass spectrum of hapten intermediate III in Example 1 of the present invention; wherein Rel. Abundance (%) represents relative abundance, and m / z represents charge-to-mass ratio.

[0077] Figure 5 This is a liquid chromatogram of hapten IV in Example 1 of the present invention; wherein, Response (mAU) represents the response value, and the unit is mAU.

[0078] Figure 6 This is the mass spectrum of hapten IV in Example 1 of the present invention; wherein Rel. Abundance (%) represents relative abundance, and m / z represents charge-to-mass ratio.

[0079] Figure 7 This is a schematic diagram of the reaction process for preparing artificial antigen VI in comparative example 1 of the present invention.

[0080] Figure 8 This is a schematic diagram of the reaction process for preparing artificial antigen VII in comparative example 2 of the present invention.

[0081] Fig. 9 Schematic diagram of the absorption spectrum of hapten IV in Example 1 of the present invention; wherein Rel.Response(%) represents the relative response value, and Wavelength represents the wavelength, and the unit is nm.

[0082] Fig.10 This is the standard curve of the homogeneous immunoenzyme test of the present invention. DETAILED DESCRIPTION

[0083] In order to make those skilled in the art better understand the scheme of the present invention, the technical scheme of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed descriptions are all exemplary and are only embodiments of a part of the present invention, rather than all embodiments.

[0084] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of the present invention.

[0085] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs. The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without specifying detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.

[0086] The main materials and reagents used in this application:

[0087] N-Boc-D-glutamic acid-1-tert-butyl ester, CAS number: 73872-71-6; triethylamine, CAS number: 121-44-8; isobutyl chloroformate, CAS number: 543-27-1; diazomethane dichloromethane, CAS number: 334-88-3: N-Boc-L-lysine methyl ester, CAS number: 3017-32-1; N-hydroxysuccinimide (NHS), CAS number: 6066-82-6; 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), CAS number: 25952-53-8; bovine serum albumin, CAS number: 9048-46-8; bovine gamma globulin, CAS number: 9007-83-4; deoxypyridinol, CAS number: 83462-55-9.

[0088] Example 1 Preparation of deoxypyridinol artificial hapten and artificial antigen

[0089] The reaction is as Figure 1 As shown, the following steps are included:

[0090] (1) Preparation of deoxypyridinol hapten

[0091] 1) 5 g of N-Boc-D-glutamic acid-1-tert-butyl ester was dissolved in 50 mL of dichloromethane, 1.9 g of triethylamine was added, and the mixture was placed in an ice-water bath for half an hour. 2.5 g of isobutyl chloroformate was added, and the mixture was reacted in an ice-water bath for 2 hours. 30 mL of a 2M diazomethane dichloromethane solution was added, and the reaction was continued in an ice-water bath for 12 hours. 20 mL of acetic acid was added to quench the mixture, 200 mL of ethyl acetate and 200 mL of water were added, and the mixture was separated and extracted. The mixture was dried over anhydrous sodium sulfate, filtered, and the organic phase was concentrated to obtain 3.9 g of a light yellow oily substance with a yield of 75%. The light yellow oily substance was subjected to mass spectrometry analysis, and the mass spectrum of the intermediate product I was shown in FIG. Figure 2 The mass-to-charge ratio (m / z) of the molecular ion peak was 328.3, which was consistent with its theoretical molecular weight of 327.2, confirming that intermediate product Ⅰ was obtained;

[0092]

[0093] 2) 3.2 g of light yellow oil A was dissolved in 40 mL of tetrahydrofuran, 1 mg of thymol blue was added as an indicator, and the mixture was stirred in an ice-water bath for half an hour. Aqueous hydrobromic acid solution was added dropwise until the solution turned purple-red, and the reaction was terminated. 1 g of sodium bicarbonate was added to quench, 200 mL of ethyl acetate and 200 mL of water were added, and the mixture was separated and extracted. The mixture was dried over anhydrous sodium sulfate, filtered, and the organic phase was concentrated. An appropriate amount of acetonitrile was added to dissolve the mixture, 15 g of silica gel was added, and the mixture was spin-dried. Column chromatography was used for purification: 2.2 g of light yellow oil was obtained as the eluent PE / EA4 / 1, and the yield was 60%. The light yellow oil was subjected to mass spectrometry analysis, and the mass spectrum of intermediate II was shown in FIG. Figure 3 The mass-to-charge ratio (m / z) of the molecular ion peak was 380.1, which was consistent with its theoretical molecular weight of 379.1, confirming that intermediate product II was obtained;

[0094]

[0095] 3) 1.8 g of light yellow oil B was dissolved in 20 mL of acetonitrile, 0.8 g of N-Boc-L-lysine methyl ester and 1.3 g of potassium carbonate were added, the mixture was stirred at room temperature for 12 hours under nitrogen protection, filtered, the filtrate was dried, 30 mL of methanol was added for dissolution, 1.4 g of DBU was added, oxygen was introduced, the mixture was stirred at room temperature for 12 hours, 200 mL of ethyl acetate and 200 mL of water were added, the mixture was separated and extracted, dried over anhydrous sodium sulfate, filtered, the organic phase was concentrated, an appropriate amount of acetonitrile was added for dissolution, 10 g of silica gel was added, the mixture was dried, and the target product was obtained by column chromatography: the eluent was PE / EA1 / 4, 2.38 g of light yellow oil, the yield was 60%, and the light yellow oil was subjected to mass spectrometry analysis. The mass spectrum of intermediate III is shown in FIG. Figure 4 The mass-to-charge ratio (m / z) of the molecular ion peak was 840.5, which was consistent with its theoretical molecular weight of 839.4, confirming that the intermediate product III was obtained;

[0096]

[0097] 4) 1 g of light yellow oil C was dissolved in 10 mL of ethanol, 0.5 g of 10% sodium hydroxide solution was added, and the mixture was stirred at room temperature for 12 hours. The pH value was adjusted to 5 with acetic acid, 100 mL of ethyl acetate and 100 mL of water were added, and the mixture was separated and extracted. The mixture was dried over anhydrous sodium sulfate, filtered, and the organic phase was concentrated and dried to obtain 0.9 g of light yellow oil. The yield was 90%. The light yellow solid was subjected to LC-MS detection to determine that the deoxypyridinol artificial hapten (IV) was obtained.

[0098] The liquid chromatogram of the artificial hapten IV of the nicardipine class is shown in Figure 5 , mass spectrum see Figure 6 .from Figure 5 It can be seen that the purity of the purified nicardipine artificial hapten reaches more than 98%. Figure 6It can be seen that the molecular ion peak of the artificial hapten obtained in this example is 826.5, which is consistent with its theoretical molecular weight of 825.5. It is preliminarily determined that the final compound obtained is the nicrazine artificial hapten IV designed by the present invention.

[0099] (2) Preparation of deoxypyridinol artificial antigen

[0100] 1) Take 120 mg of deoxypyridinol hapten and put it into a 50 mL round-bottom flask, add 5 mL of N,N-dimethylformamide (DMF), then add 75 mg of N-hydroxysuccinimide (NHS) and 135 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), stir at room temperature for reaction overnight, centrifuge after the reaction, and take the supernatant for later use.

[0101] 2) Weigh 14.5 g of disodium hydrogen phosphate dodecahydrate, 43.8 g of sodium chloride, and 1.5 g of sodium dihydrogen phosphate dihydrate, and dissolve them in distilled water to make up to 5.0 L to obtain a 0.01 M PBS buffer solution with a pH of 7.4.

[0102] 3) Weigh 0.125 g of bovine serum albumin and dissolve it in 25 mL of the PBS buffer prepared in step 2) to obtain a bovine serum albumin solution with a concentration of 5 mg / mL.

[0103] 4) Under rapid stirring, the supernatant of step 1) is slowly added dropwise to the bovine serum albumin solution, the volume ratio of the supernatant to the bovine serum albumin solution is 1:5, and the obtained mixture is stored at 4° C. overnight to obtain an artificial antigen mixture.

[0104] 5) The artificial antigen mixture was transferred into a dialysis bag and dialyzed with a pH = 2 hydrochloric acid solution for 12 hours.

[0105] 6) The artificial antigen mixture prepared in 5) was transferred into a dialysis bag and dialyzed with PBS buffer for 4 times. After the dialysis, the supernatant was centrifuged to obtain the deoxypyridinol artificial antigen (V).

[0106] Example 2 Preparation of Deoxypyridinol Antibody

[0107] The prepared deoxypyridinophenol artificial antigen was used to immunize New Zealand white rabbits, and the specific steps were as follows:

[0108] (1) diluting the above-synthesized deoxypyridinol artificial antigen to 1 mg / mL with PBS to obtain an antigen solution, and then mixing 0.5 mL of the antigen solution with 0.5 mL of Freund's complete adjuvant and injecting it into New Zealand white rabbits;

[0109] (2) 21 days later, the New Zealand white rabbits were injected with a mixture of 0.5 mL of antigen solution and 0.5 mL of Freund's complete adjuvant, and then injected every 14 days for a total of 5 injections;

[0110] (3) Blood was collected from New Zealand white rabbits, immune serum was separated, and deoxypyridinol antibodies were purified.

[0111] Example 3 Preparation of Deoxypyridinol Homogeneous Enzyme Immunoassay Reagent

[0112] Preparation of deoxypyridinol homogeneous enzyme immunoassay reagent, the specific steps are as follows:

[0113] (1) Preparation of deoxypyridinol enzyme-labeled conjugates

[0114] Deoxypyridinol artificial haptens V to VII are coupled with glucose-6-phosphate dehydrogenase to obtain:

[0115] 1) Take 100 mg of deoxypyridinol hapten and put it into a 50 mL round-bottom flask, add 5 mL of N,N-dimethylformamide (DMF), then add 75 mg of N-hydroxysuccinimide (NHS) and 135 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), stir at room temperature for reaction overnight, centrifuge after the reaction, and take the supernatant for later use.

[0116] 2) Preparation of glucose-6-phosphate dehydrogenase solution: Weigh 10 mg of glucose-6-phosphate dehydrogenase with a specification of 100 KU, dissolve it in 10 mL of a solution containing 50 mg 0.05 M Tris and 100 mg NaCl at room temperature, pH = 9.0; add 200 mg of reduced nicotinamide adenine dinucleotide, 100 mg of glucose-6-phosphate and 0.5 mL of carbitol to the solution; then add dropwise to 2 mL of DMF and set aside.

[0117] 3) Slowly drip the activated product of step 1) into the enzyme solution of step 2) and stir at 2-8°C overnight.

[0118] 4) The conjugate mixture in step 3) was transferred into a dialysis bag and dialyzed with a pH = 2 hydrochloric acid solution for 12 hours.

[0119] 5) The conjugate mixture was transferred into a dialysis bag and dialyzed with PBS buffer for 4 times. After the dialysis, the supernatant was centrifuged to obtain the deoxypyridinol enzyme-labeled conjugate.

[0120] (2) Preparation of Reagent A

[0121] The deoxypyridinol enzyme-labeled conjugate prepared in step (1) is dispersed in a 100 mM Tris buffer solution with a pH of 8.1, and the concentration of the deoxypyridinol enzyme-labeled conjugate is 0.01% to 1%.

[0122] (3) Preparation of Reagent B

[0123] 5 g of 10 mM nicotinamide adenine dinucleotide and 1.5 g of 10 mM glucose-6-phosphate were dissolved in 1 L of 50 mM, pH=8.0 Tris buffer, and the deoxypyridinol antibody prepared in Example 2 was dispersed therein. The ratio of the deoxypyridinol antibody to the enzyme substrate was 1:100 to 1:5000.

[0124] The above reagent A and reagent B are homogeneous enzyme immunoassay reagents for deoxypyridinol.

[0125] Comparative Example 1 Preparation of deoxypyridinol artificial antigen using different activation methods

[0126] Preparation method of deoxypyridinol artificial antigen (VI) (reaction formula as shown in Figure 7 ), including the following steps:

[0127] (1) Preparation of deoxypyridinol artificial hapten (IV):

[0128] The operation steps are the same as those for the preparation of the deoxypyridinol artificial hapten in Example 1.

[0129] (2) Preparation of deoxypyridinol artificial antigen (VI):

[0130] 1) 100 mg of deoxypyridinol hapten (I) was placed in a 50 mL round-bottom flask, 5 mL of N,N-dimethylformamide (DMF) was added, and then 90 mg of triethylamine and 120 mg of isobutyl chloroformate were added. The mixture was stirred at room temperature for overnight reaction. After the reaction was completed, the supernatant was centrifuged and set aside.

[0131] 2)-6) The preparation of the antigen was the same as in Example 1 to obtain the deoxypyridinol artificial antigen (VI).

[0132] Comparative Example 2 Preparation of artificial antigens using different carrier proteins

[0133] Preparation method of deoxypyridinol artificial antigen (VII) (reaction formula as shown in Figure 8 ), including the following steps:

[0134] (1) Preparation of deoxypyridinol artificial hapten (IV):

[0135] The operation steps are the same as those for the preparation of the deoxypyridinol artificial hapten in Example 1.

[0136] (2) Preparation of deoxypyridinol artificial antigen:

[0137] Bovine gamma protein (BGG) was used as a carrier protein for coupling with hapten, and the coupling steps 1)-2) were the same as those in Example 1.

[0138] 3) Weigh 0.125 g of bovine protein G and dissolve it in 25 mL of the PBS buffer prepared in step 2) to obtain a bovine protein G solution with a concentration of 5 mg / mL;

[0139] 4) under rapid stirring, slowly adding the supernatant obtained in step 1) to the bovine protein G solution, the volume ratio of the supernatant to the bovine protein G solution being 1:5, and the obtained mixed solution being stored at 4° C. overnight to obtain an artificial antigen mixed solution;

[0140] 5) The artificial antigen mixture was transferred into a dialysis bag and dialyzed with PBS buffer for 4 times. After the dialysis, the supernatant was collected by centrifugation to obtain the deoxypyridinol artificial antigen (VII).

[0141] Test Example 1 Performance Test of Deoxypyridinol Artificial Antigen

[0142] (1) Determination of molar absorption coefficient

[0143] Deoxypyridinol artificial hapten solutions with concentrations of 0 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, and 40 μg / mL were prepared with PBS buffer. Fig. 9 From the UV absorption spectrum shown, it can be seen that the maximum absorption wavelength of deoxypyridinol hapten is 244nm. The absorbance is measured at 244nm, and parallel samples are made for each concentration. The calculation formula of molar absorption coefficient is: ε = absorbance / molar concentration.

[0144] (2) Determination of the concentration of deoxypyridinol artificial antigen protein

[0145] The antigen concentration was measured by the Lowry method. 1 mL of bovine serum protein solution with concentrations of 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL was prepared with PBS buffer. Parallel samples were made for each concentration. The absorbance was detected at 650 nm using an ultraviolet spectrophotometer, and a curve of the relationship between protein concentration and absorbance was drawn. The deoxypyridinol artificial antigen prepared in Example 1 and Comparative Examples 1-2 was dissolved in PBS buffer, diluted at a ratio of 1 / 8, and the absorbance of the deoxypyridinol artificial antigen was measured at 650 nm. The corresponding protein concentration of the deoxypyridinol artificial antigen solution was read from the curve to calculate the protein concentration of the deoxypyridinol artificial antigen.

[0146] (3) Determination of coupling ratio of deoxypyridinol artificial antigen

[0147] Prepare 100 μg / mL bovine serum albumin PBS solution, dilute the deoxypyridinol artificial antigen to 100 μg / mL with PBS, measure the absorbance value A1 at 240 nm, and measure the absorbance value A2 with PBS solution as blank. The coupling ratio γ is calculated as follows: γ = [(A1-A2) / ε] / (100×10 -3 / 66000);

[0148] Wherein, ε is the molar absorption coefficient (L / mol) measured in this example, 66000 is the molecular weight of bovine serum albumin, 100×10 -3 is the bovine serum albumin concentration (g / L);

[0149] When bovine germline protein (BGG) is used as a carrier, γ = [(A1-A2) / ε] / (100×10 -3 / 150000), the molecular weight of bovine gamma protein is 1500000, 100×10 -3 is the concentration of bovine serum albumin (g / L).

[0150] The coupling ratios of the deoxypyridinol artificial antigens and the molar absorption coefficients of the corresponding haptens are shown in Table 1

[0151] Table 1 Performance test of deoxypyridinol artificial antigens V~VII

[0152] serial number Artificial Antigen Antigen concentration Molar Absorption Coefficient Coupling ratio Example 1 Ⅴ 3.42mg / mL 4856.1 27 Comparative Example 1 Ⅵ 3.21mg / mL 4759.6 16 Comparative Example 2 Ⅶ 3.19mg / mL 4812.3 20

[0153] As can be seen from Table 1, the activation method of the artificial hapten and the structure of the carrier protein have a certain influence on the coupling ratio when the hapten and the carrier protein are combined.

[0154] Test Example 2 Activity Detection of Deoxypyridinol Artificial Antigen

[0155] The titer of the immune serum prepared in Example 2 was tested by ELISA method. The test results are shown in Table 2.

[0156] Table 2 Immune serum titer

[0157] Deoxypyridinol artificial antigen Immune serum titer Ⅴ 1:80000 Ⅵ 1:19000 Ⅶ 1:26000

[0158] It can be seen from Table 2 that, compared with the artificial deoxypyridinol antigen prepared in Example 1, the titers of the immune serum obtained by immunizing animals with the artificial deoxypyridinol antigens prepared in Comparative Examples 1 and 2 are all low. However, the titer of the immune serum obtained by immunizing animals with the artificial deoxypyridinol antigen V is 1:80,000, which can be used in immunoassays, and can provide a convenient, fast and accurate way for the detection of deoxypyridinol.

[0159] Test Example 3 Deoxypyridinol homogeneous enzyme immunoassay

[0160] (1) Preparation of standard samples: Deoxypyridinol powder was dissolved in acetonitrile to prepare a 1 mg / mL stock solution, and the stock solution was diluted in blank urine to prepare standard urine samples with final concentrations of 0.0, 10.0, 25.0, 50.0, 100.0, 200.0, and 300.0 ng / mL, respectively.

[0161] (2) Standard curve: Using a fully automatic biochemical analyzer, first add the reagent B prepared in Example 3, then add the standard, and finally add the reagent A prepared in Example 3. After adding reagent A, measure the absorbance at different time points, calculate the reaction rate at different standard concentrations, and obtain a more ideal reaction standard curve, as shown in detail. Fig.10 shown.

[0162] (3) Sample testing: Repeat the measurement of 10 ng / mL, 50 ng / mL, and 200 ng / mL concentration quality control samples 5 times and measure the absorbance values. Fig.10 The test values ​​were obtained using the standard curve shown in Table 3.

[0163] Table 3 Deoxypyridinol standard sample homogeneous enzyme immunoassay results

[0164]

[0165] It can be seen from Table 3 that the homogeneous enzyme immunoassay reagent of the present invention has high accuracy and can be used for the detection of deoxypyridinol.

[0166] Test Example 4: Drug Cross-Interference Experiment

[0167] Thirty common drugs were prepared with a concentration of 50 ng / mL and tested using a homogeneous enzyme immunoassay. The test results are shown in Table 4.

[0168] Table 4 Deoxypyridinol drug cross-interference test results

[0169]

[0170] As can be seen from Table 4, the antibody immunized by the antigen of the present invention is a specific antibody for deoxypyridinol and has no cross reaction with other drugs.

Claims

1. A deoxypyridinol artificial hapten, characterized in that: The structure of the deoxypyridinol artificial hapten is shown in Formula IV:

2. The method for preparing the deoxypyridinol artificial hapten according to claim 1, characterized in that: The following steps are involved: S1: reacting N-Boc-D-glutamic acid-1-tert-butyl ester with isobutyl chloroformate and diazomethane in the presence of triethylamine, and separating and purifying to obtain an intermediate product Ⅰ as shown in formula Ⅰ; S2: reacting the intermediate product I with hydrobromic acid, separating and purifying to obtain the intermediate product II as shown in formula II; S3: The intermediate product II reacts with N-Boc-L-lysine methyl ester in the presence of a base and a catalyst, and the intermediate product III is obtained after separation and purification as shown in formula III; S4: The intermediate product III is hydrolyzed under alkaline conditions, and after separation and purification, the deoxypyridinol artificial hapten shown in formula IV is obtained.

3. A deoxypyridinol artificial antigen, characterized in that: The deoxypyridinol artificial antigen is obtained by coupling the deoxypyridinol artificial hapten as claimed in claim 1 with a carrier protein.

4. The deoxypyridinol artificial antigen according to claim 3, characterized in that: The carrier protein is bovine serum albumin.

5. The method for preparing the deoxypyridinol artificial antigen according to claim 3, characterized in that: The following steps are involved: S1: dispersing the deoxypyridinol artificial hapten as claimed in claim 1, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide in a solvent to obtain a reaction solution I; S2: dissolving the carrier protein to obtain reaction solution II; S3: Mix the reaction solution I and the reaction solution II. After the reaction is completed, filter and centrifuge, and take the supernatant to obtain the deoxypyridinol artificial antigen.

6. A deoxypyridinol antibody, characterized in that The method is prepared by immunizing experimental animals with the deoxypyridinol artificial antigen as claimed in any one of claims 3 or 4.

7. The method for preparing the deoxypyridinol antibody according to claim 6, characterized in that: The following steps are involved: (1) mixing a 1 mg / mL deoxypyridinol artificial antigen PBS solution with Freund's complete adjuvant to obtain a mixed solution; (2) injecting the mixed solution obtained in step (1) into an immunized animal; (3) Blood is collected from the immunized animal, immune serum is separated from the blood, and deoxypyridinol antibodies are separated.

8. Use of the artificial hapten of deoxypyridinol as claimed in claim 1, the artificial antigen of deoxypyridinol as claimed in any one of claims 3 or 4, or the antibody of deoxypyridinol as claimed in claim 6 in the preparation of a reagent for detecting deoxypyridinol.

9. A homogeneous enzyme immunoassay reagent for deoxypyridinol, characterized in that: The homogeneous enzyme immunoassay reagent comprises an anti-deoxypyridinol specific antibody and an enzyme reagent; The anti-deoxypyridinol specific antibody is the deoxypyridinol antibody according to claim 6; The enzyme reagent consists of a deoxypyridinol enzyme-labeled conjugate and an enzyme substrate. The deoxypyridinol enzyme-labeled conjugate is obtained by coupling the deoxypyridinol artificial hapten described in claim 1 with glucose-6-phosphate dehydrogenase, and the enzyme substrate is glucose-6-phosphate.

10. A method for preparing the deoxypyridinol homogeneous enzyme immunoassay reagent according to claim 9, characterized in that: The following steps are involved: (1) Preparation of reagent A: The deoxypyridinol artificial hapten and glucose-6-phosphate dehydrogenase are coupled to obtain a deoxypyridinol enzyme-labeled conjugate; the prepared deoxypyridinol enzyme-labeled conjugate is dispersed in a Tris buffer; (2) Preparation of reagent B: Disperse glucose-6-phosphate, nicotinamide adenine dinucleotide, and deoxypyridinol antibody in Tris buffer; The reagent A and the reagent B are deoxypyridinol homogeneous enzyme immunoassay reagents.

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