CN105037532A - 17-ketosteroid immunogen, antibody and detection reagent and preparation method thereof - Google Patents
17-ketosteroid immunogen, antibody and detection reagent and preparation method thereof Download PDFInfo
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- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- 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
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
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- 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
- G01N33/534—Production of labelled immunochemicals with radioactive label
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- 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
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- 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
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
Abstract
The invention discloses a 17-ketosteroid immunogen, antibody and detection reagent and a preparation method thereof. The prepared 17-ketosteroid immunogen is high in immunogenicity, can conduct induction to obtain the high-potency antibody with the 17-ketosteroid resistance characteristic, and has no cross reaction with frequently-seen 62 types of medicine; by means of the 17-ketosteroid detection reagent prepared from the antibody, the content of 17-ketosteroid in biological samples such as urine can be accurately and rapidly confirmed. Compared with an existing detection reagent on the market, the detection reagent has the advantages of being easy and convenient to operate, high in flexibility, high in specificity, accurate in result and the like, can effectively reduce the detection cost of 17-ketosteroid and is beneficial to large-scale clinical application and popularization.
Description
Technical field
The invention belongs to biological technical field, relate to 17-KS immunogen, anti-17-KS specific antibody and 17-KS detection reagent.
Background technology
17-KS (17-ketosteroids), its structural formula is as shown in formula III:
17-KS refers to and the person that has ketone group in cortin and sexual hormoue on 17 carbon potentials comprises androsterone, epiandrosterone, 17-Hormoforin, 11-oxygen androsterone, 11-hydroxyandrosterone etc., and major part exists with combining form.The androgenic meta-bolites of 17-KS mainly suprarenal gland and testicular secretion, 17-KS in women and children's urine is mainly derived from adrenal cortex, and the 17-KS in Male urine about 2/3 derives from adrenal cortex, 1/3 and derives from testis.Urine 17-ketosteroid can react the general status of adrenocortical hormone, glucocorticosteroid and sexual gland secretion, has larger value for the androgenic function of evaluation acth secretion.
Classical 17-KS detection method is colorimetric method, this method principle be based on this type of sterid on 17 carbon atoms with ketone group in basic solution and a position dinitrobenzene react, generating quinoid compound is red-purple, and carries out the same reference liquid colorimetric analysis processed and can try to achieve content.But this method technology comparatively backwardness, less stable, has been not suitable for using in enormous quantities clinically.The 17-KS detection reagent of good, highly sensitive, the high specificity of deficient in stability in the market, the especially measured Automated inspection reagent of matter.Therefore, research and develop that a kind of quality reaches clinical requirement, practical, cost performance is high, the 17-KS detection reagent that can be applicable to automatic clinical chemistry analyzer is imperative.
Summary of the invention
The defect that the present invention exists to overcome prior art, adopt unique 17-KS derivative to prepare the strong 17-KS immunogen of immunogenicity and antibody thereof, the 17-KS homogeneous enzyme immunoassay detection reagent prepared with this antibody can be implemented on automatic clinical chemistry analyzer 17-KS high-throughput, rapid detection.This detection reagent has the advantages such as easy and simple to handle, highly sensitive, high specificity, result are accurate, effectively can also reduce 17-KS testing cost, is conducive to clinical expansion and uses.
One object of the present invention is the 17-KS immunogen providing a kind of immunogenicity strong.
Another object of the present invention is to provide a kind of 17-KS immunogenic preparation method.
Another object of the present invention is to provide the anti-17-KS specific antibody of the high specificity using 17-KS immunogen of the present invention to prepare.
Another object of the present invention is to provide a kind of 17-KS detection reagent and preparation method thereof.
17-KS immunogen of the present invention, immunogenicity is high, can induce the anti-17-KS specific antibody obtaining high-titer.This antibodies specific is high, strong with the bonding force of 17-KS.The 17-KS detection reagent prepared by this antibody, can determine the 17-KS content in sample quickly and accurately.The present invention is achieved by the following technical solutions:
A kind of 17-KS immunogen, its structural formula is as shown in formula I:
Carrier, for having immunogenic protein or polypeptide, is preferably serum protein, hemocyanin and thyroglobulin, is more preferably serum albumin, more preferably bovine serum albumin.
Described 17-KS immunogen is formed by connecting by 17-KS derivative and above-mentioned carrier, and the chemical structure of 17-KS derivative is as shown in formula II:
The immunogenic concrete route of synthesis of this 17-KS and method as follows:
(1) carrier proteins 100 ~ 300mg is dissolved in 25 ~ 75ml0.2M, in the phosphoric acid buffer of pH8.5;
(2) following chemical is joined stirring and dissolving in small beaker: 100 ~ 300mg17-ketosteroid derivative, 1.75 ~ 5.25ml dimethyl formamide, 1.75 ~ 5.25ml ethanol, 3.5 ~ 10.5ml10mM, the potassium phosphate buffer of pH5.0,100 ~ 300mg1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 25 ~ 75mgN-hydroxy thiosuccinimide, by these chemical at room temperature stirring and dissolving reaction 30 ~ 60min;
(3) solution dissolved is dropped in carrier protein solution, and stir at 2 ~ 8 DEG C and spend the night, obtain antigen; Synthetic antigen is carried out purifying through dialysis, obtains 17-KS immunogen.
A kind of anti-17-KS specific antibody, obtains by producing after above-mentioned 17-KS immunogen immune animal.
Described anti-17-KS specific antibody adopts ordinary method inoculation experiments animal by above-mentioned obtained 17-KS immunogen, and get antiserum(antisera) after booster immunization, concrete steps are as follows:
(1) with PBS, the BSA-17-ketosteroid 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 the identical antigenic solution of 0.5 ~ 5.0ml and equivalent Freund's incomplete adjuvant, above-mentioned laboratory animal is injected once, afterwards every surrounding injection once, amount to injection 3 ~ 6 times;
(3) get blood to above-mentioned laboratory animal, separation and purification obtains the anti-17-KS specific antibody of tiring as 1:30000 ~ 1:50000.
Anti-17-KS specific antibody of the present invention is complete antibody molecule, also comprises and retaining and the antibody fragment of 17-KS specific binding capacity or antibody derivatives.
The polyclonal antibody that antibody of the present invention obtains animal booster immunization for 17-KS immunogen that employing is single, or for after immunity through monoclonal antibody that somatic hybridization obtains; Described laboratory animal is the one of rabbit, goat, mouse, sheep, cavy or horse, is preferably rabbit.
The invention provides a kind of 17-KS detection reagent, containing above-mentioned anti-17-KS specific antibody and indicator.
Indicator of the present invention is selected from enzyme reagent, radio isotope reagent, fluorescent reagent, luminescence reagent.Preferably, indicator is enzyme reagent, is made up of the substrate of 17-KS enzyme mark conjugate and enzyme.
Above-mentioned enzyme mark conjugate is glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate; The substrate of above-mentioned enzyme is G-6-P.
17-KS homogeneous enzyme immunoassay detection reagent 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-17-KS specific antibody are mixed.Therefore, 17-KS homogeneous enzyme immunoassay detection reagent comprises two class reagent:
(1) reagent A is mixed by anti-17-KS specific antibody and homogeneous phase enzyme substrates, and concrete preparation process is as follows:
1) Reduced nicotinamide-adenine dinucleotide (NAD) of 2.018 ~ 8.072g (5.625 ~ 22.50mM) oxidation state, 0.856 ~ 3.422g (5.625 ~ 22.50mM) G-6-P (G-6-P) the Tris buffer solution of 0.5 ~ 2L55mM, pH=8.0 are made homogeneous phase enzyme substrates;
2) be added in above-mentioned homogeneous phase enzyme substrates by the anti-17-KS specific antibody of preparation, the volume ratio of antibody and homogeneous phase enzyme substrates is 1:100 ~ 1:10000, is preferably 1:1000;
(2) reagent B is mixed by glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing and Tris damping fluid, and preparation method is as follows:
1) preparation of glucose-6-phosphate dehydrogenase (G6PD) (G6PDH) solution:
A. take the G6PDH that 7.5 ~ 22.5mg specification is 100KU, room-temperature dissolution contains 72.6mg (0.05M) Tris, 8mgMgCl in 6 ~ 18mL
2(3.3mM) with in the solution of 100mgNaCl, these pH value of solution=9.0;
B. the Reduced nicotinamide-adenine dinucleotide (NADH) of 112.5 ~ 337.5mg reduction-state is added, 67.5 ~ 202.5mg G-6-P (G-6-P) and 0.375 ~ 1.125mL Trivalin SF;
C. 1 ~ 3mL dimethyl sulfoxide (DMSO) is dropwise added;
2) activation of 17-KS derivative:
A. take 5 ~ 15mg17-ketosteroid derivative under anhydrous conditions, be dissolved in 300 ~ 900 μ LDMF;
B. above-mentioned solution temperature is made to drop to-2 ~-8 DEG C;
C. 1.5 ~ 4.5 μ L Tributylamines are added;
D. 0.75 ~ 2.25 μ L isobutyl chlorocarbonate is added;
E.-2 ~-8 DEG C are stirred 30 ~ 60 minutes;
3) connection of G6PDH and 17-KS derivative:
A. the 17-KS derivative solution of above-mentioned activation is dropwise joined in the G6PDH solution of above-mentioned dissolving;
B.2-8 a DEG C stirring is spent the night;
4) purified product:
Connect product by G-25 gel chromatography column purification, the final product of acquisition is glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing, stores at 2-8 DEG C.
5) be added in the Tris damping fluid of 120mM, pH=8.2 by the glucose-6-phosphate dehydrogenase (G6PD) of preparation-hapten conjugation thing, the volume ratio of above-mentioned conjugate and Tris damping fluid is 1:100 ~ 1:10000, is preferably 1:3000.
17-KS immunogens of the present invention is strong, immunogenicity is high, the anti-17-KS specific antibody high specificity prepared, height of tiring, and with common 62 kinds of medicines without any cross reaction; Homogeneous enzyme immunoassay detection reagent containing above-mentioned anti-17-KS specific antibody can determine the 17-KS content in the biological samples such as urine easily and fast, exactly, and can on automatic clinical chemistry analyzer the multiple sample of Simultaneously test, realize the rapid mensuration of high-throughput of 17-KS, accuracy is high, high specificity, tolerance range is all enhanced before comparing with detection efficiency, achieve the full-automation of testing process simultaneously, less demanding to testing staff, is easy to realize and promote the use of.
Accompanying drawing explanation
Fig. 1 is the ELISA detection reaction curve of 17-KS;
Fig. 2 is the homogeneous enzyme immunoassay response curve of 17-KS;
Fig. 3 is 17-KS homogeneous enzyme immunoassay correlation analysis figure.
Embodiment
The immunogenic synthesis of embodiment one 17-KS
17-KS immunogen is by the 17-KS derivative shown in bovine serum albumin (BovineSerumAlbumin, BSA) Yu formula II
group is formed by connecting, and concrete steps are as follows:
1. bovine serum albumin 200mg is dissolved in 50ml0.2M, in the phosphoric acid buffer of pH8.5;
2. following chemical is joined stirring and dissolving in small beaker: 200mg17-ketosteroid derivative, 3.5ml dimethyl formamide, 3.5ml ethanol, 7.0ml10mM, the potassium phosphate buffer of pH5.0,200mg1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 50mgN-hydroxy thiosuccinimide, by these chemical at room temperature stirring and dissolving reaction 30min;
3. the solution dissolved is dropped in BSA solution, and stir at 2 ~ 8 DEG C and spend the night, obtain antigen; Synthetic antigen is carried out purifying through dialysis, obtains 17-KS immunogen.
Embodiment two: the preparation of anti-17-KS specific antibody
17-KS immunogen embodiment one prepared adopts ordinary method inoculation experiments animal rabbit, and get antiserum(antisera) after booster immunization, concrete steps are as follows:
1. with PBS, the 17-KS 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 the identical antigenic solution of 1.0ml and equivalent Freund's incomplete adjuvant, above-mentioned experimental animal rabbit is injected once, afterwards every surrounding injection once, amount to injection 4 times.
3. the experimental animal rabbit of pair step 2 gets blood, and separation and purification obtains the anti-17-KS specific antibody of tiring as 1:30000 ~ 1:50000.
Embodiment three: the ELISA inspection of 17-KS
The foundation of the ELISA examination criteria curve of 1.17-ketosteroid
(1) preparation of standard substance
17-KS powder (being purchased from Sigma company) is dissolved in methanol solution, is prepared into the storage liquid of 1mg/ml.Storage liquid being diluted successively with ELISA damping fluid is the standardized solution of 20.00mg/L, 10.00mg/L, 5.00mg/L, 2.50mg/L, 1.25mg/L and 0.00mg/L.Wherein, ELISA damping fluid contains 50.0mMTris, the BSA of 145mMNaCl and 0.25%.
(2) the ELISA method of inspection preparation standard curve of 17-KS is utilized
With PBS, anti-17-KS specific antibody prepared in embodiment two is diluted to the final concentration solution of 1:8000,100 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-17-KS antibody being washed 3 times with PBS, add the BSA solution of 0.5% of 200 μ L/ holes, 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-17-ketosteroid conjugate of 100 μ L/ hole working concentrations again; 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 sulfuric acid) again.Measure the light absorption value of 450nm.The light absorption value calibration of the 450nm corresponding to each standard substance, production standard curve, result as shown in Figure 2.
2. the detection of 17-KS content in testing sample
(1) testing sample is made
Preparation method: 17-KS powder (being purchased from Sigma company) is dissolved in the storage liquid that methanol solution makes 1000mg/L, and this storage liquid is diluted in blank diaper, 0.00 is respectively to final concentration, 1.50,8.00,20.00mg/L, forms urine specimen that is blank, basic, normal, high concentration.This blank diaper is not containing the Healthy People urine of 17-KS.
(2) testing method
Utilize the ELISA method of inspection of above-mentioned 17-KS, the urine specimen of above-mentioned blank, basic, normal, high concentration replaced standard substance, test above-mentioned blank, basic, normal, high concentration urine specimen at the light absorption value of 450nm.
(3) test result
The typical curve of the ELISA inspection of the 17-KS of contrast shown in Fig. 1, calculate 17-KS content in each sample, and 3 multiple holes mensuration are carried out to each sample, the actual content according to 17-KS in above-mentioned sample calculates the rate of recovery, and result is as shown in table 1.
The ELISA of table 117-ketosteroid detects recovery experiment
| Urine sample | Blank | Low | In | High |
| Sample concentration (mg/L) | 0.00 | 1.50 | 8.00 | 20.00 |
| Test 1 | 0.02 | 1.41 | 7.99 | 20.13 |
| Test 2 | 0.04 | 1.55 | 8.12 | 19.81 |
| Test 3 | 0.03 | 1.58 | 8.05 | 20.25 |
| Mean value (mg/L) | 0.01 | 1.51 | 8.05 | 20.06 |
| The rate of recovery (%) | - | 101.0 | 100.6 | 100.3 |
From result in table 1: the 17-KS rate of recovery adopting the ELISA detection reagent of 17-KS of the present invention to measure in different concns sample is all higher, equal > 90%, illustrate that anti-17-KS specific antibody of the present invention may be used for the detection of 17-KS 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 take the G6PDH that 15mg specification is 100KU, room-temperature dissolution contains 72.6mg (0.05M) Tris, 8mgMgCl in 12mL
2(3.3mM) with in the solution of 100mgNaCl, these pH value of solution=9.0, this step is carried out in beaker C.
(2) in above-mentioned beaker C, the Reduced nicotinamide-adenine dinucleotide (NADH) of 225mg reduction-state is added, 135mg G-6-P (G-6-P) and 0.75mL Trivalin SF (Carbitol).
(3) in above-mentioned beaker C, 2mL dimethyl sulfoxide (DMSO) (dimethysulfoxide, DMSO) is dropwise added again.
The activation of 2.17-ketosteroid derivative:
(1) take the above-mentioned 17-KS derivative 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 Tributylamines (tributylamine) are added.
(4) 1.5 μ L isobutyl chlorocarbonates (isobutylchloroformate) are added.
(5)-2 ~-8 DEG C are stirred 30 minutes.
The connection of 3.G6PDH and 17-KS derivative:
(1) the 17-KS derivative solution of above-mentioned activation is dropwise joined in the G6PDH solution of above-mentioned dissolving.
(2) 2-8 DEG C of stirring is spent the night.
4. purified product:
By the solution in G-25 gel chromatography column purification step 3, the final product of acquisition is glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing, stores at 2-8 DEG C.
Embodiment five: the preparation of 17-KS homogeneous enzyme immunoassay detection reagent
1. the preparation of reagent A: the Reduced nicotinamide-adenine dinucleotide (NAD) of 4.036g (11.25mM) oxidation state, 1.711g (11.25mM) G-6-P (G-6-P) are placed in beaker D, make homogeneous phase enzyme substrates with the Tris buffer solution of 1L55mM, pH=8.0; Be added in above-mentioned homogeneous phase enzyme substrates by the anti-17-KS specific antibody of above-mentioned preparation, the volume ratio of antibody and homogeneous phase enzyme substrates is 1:1000.
2. the preparation of reagent B: glucose-6-phosphate dehydrogenase (G6PD) embodiment four prepared-hapten conjugation thing is added in the Tris damping fluid of 120mM, pH=8.2, the volume ratio of above-mentioned conjugate and Tris damping fluid is 1:3000.
Embodiment six: the inspection of 17-KS homogeneous enzyme immunoassay and result
1. obtain typical curve:
(1) auspicious BS-480 automatic clinical chemistry analyzer reaction parameter (see table 2) advanced in years is set.
(2) operation steps is: first reagent adding A, then adds standard substance, finally adds reagent B.After adding reagent B, measure the OD of different time points
340light absorption value, calculates speed of reaction during different standards product concentration, needs the volume ratio constantly adjusting reagent A and reagent B in actual mechanical process, adjusts light-metering point simultaneously, finally draws comparatively ideal reaction normal graphic representation, as shown in Figure 3.
Table 2 steps auspicious BS-480 automatic clinical chemistry analyzer reaction parameter
2. pattern detection: the typical curve obtained by homogeneous enzyme immunoassay detection reagent of the present invention, replication basic, normal, high concentration Quality Control sample 10 times, above-mentioned Quality Control sample is: be dissolved in human urine by 17-KS standard substance, be respectively 1.50 to concentration, 8.00,20.00mg/L.Detection data and data analysis are in table 3.
Table 3 sample determination and precision and rate of recovery assessment
| Urine sample | Low | In | High |
| Sample concentration (mg/L) | 1.50 | 8.00 | 20.00 |
| 1 | 1.52 | 8.21 | 20.61 |
| 2 | 1.61 | 8.22 | 21.17 |
| 3 | 1.54 | 8.15 | 19.78 |
| 4 | 1.46 | 8.07 | 20.75 |
| 5 | 1.59 | 7.96 | 19.11 |
| 6 | 1.63 | 8.09 | 20.85 |
| 7 | 1.55 | 8.04 | 19.14 |
| 8 | 1.62 | 8.11 | 20.64 |
| 9 | 1.49 | 7.94 | 20.31 |
| 10 | 1.58 | 8.04 | 20.74 |
| Mean value (mg/L) | 1.56 | 8.08 | 20.31 |
| Standard deviation (SD) | 0.057 | 0.094 | 0.723 |
| Precision (CV%) | 3.66 | 1.16 | 3.56 |
| Rate of recovery % | 104.0 | 101.0 | 101.6 |
Detected result: the accuracy that homogeneous enzyme immunoassay detection reagent of the present invention measures is high, and the rate of recovery reaches 95%-105%, and precision is high, and CV is all lower than 5%.
Embodiment seven: interfering effects of drug is tested
Choose 62 kinds of Common drugs and carry out Interference Detection, adjustment concentration, to 1.00mg/L, adopts the homogeneous enzyme immunoassay method of embodiment six to measure:
1. by reagent A contact reacts prepared by interference medicament to be measured and embodiment five, then add reagent B;
2. detect the OD of above-mentioned mixing solutions
340light absorption value, obtains the concentration of respective substance according to the typical curve of embodiment six.
62 kinds of common medicine names and measurement result are specifically see table 4.
Table 4 common interference drug determination result
Measurement result shows: the concentration that above-mentioned 62 kinds of Common drugs are equivalent to 17-KS is all less than 0.01mg/L.As can be seen here, antibody of the present invention is the specific antibody of anti-17-KS, with other medicines no cross reaction.
Embodiment eight: correlation analysis
Use Spain's biosystem (chromatogram-spectrophotometry) and homogeneous enzyme immunoassay reagent of the present invention to carry out correlation analysis respectively to 100 routine clinical samples, the data of mensuration are see table 5.
Table 5 clinical sample measured value
Map to above-mentioned data, see Fig. 3, the linear equation obtained is: y=1.0113x-0.1401, coefficient R
2=0.9907, show that the accuracy of detection reagent of the present invention mensuration 17-KS 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 utilize specification sheets of the present invention and accompanying drawing content to do equivalent structure or equivalent flow process conversion; or be directly or indirectly used in other correlative technology fields, be all in like manner included in scope of patent protection of the present invention.
Claims (9)
1. a 17-KS immunogen, its structural formula is as shown in formula I:
Carrier, for having immunogenic protein or polypeptide, is selected from the one in serum protein, hemocyanin or thyroglobulin, is preferably serum protein, is more preferably bovine serum albumin.
2. the immunogenic preparation method of 17-KS as claimed in claim 1, is characterized in that comprising following steps:
(1) carrier proteins 100 ~ 300g is dissolved in 25 ~ 75ml0.2M, in the phosphoric acid buffer of pH8.5;
(2) following chemical is joined stirring and dissolving in small beaker: 100 ~ 300mg17-ketosteroid derivative, 1.75 ~ 5.25ml dimethyl formamide, 1.75 ~ 5.25ml ethanol, 3.5 ~ 10.5ml10mM, the potassium phosphate buffer of pH5.0,100 ~ 300mg1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 25 ~ 75mgN-hydroxy thiosuccinimide, by above-mentioned chemical at room temperature stirring and dissolving reaction 30 ~ 60min;
(3) solution dissolved is dropped in carrier protein solution, and stir at 2 ~ 8 DEG C and spend the night, obtain antigen; Synthetic antigen is carried out purifying through dialysis, obtains 17-KS immunogen.
3. 17-KS immunogen preparation method according to claim 2, is characterized in that described 17-KS derivant structure formula is as shown in formula II:
4. an anti-17-KS specific antibody, by the complete antibody molecule produced after the 17-KS immunogen immune laboratory animal described in claim 1-2 any one, or for retaining and the antibody fragment of 17-KS specific binding capacity or antibody derivatives.
5. the anti-17-KS specific antibody of one according to claim 4, it is characterized in that described complete antibody molecule, antibody fragment or antibody derivatives, for the polyclonal antibody adopting single 17-KS immunogen to obtain animal booster immunization, or it is the monoclonal antibody obtained through somatic hybridization after immunity; Described laboratory animal is the one of rabbit, goat, mouse, sheep, cavy or horse, is preferably rabbit.
6. a preparation method for the anti-17-KS specific antibody according to any one of claim 4-5, is characterized in that comprising following steps:
(1) with PBS, 17-KS 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 the identical antigenic solution of 0.5 ~ 5.0ml and equivalent Freund's incomplete adjuvant, above-mentioned laboratory animal is injected once, afterwards every surrounding injection once, amount to injection 3 ~ 6 times;
(3) get blood to the laboratory animal of step (2), separation and purification obtains the anti-17-KS specific antibody of tiring as 1:30000 ~ 1:50000.
7. a 17-KS detection reagent, containing the anti-17-KS specific antibody described in claim 4-6 and indicator, described indicator is selected from the one in enzyme reagent, radio isotope reagent, fluorescent reagent or luminescence reagent, is preferably enzyme reagent; Described enzyme reagent is made up of the substrate of 17-KS enzyme mark conjugate and enzyme, and enzyme mark conjugate is preferably glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate, and the substrate of enzyme is preferably G-6-P.
8. a preparation method for 17-KS detection reagent as claimed in claim 7, is characterized in that comprising following steps:
(1) reagent A: the Tris buffer solution of the Reduced nicotinamide-adenine dinucleotide of 2.018 ~ 8.072g, 5.625 ~ 22.50mM oxidation state and 0.856 ~ 3.422g, 5.625 ~ 22.50mM G-6-P, 0.5 ~ 2L55mM, pH=8.0 is made homogeneous phase enzyme substrates; Be added in above-mentioned homogeneous phase enzyme substrates by the anti-17-KS specific antibody according to any one of claim 4-6, the volume ratio of anti-17-KS specific antibody and homogeneous phase enzyme substrates is 1:100 ~ 1:10000;
(2) reagent B: 17-KS enzyme mark conjugate is added in the Tris damping fluid of 120mM, pH=8.2, the volume ratio of 17-KS enzyme mark conjugate and Tris damping fluid is 1:100 ~ 1:10000;
Described anti-17-KS specific antibody and the volume ratio of homogeneous phase enzyme substrates are preferably 1:1000;
Described 17-KS enzyme mark conjugate and the volume ratio of Tris damping fluid are preferably 1:3000.
9. the 17-KS detection reagent according to any one of claim 7-8, is characterized in that the preparation method of described 17-KS enzyme mark conjugate comprises following steps:
(1) preparation of glucose-6-phosphate dehydrogenase (G6PD) solution: take 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 ~ 18mL
2with in the solution of 100mgNaCl, pH=9.0; Add the Reduced nicotinamide-adenine dinucleotide of 112.5 ~ 337.5mg reduction-state, 67.5 ~ 202.5mg G-6-P and 0.375 ~ 1.125mL Trivalin SF in the solution; Dropwise add 1 ~ 3mL dimethyl sulfoxide (DMSO) again;
(2) activation of 17-KS derivative: take 5 ~ 15mg17-ketosteroid derivative under anhydrous conditions, is dissolved in 300 ~ 900 μ L dimethyl formamides; Above-mentioned solution temperature is made to drop to-2 ~-8 DEG C; Add 1.5 ~ 4.5 μ L Tributylamines; 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 17-KS derivative: the 17-KS derivative solution that step (2) activates dropwise is joined in the glucose-6-phosphate dehydrogenase (G6PD) solution that step (1) dissolves; 2-8 DEG C of stirring is spent the night;
(4) purified product: connect product by G-25 gel chromatography column purification, the final product of acquisition is glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing, stores at 2-8 DEG C.
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