CN105801688A - Deoxy pyridinoline immunogen, antibody, detection reagent and preparing method - Google Patents
Deoxy pyridinoline immunogen, antibody, detection reagent and preparing method Download PDFInfo
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- CN105801688A CN105801688A CN201610231728.4A CN201610231728A CN105801688A CN 105801688 A CN105801688 A CN 105801688A CN 201610231728 A CN201610231728 A CN 201610231728A CN 105801688 A CN105801688 A CN 105801688A
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- Prior art keywords
- deoxypyridinoline
- antibody
- solution
- immunogen
- reagent
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- 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
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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Abstract
The invention discloses deoxy pyridinoline immunogen, an antibody, a detection reagent and a preparing method.The prepared deoxy pyridinoline immunogen is high in immunogenicity and capable of obtaining high-potency specific antibody resisting deoxy pyridinoline through induction and has no cross reaction with 62 common dugs; the deoxy pyridinoline detection reagent prepared from the antibody can accurately and fast determine the content of deoxy pyridinoline in urine and other biological biological samples.Compared with existing detection reagents in the market, the detection reagent has the advantages of being easy to operate, high in sensitivity and specificity, accurate in result, capable of effectively reducing detection cost of deoxy pyridinoline and beneficial for large-scale clinical utilization and popularization.
Description
Technical field
The invention belongs to biological technical field, relate to deoxypyridinoline immunogen, anti-deoxypyridinoline specific antibody and deoxypyridinoline detectable and preparation method thereof.
Background technology
Deoxypyridinoline (Deoxypyridinoline, DPD), its structural formula is such as shown in formula II:
Formula II
Deoxypyridinoline exists only in the NTx fiber of skeleton, and the NTx fiber molecule being ripe constitutes molecule and intermolecular commissure thing during collagen fiber, acts the effect stablizing collagen chain.During bone resorption, DPD is as NTx fiber degradation product, is released into entrance blood circulation with the form that free or peptide combine, and without the degraded of liver directly through renal excretion, excretes with urine.DPD, by food effect, may alternatively appear in before bone density there is no change.Research shows: within 35～40 years old, be people's age bracket that bone density is the highest in life, hereafter bone density begins to decline, enter the bone loss phase, that is this age bracket is behaved and is entered the early stage in bone loss stage, and before 40 years old, occur that DPD is significantly raised, the balance then indicated between internal bone formation and bone resorption is damaged, and abnormal loss occurs in bone amount, and osteoporotic early symptom likely occurs in prompting.Along with the increase of the raising of people's living standard and average life, aged's sharp increase, and primary osteoporosis has become the degenerative disease that old people is common.The common method making a definite diagnosis osteoporosis at present is that X ray borne densitometers (DEXA) measures bone density (BMD), but because it has radioactivity, human body side effect is bigger, and early stage bone amount is reduced the sensitivity that shortage is enough by borne densitometers, the metabolic activity that skeleton is real-time can not be reflected.DPD is special biochemical indicator as reflection one sensitivity of bone metabolism, both can early diagnosis osteoporosis, reduce incidence of fracture, can be used for again assessing the prognosis of osteoporosis, for monitoring with prevent and treat bone amount and run off in advance and provide simple and easy to do, high specificity, a highly sensitive clinical indices.Additionally, DPD is significant in reflection trimester of pregnancy and bone resorption increase age of sucking.DPD has played great function in optimum osseous lesion diagnoses, and specificity is higher compared with traditional biochemical indicator, sensitivity is higher.Current research is pointed out: DPD can as the sensitive osteolytic mark of malignant tumor, and at malignant metastatic tumor of bone early diagnosis, monitoring Bone tumour therapeutic effect aspect has certain effect, and effect is better than the imaging diagnosis methods such as traditional X-ray, ECT.
DPD content in urine can adopt multiple method to detect, and the method for early stage has: molecular sieve chromatography, ion-exchange chromatography method etc., currently used more mainly having: high performance liquid chromatography, enzyme-linked immunosorbent assay, science of law luminescent immunoassay etc..But these methods length consuming time, cost are high, detection small scale, can not meet the requirement of DPD content high flux, rapid detection in a large amount of clinical patients urine.Deficient in stability is good, highly sensitive in the market, the DPD detectable of high specificity, the especially measured Automated inspection reagent of matter.Therefore, research and development are a kind of, and quality reaches Clinical Laboratory requirement, practical, cost performance is high, can be applicable to the DPD detectable of automatic clinical chemistry analyzer imperative.
Summary of the invention
The defect that the present invention exists to overcome prior art, adopting unique deoxypyridinoline to prepare the strong deoxypyridinoline immunogen of immunogenicity and antibody thereof, the deoxypyridinoline homogeneous enzyme immunoassay detectable prepared with this antibody can be implemented on automatic clinical chemistry analyzer deoxypyridinoline high flux, rapid detection.This detectable has the advantages such as easy and simple to handle, highly sensitive, high specificity, result are accurate, moreover it is possible to effectively reduces deoxypyridinoline testing cost, is conducive to clinical expansion to use.
It is an object of the present invention to provide the deoxypyridinoline immunogen that a kind of immunogenicity is strong.
A kind of immunogenic preparation method of deoxypyridinoline of offer is provided.
Another purpose of the present invention is in that to provide the anti-deoxypyridinoline specific antibody of the high specificity using deoxypyridinoline immunogen of the present invention to prepare.
It is yet a further object of the present invention to provide a kind of deoxypyridinoline detectable and preparation method thereof.
The deoxypyridinoline immunogen of the present invention, immunogenicity is high, it is possible to induction obtains the anti-deoxypyridinoline specific antibody of high-titer.This antibody specificity is high, strong with the adhesion of deoxypyridinoline.The deoxypyridinoline detectable prepared by this antibody, it is possible to determine the deoxypyridinoline content in sample quickly and accurately.The present invention is achieved by the following technical solutions:
A kind of deoxypyridinoline immunogen, its structural formula is such as shown in formula I:
Formula I
Carrier for having immunogenic protein or polypeptide, the one in serum albumin, hemocyanin or Elityran.It is more preferably serum albumin, more preferably bovine serum albumin.
Described deoxypyridinoline immunogen is formed by connecting by deoxypyridinoline and above-mentioned carrier, and the chemical constitution of deoxypyridinoline is such as shown in formula II:
Formula II
The immunogenic preparation method of this deoxypyridinoline is as follows:
(1) carrier protein 100～300mg is dissolved in the phosphate buffer of 25～75ml0.2M, pH8.5；
(2) following chemicals is joined stirring and dissolving in small beaker: 100～300mg deoxypyridinoline, 1.75～5.25ml dimethylformamide, 1.75～5.25ml ethanol, 3.5～10.5ml10mM, the kaliumphosphate buffer of pH5.0,100～300mg1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 25～75mgN-hydroxy thiosuccinimide, react these chemicals at room temperature stirring and dissolving to 30～60min；
(3) solution dissolved is dropped in carrier protein solution, and stir at 2～8 DEG C overnight, obtain antigen；Synthetic antigen is purified through dialysis, obtains deoxypyridinoline immunogen.
A kind of anti-deoxypyridinoline specific antibody, is by the complete antibody molecule produced after above-mentioned deoxypyridinoline immunogen immune laboratory animal, or is antibody fragment or the antibody derivatives of reservation and deoxypyridinoline specific binding capacity.
Described complete antibody molecule, antibody fragment or antibody derivatives, for the polyclonal antibody adopting single deoxypyridinoline immunogen that animal booster immunization is obtained, or the monoclonal antibody for obtaining through somatic hybridization after immunity.Described laboratory animal is the one of rabbit, goat, mice, sheep, Cavia porcellus or horse, it is preferred to rabbit.
Described anti-deoxypyridinoline specific antibody is adopted conventional method inoculation experiments animal by above-mentioned prepared deoxypyridinoline immunogen, takes antiserum after booster immunization.
The preparation method of anti-deoxypyridinoline specific antibody, specifically comprises the following steps that
(1) with PBS, the BSA-deoxypyridinoline immunogen of above-mentioned synthesis is diluted to 0.1～3.0mg/ml, obtains antigenic solution, then mix with equivalent Freund's complete adjuvant with 0.5～5.0ml antigenic solution, laboratory animal is injected；
After (2) 2～3 weeks, then with equivalent incomplete Freund's adjuvant, above-mentioned laboratory animal is injected once with antigenic solution identical for 0.5～5.0ml, inject once every surrounding afterwards, amount to injection 3～6 times；
(3) above-mentioned laboratory animal is taken blood, separate purification and obtain the anti-deoxypyridinoline specific antibody that titer is 1:30000～1:50000.
The present invention provides a kind of deoxypyridinoline detectable, containing above-mentioned anti-deoxypyridinoline specific antibody and indicator, described indicator one in enzyme reagent, radiosiotope reagent, fluorometric reagent or luminescence reagent；Described enzyme reagent is made up of the substrate of deoxypyridinoline enzyme mark conjugate and enzyme, and enzyme mark conjugate is glucose-6-phosphate dehydrogenase (G6PD)-hapten enzyme mark conjugate, and the substrate of enzyme is G-6-P.
The preparation method of deoxypyridinoline detectable, it is characterised in that comprise the steps of
(1) reagent A: 2.018～8.072g, the nicotinamide adenine dinucleotide of 5.625～22.50mM oxidation state and 0.856～3.422g, 5.625～22.50mM G-6-P Tris buffer solution of 0.5～2L55mM, pH=8.0 are made homogeneous zymolyte；Being added in above-mentioned homogeneous zymolyte by described anti-deoxypyridinoline specific antibody, the volume ratio of anti-deoxypyridinoline specific antibody and homogeneous zymolyte is 1:100～1:10000；
(2) volume ratio of reagent B: be added in the Tris buffer of 120mM, pH=8.2 by deoxypyridinoline enzyme mark conjugate, deoxypyridinoline enzyme mark conjugate and Tris buffer is 1:100～1:10000.
The volume ratio of described anti-deoxypyridinoline specific antibody and homogeneous zymolyte is preferably 1:550；
The volume ratio of described deoxypyridinoline enzyme mark conjugate and Tris buffer is preferably 1:1500.
The preparation method of described deoxypyridinoline enzyme mark conjugate comprises the steps of
(1) preparation of glucose-6-phosphate dehydrogenase (G6PD) solution: weighing the glucose-6-phosphate dehydrogenase (G6PD) that 7.5～22.5mg specification is 100KU, room-temperature dissolution contains 72.6mg0.05MTris, 8mg3.3mMMgCl in 6～18mL2With in the solution of 100mgNaCl, pH=9.0；Add the nicotinamide adenine dinucleotide of 112.5～337.5mg reduction-state, 67.5～202.5mg G-6-P and 0.375～1.125mL carbitol in the solution；It is added dropwise over 1～3mL dimethyl sulfoxide again；
(2) activation of deoxypyridinoline: weigh 5～15mg deoxypyridinoline under anhydrous conditions, is dissolved in 300～900 μ L dimethylformamides；Above-mentioned solution temperature is made to drop to-2 ~-8 DEG C；Add 1.5～4.5 μ L tri-n-butylamines；Add 0.75～2.25 μ L isobutyl chlorocarbonate；-2 ~-8 DEG C are stirred 30～60 minutes；
(3) connection of glucose-6-phosphate dehydrogenase (G6PD) and deoxypyridinoline: the deoxypyridinoline dropwise that step (2) activates is joined in the glucose-6-phosphate dehydrogenase (G6PD) solution that step (1) is dissolved；2-8 DEG C of stirring is overnight；
(4) purified product: by G-25 gel chromatography column purification connect product, it is thus achieved that end product be glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing, at 2-8 DEG C store.
Deoxypyridinoline homogeneous enzyme immunoassay detectable is before the use, in order to avoid the substrate of the enzyme mark conjugate in indicator and enzyme reacts, the substrate of enzyme mark conjugate and enzyme is unmixed and separated, so the substrate of enzyme and above-mentioned anti-deoxypyridinoline specific antibody being mixed.
The deoxypyridinoline immunogens of the present invention is strong, immunogenicity is high, and anti-deoxypyridinoline specific antibody high specificity, the titer prepared are high, and with 62 kinds of common medicines without any cross reaction；Homogeneous enzyme immunoassay detectable containing above-mentioned anti-deoxypyridinoline specific antibody can easily and fast, the deoxypyridinoline content that accurately determines in the biological samples such as urine, and multiple sample can be measured on automatic clinical chemistry analyzer simultaneously, realize the rapid mensuration of high flux of deoxypyridinoline, accuracy is high, high specificity, degree of accuracy is all enhanced before comparing with detection efficiency, it is simultaneously achieved the full-automation of detection process, less demanding to testing staff, it is easy to accomplish with promote the use of.
Accompanying drawing explanation
The ELISA that Fig. 1 is deoxypyridinoline detects response curve；
Fig. 2 is the homogeneous enzyme immunoassay response curve of deoxypyridinoline；
Fig. 3 is deoxypyridinoline homogeneous enzyme immunoassay correlation analysis figure.
Detailed description of the invention
The immunogenic synthesis of embodiment one deoxypyridinoline
Deoxypyridinoline immunogen is by bovine serum albumin (BovineSerumAlbumin, BSA) and the deoxypyridinoline shown in formula IIGroup is formed by connecting, and specifically comprises the following steps that
1. bovine serum albumin 200mg is dissolved in the phosphate buffer of 50ml0.2M, pH8.5；
2. following chemicals is joined stirring and dissolving in small beaker: 200mg deoxypyridinoline, 3.5ml dimethylformamide, 3.5ml ethanol, 7.0ml10mM, the kaliumphosphate buffer of pH5.0,200mg1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 50mgN-hydroxy thiosuccinimide, react these chemicals at room temperature stirring and dissolving to 30min；
3. the solution dissolved is dropped in BSA solution, and stir at 2～8 DEG C overnight, obtain antigen；Synthetic antigen is purified through dialysis, obtains deoxypyridinoline immunogen.
Embodiment two: the preparation of anti-deoxypyridinoline specific antibody
Deoxypyridinoline immunogen embodiment one prepared adopts conventional method inoculation experiments animal rabbit, takes antiserum, specifically comprise the following steps that after booster immunization
1. with PBS, the deoxypyridinoline immunogen of above-mentioned synthesis is diluted to 1.0mg/ml, obtains antigenic solution, then mix with equivalent Freund's complete adjuvant with 1.0ml antigenic solution, experimental animal rabbit is injected.
After 2.2～3 weeks, then with equivalent incomplete Freund's adjuvant, above-mentioned experimental animal rabbit is injected once with antigenic solution identical for 1.0ml, inject once every surrounding afterwards, amount to injection 4 times.
3. the experimental animal rabbit of pair step 2 takes blood, separates purification and obtains the anti-deoxypyridinoline specific antibody that titer is 1:30000～1:50000.
Embodiment three: the ELISA inspection of deoxypyridinoline
1. the foundation of the ELISA examination criteria curve of deoxypyridinoline
(1) preparation of standard substance
Deoxypyridinoline powder (being purchased from Sigma company) is dissolved in methanol solution, prepares into the storage liquid of 1000 μm of ol/L.It is the standard solution of 300.00nmol/L, 100.00nmol/L, 30.00nmol/L, 10.00nmol/L, 3.00nmol/L and 0.00nmol/L with ELISA buffer by storing that liquid dilutes successively.Wherein, ELISA buffer contains 50.0mMTris, the BSA of 145mMNaCl and 0.25%.
(2) the ELISA method of inspection utilizing deoxypyridinoline prepares standard curve
Anti-deoxypyridinoline specific antibody prepared in embodiment two is diluted to the final concentration solution of 1:5000 with PBS, and 100 μ L/ holes are coated on 96 hole elisa plates, place 12-24h for 4 DEG C；After the above-mentioned 96 hole elisa plates being coated with anti-deoxypyridinoline antibody being washed 3 times with PBS, add 200 μ L/ holes 0.5% BSA solution, close for 4 DEG C and place 8-16h.Then wash 3 times with PBS, add the standard substance in 20 μ L/ holes.Add the HRP-deoxypyridinoline conjugate of 100 μ L/ hole working concentrations；After incubated at room temperature 30min, PBS washes plate 5 times；Then every hole adds 100 μ LTMB substrates, incubated at room 30min.Every hole adds 100 μ L stop buffers (2M sulphuric acid) again.Measure the light absorption value of 450nm.The light absorption value calibration of the 450nm corresponding to each standard substance, makes standard curve, and result is as shown in Figure 2.
2. the detection of deoxypyridinoline content in testing sample
(1) testing sample is made
Preparation method: deoxypyridinoline powder (being purchased from Sigma company) is dissolved in methanol solution and makes the storage liquid of 1000 μm of ol/L, and this storage liquid is diluted in blank diaper, to final concentration respectively 0.00,5.00,80.00,250.00nmol/L, form the urine specimen of concentration blank, basic, normal, high.This blank diaper is the Healthy People urine without deoxypyridinoline.
(2) method of testing
Utilize the ELISA method of inspection of above-mentioned deoxypyridinoline, the urine specimen of above-mentioned blank, basic, normal, high concentration is replaced standard substance, test the urine specimen light absorption value at 450nm of above-mentioned blank, basic, normal, high concentration.
(3) test result
The standard curve of the ELISA inspection of comparison deoxypyridinoline shown in Fig. 1, calculate deoxypyridinoline content in each sample, and each sample is carried out 3 multiple holes mensuration, and calculating the response rate according to the actual content of deoxypyridinoline in above-mentioned sample, result is as shown in table 1.
The ELISA of table 1 deoxypyridinoline detects recovery experiment
| Urine sample | Blank | Low | In | High |
| Sample concentration (nmol/L) | 0.00 | 5.00 | 80.00 | 250.00 |
| Test 1 | 0.03 | 4.96 | 81.23 | 255.52 |
| Test 2 | 0.00 | 5.31 | 80.79 | 249.49 |
| Test 3 | 0.03 | 5.15 | 82.34 | 258.30 |
| Meansigma methods (nmol/L) | 0.02 | 5.14 | 81.45 | 254.44 |
| The response rate (%) | - | 102.8% | 101.80% | 101.80% |
From result in table 1: adopt the deoxypyridinoline response rate that the ELISA detectable of deoxypyridinoline of the present invention measures in variable concentrations sample all higher, equal > 90%, illustrate that anti-deoxypyridinoline specific antibody of the present invention may be used for the detection of deoxypyridinoline in sample, and result precision is high.
Embodiment four: the preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing
1. the preparation of glucose-6-phosphate dehydrogenase (G6PD) (G6PDH) solution:
(1) accurately weighing the G6PDH that 15mg specification is 100KU, room-temperature dissolution contains 72.6mg(0.05M in 12mL) Tris, 8mgMgCl2(3.3mM) with in the solution of 100mgNaCl, these pH value of solution=9.0, this step carries out in beaker C.
(2) in above-mentioned beaker C, add the nicotinamide adenine dinucleotide (NADH) of 225mg reduction-state, 135mg G-6-P (G-6-P) and 0.75mL carbitol (Carbitol).
(3) in above-mentioned beaker C, 2mL dimethyl sulfoxide (dimethysulfoxide, DMSO) it is added dropwise over again.
2. the activation of deoxypyridinoline:
(1) weigh the above-mentioned deoxypyridinoline of 10mg under anhydrous conditions, be dissolved in 600 μ LDMF.
(2) above-mentioned solution temperature is made to drop to-2 ~-8 DEG C.
(3) 3 μ L tri-n-butylamine (tributylamine) are added.
(4) 1.5 μ L isobutyl chlorocarbonate (isobutylchloroformate) are added.
(5)-2 ~-8 DEG C are stirred 30 minutes.
The connection of 3.G6PDH and deoxypyridinoline:
(1) the deoxypyridinoline dropwise of above-mentioned activation is joined in the G6PDH solution of above-mentioned dissolving.
(2) 2-8 DEG C of stirring is overnight.
4. purified product:
By the solution in G-25 gel chromatography column purification step 3, it is thus achieved that end product be glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing, at 2-8 DEG C store.
Embodiment five: the preparation of deoxypyridinoline homogeneous enzyme immunoassay detectable
1. the preparation of reagent A: by 4.036g(11.25mM) nicotinamide adenine dinucleotide (NAD) of oxidation state, 1.711g(11.25mM) G-6-P (G-6-P) is placed in beaker D, makes homogeneous zymolyte with the Tris buffer solution of 1L55mM, pH=8.0；Being added in above-mentioned homogeneous zymolyte by the anti-deoxypyridinoline specific antibody of above-mentioned preparation, the volume ratio of antibody and homogeneous zymolyte is 1:550.
2. the volume ratio of the preparation of reagent B: glucose-6-phosphate dehydrogenase (G6PD) embodiment four prepared-hapten conjugation thing is added in the Tris buffer of 120mM, pH=8.2, above-mentioned conjugate and Tris buffer is 1:1500.
Embodiment six: the inspection of deoxypyridinoline homogeneous enzyme immunoassay and result
1. obtain standard curve:
(1) auspicious BS-480 automatic clinical chemistry analyzer response parameter (see table 2) advanced in years is set.
(2) operating procedure is: first reagent adding A, adds standard substance, is eventually adding reagent B.After adding reagent B, measure the OD of different time points340Light absorption value, calculates reaction rate during various criterion product concentration, needs constantly to adjust the volume ratio of reagent A and reagent B in actual mechanical process, adjusts light-metering point simultaneously, finally draws comparatively ideal reaction normal curve chart, as shown in Figure 3.
Table 2 steps auspicious BS-480 automatic clinical chemistry analyzer response parameter
2. pattern detection: the standard curve obtained by the homogeneous enzyme immunoassay detectable of the present invention, the basic, normal, high concentration Quality Control sample of replication 10 times, above-mentioned Quality Control sample is: be dissolved in human urine by deoxypyridinoline standard substance, to concentration respectively 5.00,80.00,250.00nmol/L.Detection data and data analysis are in Table 3.
Table 3 sample determination and precision and the response rate are assessed
| Urine sample | Low | In | High |
| Sample concentration (nmol/L) | 5.00 | 80.00 | 250.00 |
| 1 | 5.26 | 79.81 | 255.45 |
| 2 | 4.91 | 82.14 | 249.63 |
| 3 | 5.82 | 83.20 | 258.70 |
| 4 | 5.09 | 81.06 | 253.59 |
| 5 | 4.85 | 84.23 | 255.31 |
| 6 | 5.40 | 79.95 | 241.74 |
| 7 | 5.28 | 83.55 | 257.98 |
| 8 | 5.37 | 81.79 | 250.22 |
| 9 | 5.53 | 78.00 | 255.00 |
| 10 | 4.92 | 80.29 | 251.11 |
| Meansigma methods (nmol/L) | 5.24 | 81.40 | 252.87 |
| Standard deviation (SD) | 0.31 | 1.95 | 4.97 |
| Precision (CV%) | 5.92% | 2.40% | 1.97% |
| Response rate % | 104.80% | 101.75% | 101.15% |
Testing result: the accuracy that the homogeneous enzyme immunoassay detectable of the present invention measures is high, and the response rate reaches 95%-105%, precision is high, and CV is below 6%.
Embodiment seven: interfering effects of drug is tested
Choose 62 kinds of Common drugs and carry out Interference Detection, adjust concentration to 1.00nmol/L, adopt the homogeneous enzyme immunoassay method of embodiment six to be measured:
1. reagent A haptoreaction interference medicament to be measured and embodiment five prepared, adds reagent B；
2. detect the OD of above-mentioned mixed solution340Light absorption value, obtains the concentration of respective substance according to the standard curve of embodiment six.
62 kinds of common medicine names and measurement result are referring specifically to table 4.
Table 4 common interference drug monitoring result
| ID# | Compound title | It is equivalent to the concentration (nmol/L) of deoxypyridinoline | ID# | Compound title | It is equivalent to the concentration (nmol/L) of deoxypyridinoline |
| 1 | Aspirin | 0.0 | 32 | Phenylpropanolamine | 0.0 |
| 2 | β-phenyl-ethylamine | 0.0 | 33 | Procainamide | 0.0 |
| 3 | Amphetamines | 0.0 | 34 | Procaine | 0.0 |
| 4 | Ampicillin | 0.0 | 35 | Quinidine | 0.0 |
| 5 | Bent | 0.0 | 36 | Zomepirac | 0.0 |
| 6 | Chlorpromazine | 0.0 | 37 | Phyenlephrinium | 0.0 |
| 7 | Clorazepate | 0.0 | 38 | Cinnamyl Ai Kening | 0.0 |
| 8 | Gemfibrozil | 0.0 | 39 | Ecgonine | 0.0 |
| 9 | Fenoprofen | 0.0 | 40 | The West, ground | 0.0 |
| 10 | Methamphetamine | 0.0 | 41 | Cotinine | 0.0 |
| 11 | Gentisic acid | 0.0 | 42 | Atenolol | 0.0 |
| 12 | Gemfibrozil | 0.0 | 43 | Propranolol | 0.0 |
| 13 | Hydrocodone | 0.0 | 44 | Glutethimide | 0.0 |
| 14 | Ibuprofen | 0.0 | 45 | Bute | 0.0 |
| 15 | Imipramine | 0.0 | 46 | Lysergic acid diethylamine | 0.0 |
| 16 | DADPS | 0.0 | 47 | Cannabinol | 0.0 |
| 17 | Naproxen | 0.0 | 48 | Loperamide | 0.0 |
| 18 | Hydrochlorothiazide | 0.0 | 49 | Isoxsuprine | 0.0 |
| 19 | Pethidine | 0.0 | 50 | Phenylalanine | 0.0 |
| 20 | Naloxone | 0.0 | 51 | Fluoxetine Hydrochloride | 0.0 |
| 21 | Ephedrine | 0.0 | 52 | Salbutamol | 0.0 |
| 22 | Nicotiamide | 0.0 | 53 | Penicillin | 0.0 |
| 23 | Ranitidine | 0.0 | 54 | Methyl diethanolamine | 0.0 |
| 24 | Amobarbital | 0.0 | 55 | Dimethylene dioxygen amfetamine | 0.0 |
| 25 | Methylenedioxyamphetamine | 0.0 | 56 | Doxylamine succinate | 0.0 |
| 26 | Tetrahydrocannabinol | 0.0 | 57 | Nalbuphine | 0.0 |
| 27 | Nystatin | 0.0 | 58 | Normorphine | 0.0 |
| 28 | Acetylmorphine | 0.0 | 59 | Oxycodone | 0.0 |
| 29 | Benzfetamine | 0.0 | 60 | KET | 0.0 |
| 30 | Promethazine | 0.0 | 61 | Diphenhydramine | 0.0 |
| 31 | Aspartame | 0.0 | 62 | Duromine | 0.0 |
Measurement result shows: the concentration that above-mentioned 62 kinds of Common drugs are equivalent to deoxypyridinoline is respectively less than 0.01nmol/L.As can be seen here, the antibody of the present invention is the specific antibody of anti-deoxypyridinoline, with other medicines no cross reaction.
Embodiment eight: correlation analysis
The homogeneous enzyme immunoassay reagent that 100 example clinical samples use high performance liquid chromatography and the present invention respectively carries out correlation analysis, and the data of mensuration are referring to table 5.
Table 5 clinical sample measured value
| Catalogue number(Cat.No.) | Homogeneous enzyme immunoassay method measured value (nmol/L) | High effective liquid chromatography for measuring value (nmol/L) 9--> |
| 1 | 6.31 | 6.73 |
| 2 | 0.93 | 0.91 |
| 3 | 1.66 | 1.75 |
| 4 | 3.20 | 3.09 |
| 5 | 2.35 | 2.43 |
| 6 | 4.27 | 4.10 |
| 7 | 2.35 | 2.46 |
| 8 | 1.52 | 1.57 |
| 9 | 0.96 | 1.01 |
| 10 | 3.00 | 3.03 |
| 11 | 4.94 | 5.12 |
| 12 | 3.35 | 3.37 |
| 13 | 1.65 | 1.76 |
| 14 | 5.03 | 5.52 |
| 15 | 1.32 | 1.45 |
| 16 | 2.10 | 2.15 |
| 17 | 4.65 | 4.44 |
| 18 | 3.42 | 3.66 |
| 19 | 4.03 | 4.25 |
| 20 | 1.67 | 1.71 |
| 21 | 1.38 | 1.42 |
| 22 | 0.92 | 0.95 |
| 23 | 0.46 | 0.65 |
| 24 | 3.81 | 3.88 |
| 25 | 1.41 | 1.43 |
| 26 | 2.12 | 2.19 |
| 27 | 1.25 | 1.39 |
| 28 | 4.40 | 4.00 |
| 29 | 5.66 | 5.45 |
| 30 | 5.68 | 5.79 |
| 31 | 2.37 | 2.46 |
| 32 | 1.69 | 1.63 |
| 33 | 4.08 | 4.27 |
| 34 | 2.50 | 2.68 |
| 35 | 1.54 | 1.56 |
| 36 | 6.77 | 7.01 |
| 37 | 1.99 | 2.06 |
| 38 | 3.32 | 3.53 |
| 39 | 7.00 | 7.26 10 --> |
| 40 | 2.17 | 2.14 |
| 41 | 2.38 | 2.55 |
| 42 | 1.20 | 1.22 |
| 43 | 5.43 | 5.03 |
| 44 | 3.92 | 3.76 |
| 45 | 1.35 | 1.29 |
| 46 | 0.80 | 0.93 |
| 47 | 2.54 | 2.68 |
| 48 | 6.59 | 6.35 |
| 49 | 3.73 | 3.97 |
| 50 | 5.67 | 5.40 |
| 51 | 1.21 | 1.28 |
| 52 | 1.02 | 1.06 |
| 53 | 2.55 | 2.73 |
| 54 | 1.00 | 0.99 |
| 55 | 3.47 | 3.69 |
| 56 | 5.08 | 4.80 |
| 57 | 1.73 | 1.75 |
| 58 | 0.95 | 1.02 |
| 59 | 2.83 | 2.69 |
| 60 | 1.91 | 1.87 |
| 61 | 2.24 | 2.32 |
| 62 | 2.66 | 2.63 |
| 63 | 5.51 | 5.17 |
| 64 | 3.86 | 3.83 |
| 65 | 2.81 | 2.67 |
| 66 | 4.60 | 4.49 |
| 67 | 1.99 | 2.03 |
| 68 | 1.20 | 1.19 |
| 69 | 4.43 | 4.63 |
| 70 | 5.96 | 6.21 |
| 71 | 1.65 | 1.60 |
| 72 | 1.39 | 1.48 |
| 73 | 3.89 | 3.49 |
| 74 | 5.14 | 5.22 |
| 75 | 2.72 | 2.57 |
| 76 | 4.08 | 4.23 |
| 77 | 0.99 | 1.06 |
| 78 | 2.08 | 2.12 11 --> |
| 79 | 1.91 | 2.00 |
| 80 | 4.50 | 4.71 |
| 81 | 3.64 | 3.73 |
| 82 | 2.98 | 2.92 |
| 83 | 3.83 | 3.75 |
| 84 | 1.45 | 1.41 |
| 85 | 5.00 | 4.90 |
| 86 | 2.18 | 2.34 |
| 87 | 5.69 | 5.92 |
| 88 | 3.05 | 2.94 |
| 89 | 1.44 | 1.51 |
| 90 | 6.17 | 5.94 |
| 91 | 2.91 | 2.90 |
| 92 | 0.79 | 0.77 |
| 93 | 1.55 | 1.51 |
| 94 | 5.05 | 5.29 |
| 95 | 2.36 | 2.45 |
| 96 | 1.87 | 1.96 |
| 97 | 2.82 | 2.85 |
| 98 | 0.97 | 1.00 |
| 99 | 3.45 | 3.33 |
| 100 | 5.81 | 6.02 |
Above-mentioned data are mapped, and referring to Fig. 3, the linear equation obtained is: y=0.9946x+0.0482, coefficient R2=0.9906, it was shown that the accuracy that the detectable of the present invention measures deoxypyridinoline clinical samples is high.
It should be noted that; the foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every equivalent structure utilizing description of the present invention and accompanying drawing content to do or equivalence flow process conversion; or directly or indirectly it is used in other correlative technology fields, all in like manner include in the scope of patent protection of the present invention.
Claims (8)
1. a deoxypyridinoline immunogen, its structural formula is such as shown in formula I:
Formula I
Carrier for having immunogenic protein or polypeptide, the one in serum albumin, hemocyanin or Elityran.
2. the immunogenic preparation method of deoxypyridinoline as claimed in claim 1, it is characterised in that comprise the steps of
(1) carrier protein 100～300g is dissolved in the phosphate buffer of 25～75ml0.2M, pH8.5；
(2) following chemicals is joined stirring and dissolving in small beaker: 100～300mg deoxypyridinoline, 1.75～5.25ml dimethylformamide, 1.75～5.25ml ethanol, 3.5～10.5ml10mM, the kaliumphosphate buffer of pH5.0,100～300mg1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 25～75mgN-hydroxy thiosuccinimide, react above-mentioned chemicals at room temperature stirring and dissolving to 30～60min；
(3) solution dissolved is dropped in carrier protein solution, and stir at 2～8 DEG C overnight, obtain antigen；Synthetic antigen is purified through dialysis, obtains deoxypyridinoline immunogen.
3. an anti-deoxypyridinoline specific antibody, the complete antibody molecule produced after the deoxypyridinoline immunogen immune laboratory animal described in claim 1, or be antibody fragment or the antibody derivatives of reservation and deoxypyridinoline specific binding capacity.
4. the anti-deoxypyridinoline specific antibody of one according to claim 3, it is characterized in that described complete antibody molecule, antibody fragment or antibody derivatives, for the polyclonal antibody adopting single deoxypyridinoline immunogen that animal booster immunization is obtained, or the monoclonal antibody for obtaining through somatic hybridization after immunity.
5. the preparation method of the anti-deoxypyridinoline specific antibody as according to any one of claim 3-4, it is characterised in that comprise the steps of
(1) with PBS, deoxypyridinoline immunogen is diluted to 0.1～3.0mg/ml, obtains antigenic solution, then mix with equivalent Freund's complete adjuvant with 0.5～5.0ml antigenic solution, laboratory animal is injected；
After (2) 2～3 weeks, then with equivalent incomplete Freund's adjuvant, above-mentioned laboratory animal is injected once with antigenic solution identical for 0.5～5.0ml, inject once every surrounding afterwards, amount to injection 3～6 times；
(3) laboratory animal of step (2) is taken blood, separate purification and obtain the anti-deoxypyridinoline specific antibody that titer is 1:30000～1:50000.
6. a deoxypyridinoline detectable, containing the anti-deoxypyridinoline specific antibody described in claim 3 or 4 and indicator, described indicator one in enzyme reagent, radiosiotope reagent, fluorometric reagent or luminescence reagent；Described enzyme reagent is made up of the substrate of deoxypyridinoline enzyme mark conjugate and enzyme, and enzyme mark conjugate is glucose-6-phosphate dehydrogenase (G6PD)-hapten enzyme mark conjugate, and the substrate of enzyme is G-6-P.
7. the preparation method of a deoxypyridinoline detectable as claimed in claim 6, it is characterised in that comprise the steps of
(1) reagent A: 2.018～8.072g, the nicotinamide adenine dinucleotide of 5.625～22.50mM oxidation state and 0.856～3.422g, 5.625～22.50mM G-6-P Tris buffer solution of 0.5～2L55mM, pH=8.0 are made homogeneous zymolyte；Being added in above-mentioned homogeneous zymolyte by the anti-deoxypyridinoline specific antibody described in claim 3 or 4, the volume ratio of anti-deoxypyridinoline specific antibody and homogeneous zymolyte is 1:100～1:10000；
(2) volume ratio of reagent B: be added in the Tris buffer of 120mM, pH=8.2 by deoxypyridinoline enzyme mark conjugate, deoxypyridinoline enzyme mark conjugate and Tris buffer is 1:100～1:10000.
8. the preparation method of deoxypyridinoline detectable according to claim 7, it is characterised in that the preparation method of described deoxypyridinoline enzyme mark conjugate comprises the steps of
(1) preparation of glucose-6-phosphate dehydrogenase (G6PD) solution: weighing the glucose-6-phosphate dehydrogenase (G6PD) that 7.5～22.5mg specification is 100KU, room-temperature dissolution contains 72.6mg0.05MTris, 8mg3.3mMMgCl in 6～18mL2With in the solution of 100mgNaCl, pH=9.0；Add the nicotinamide adenine dinucleotide of 112.5～337.5mg reduction-state, 67.5～202.5mg G-6-P and 0.375～1.125mL carbitol in the solution；It is added dropwise over 1～3mL dimethyl sulfoxide again；
(2) activation of deoxypyridinoline: weigh 5～15mg deoxypyridinoline under anhydrous conditions, is dissolved in 300～900 μ L dimethylformamides；Above-mentioned solution temperature is made to drop to-2 ~-8 DEG C；Add 1.5～4.5 μ L tri-n-butylamines；Add 0.75～2.25 μ L isobutyl chlorocarbonate；-2 ~-8 DEG C are stirred 30～60 minutes；
(3) connection of glucose-6-phosphate dehydrogenase (G6PD) and deoxypyridinoline: the deoxypyridinoline dropwise that step (2) activates is joined in the glucose-6-phosphate dehydrogenase (G6PD) solution that step (1) is dissolved；2-8 DEG C of stirring is overnight；
(4) purified product: by G-25 gel chromatography column purification connect product, it is thus achieved that end product be glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing, at 2-8 DEG C store.
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Application publication date: 20160727 |