CN105037532B - 17 ketosteroid immunogenes, antibody and detection reagent and preparation method - Google Patents

17 ketosteroid immunogenes, antibody and detection reagent and preparation method Download PDF

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CN105037532B
CN105037532B CN201510444072.XA CN201510444072A CN105037532B CN 105037532 B CN105037532 B CN 105037532B CN 201510444072 A CN201510444072 A CN 201510444072A CN 105037532 B CN105037532 B CN 105037532B
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虞留明
岳育红
张顺利
梁玉芳
王清涛
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SUZHOU EVERMED CO Ltd
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Abstract

The invention discloses 17 ketosteroid immunogenes, antibody and detection reagent and preparation method.17 ketosteroid immunogenes prepared by the present invention, immunogenicity is high, can induce to obtain the anti-17 ketosteroid specific antibodies of high-titer, and with 62 kinds of common medicines without any cross reaction；The 17 ketosteroid detection reagents obtained by the Antibody preparation, it can accurately and quickly determine 17 ketosteroid contents in the biological samples such as urine.Compared with the existing detection reagent of in the market, detection reagent of the present invention has the advantages that easy to operate, high sensitivity, high specificity, result are accurate, moreover it is possible to effectively reduces by 17 ketosteroid testing costs, is advantageous to clinical large-scale promotion and uses.

Description

17-KS immunogene, antibody and detection reagent and preparation method
Technical field
The invention belongs to biological technical field, is related to 17-KS immunogene, anti-17-KS specific antibody With 17-KS detection reagent.
Background technology
17-KS (17-ketosteroids), shown in its structural formula such as formula (III)：
17-KS refers in cortin and sex hormone to have ketone group on 17 carbon potentials person, including androsterone, epiandrosterone, 17-Hormoforin, 11- oxygen androsterone, 11- hydroxyandrosterones etc., it is most of to exist with combining form.17-KS is mainly adrenal gland And the metabolite of the androgen of testicular secretion, the 17-KS in women and children's urine are mainly derived from adrenal gland skin Matter, the 17-KS about 2/3 in Male urine derive from testis from adrenal cortex, 1/3.Urine 17-ketosteroid can The general status of cortex hormone of aadrenaline, glucocorticoid and sexual gland secretion is reacted, for evaluation acth secretion androgen Function has larger value.
Classical 17-KS detection method is colorimetric method, and the method principle is based on such in 17 carbon originals The sterid with ketone group reacts in alkaline solution with meta dinitro benzene on son, and generation quinoid compound is in purple Red, content can be tried to achieve with the titer colorimetric analysis equally handled.But the method technology more fall behind, stability compared with Difference, clinically high-volume is not suitable for and has used.Deficient in stability is good in the market, high sensitivity, the 17- ketones of high specificity The measured Automated inspection reagent of sterol detection reagent, especially matter.Therefore, research and develop a kind of quality and reach clinical requirement, practicality Property is strong, cost-effective, and the 17-KS detection reagent that can be applied to automatic clinical chemistry analyzer is imperative.
The content of the invention
The present invention using unique 17-KS derivative in order to overcome the shortcomings of the prior art, prepared immune The strong 17-KS immunogene of originality and its antibody, with the 17-KS homogeneous enzyme immunoassay detection reagent of the Antibody preparation It can realize on automatic clinical chemistry analyzer to 17-KS high flux, rapid detection.The detection reagent has behaviour The advantages that making simplicity, high sensitivity, high specificity, accurate result, moreover it is possible to effectively reduce 17-KS testing cost, favorably Used in clinical expansion.
It is an object of the present invention to provide a kind of strong 17-KS immunogene of immunogenicity.
It is another object of the present invention to provide a kind of preparation method of 17-KS immunogene.
A further object of the present invention is to provide the specificity being prepared using 17-KS immunogene of the present invention Strong anti-17-KS specific antibody.
It is yet a further object of the present invention to provide a kind of 17-KS detection reagent and preparation method thereof.
The 17-KS immunogene of the present invention, immunogenicity is high, and the anti-17- ketones that can induce to obtain high-titer are consolidated Alcohol specific antibody.The antibody specificity is high, strong with the adhesion of 17-KS.The 17- ketones obtained by the Antibody preparation Sterol detection reagent, it can quickly and accurately determine the 17-KS content in sample.The present invention is by following technology What scheme was realized：
A kind of 17-KS immunogene, shown in its structural formula such as formula (I)：
Carrier is protein or polypeptide with immunogenicity, preferably haemocyanin, hemocyanin and thyroid gland ball egg In vain, more preferably seralbumin, more preferably bovine serum albumin(BSA).
Described 17-KS immunogene is formed by connecting by 17-KS derivative and above-mentioned carrier, 17- ketones Shown in the chemical constitution of steroid derivatives such as formula (II)：
The specific route of synthesis and method of the 17-KS immunogene are as follows：
(1) 100~300mg of carrier protein is dissolved in 25~75ml 0.2M, in pH 8.5 phosphate buffer；
(2) following chemicals is added to stirring and dissolving in small beaker：100~300mg17- ketosteroids derivative, 1.75~5.25ml dimethylformamides, 1.75~5.25ml ethanol, 3.5~10.5ml 10mM, pH 5.0 potassium phosphate buffering Liquid, 100~300mg 1- ethyls -3- (- 3- dimethylaminopropyls) carbodiimide, 25~75mg N- hydroxy thiosuccinimides, 30~60min of dissolving reaction is stirred at room temperature in these chemicals；
(3) solution dissolved is added dropwise in carrier protein solution, and be stirred overnight at 2~8 DEG C, obtain antigen； Synthetic antigen is purified by dialysis, obtains 17-KS immunogene.
A kind of anti-17-KS specific antibody, by being produced after above-mentioned 17-KS immunogen immune animal Arrive.
Described anti-17-KS specific antibody uses routine side by 17-KS immunogene obtained above Method inoculation experiments animal, antiserum is taken after booster immunization, is comprised the following steps that：
(1) the BSA-17- ketosteroid immunogenes of above-mentioned synthesis are diluted to 0.1~3.0mg/ml with PBS, obtain antigen Solution, then mixed with 0.5~5.0ml antigenic solutions with equivalent Freund's complete adjuvant, experimental animal is injected；
After (2) 2~3 weeks, then with 0.5~5.0ml identicals antigenic solution and equivalent incomplete Freund's adjuvant to above-mentioned reality Test animal injection once, afterwards every surrounding injection once, inject 3~6 times altogether；
(3) blood is taken to above-mentioned experimental animal, isolates and purifies to obtain potency as 1:30000~1:50000 anti-17- ketones are consolidated Alcohol specific antibody.
The anti-17-KS specific antibody of the present invention is complete antibody molecule, also includes retaining and consolidates with 17- ketones The antibody fragment or antibody derivatives of alcohol specific binding capacity.
The antibody of the present invention is polyclonal to be obtained using single 17-KS immunogene to animal booster immunization Antibody, or be the monoclonal antibody obtained after being immunized through somatic hybridization；Described experimental animal is rabbit, goat, mouse, silk floss One kind of sheep, cavy or horse, preferably rabbit.
The present invention provides a kind of 17-KS detection reagent, containing above-mentioned anti-17-KS specific antibody and refers to Show reagent.
Indicator of the present invention is selected from enzymatic reagent, radio isotope reagent, fluorometric reagent, luminescence reagent.Preferably, refer to It is enzymatic reagent to show 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 Portugal Grape sugar -6- phosphoric acid.
17-KS homogeneous enzyme immunoassay detection reagent before the use, in order to avoid in indicator enzyme mark coupling The substrate of thing and enzyme reacts, and the substrate of enzyme mark conjugate and enzyme is unmixed and separated, so the substrate by enzyme Mixed with above-mentioned anti-17-KS specific antibody.Therefore, 17-KS homogeneous enzyme immunoassay detection reagent bag Include two class reagents：
(1) reagent A is mixed by anti-17-KS specific antibody and homogeneous zymolyte, and specific preparation process is such as Under：
1) by the NADH (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), 0.5~2L 55mM, pH=8.0 Tris Homogeneous zymolyte is made in buffer solution；
2) the anti-17-KS specific antibody of preparation is added in above-mentioned homogeneous zymolyte, antibody and homogeneous enzyme bottom The volume ratio of thing is 1:100~1:10000, preferably 1:1000；
(2) reagent B is mixed by glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing with Tris buffer solutions, preparation side Method is as follows：
1) preparation of glucose-6-phosphate dehydrogenase (G6PD) (G6PDH) solution：
A. the G6PDH that 7.5~22.5mg specifications are 100KU is weighed, room-temperature dissolution contains 72.6mg in 6~18mL (0.05M)Tris、8mg MgCl2In (3.3mM) and 100mg NaCl solution, pH value of solution=9.0；
B. the NADH (NADH) of 112.5~337.5mg reduction-states, 67.5~202.5mg are added G-6-P (G-6-P) and 0.375~1.125mL carbitols；
C. 1~3mL dimethyl sulfoxide (DMSO)s are added dropwise；
2) activation of 17-KS derivative：
A. 5~15mg17- ketosteroid derivatives are weighed under anhydrous conditions, are 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 tri-n-butylamines are added；
D. 0.75~2.25 μ L isobutyl chlorocarbonates are added；
Stir 30~60 minutes e.-2~-8 DEG C；
3) G6PDH and 17-KS derivative connection：
A. the 17-KS derivative solution of above-mentioned activation is added dropwise in the G6PDH solution of above-mentioned dissolving；
B.2-8 DEG C it is stirred overnight；
4) purified product：
Connection product is purified by G-25 gel chromatography columns, the final product of acquisition be glucose-6-phosphate dehydrogenase (G6PD)- Hapten conjugation thing, stored at 2-8 DEG C.
5) Tris that the glucose-6-phosphate dehydrogenase (G6PD) of preparation-hapten conjugation thing is added to 120mM, pH=8.2 is buffered In liquid, the volume ratio of above-mentioned conjugate and Tris buffer solutions is 1:100~1:10000, preferably 1:3000.
17-KS immunogens of the invention are strong, immunogenicity is high, and the anti-17-KS prepared is special Heterogenetic antibody high specificity, potency are high, and with 62 kinds of common medicines without any cross reaction；Contain above-mentioned anti-17- ketones The homogeneous enzyme immunoassay detection reagent of sterol specific antibody can easily and fast, accurately determine in the biological samples such as urine 17-KS content, and multiple samples can be determined simultaneously on automatic clinical chemistry analyzer, realize 17-KS The rapid measure of high flux, the degree of accuracy is high, high specificity, and accuracy compare with detection efficiency has larger carry before Height, while the full-automation of detection process is realized, to the less demanding of testing staff, it is easy to accomplish and promote the use of.
Brief description of the drawings
Fig. 1 is the ELISA detection response curves 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 synthesis of the 17-KS immunogene of embodiment one
17-KS immunogene is shown in bovine serum albumin(BSA) (Bovine Serum Albumin, BSA) and formula (II) 17-KS derivativeGroup is formed by connecting, and comprises the following steps that：
1. bovine serum albumin(BSA) 200mg is dissolved in 50ml 0.2M, in pH 8.5 phosphate buffer；
2. following chemicals is added to stirring and dissolving in small beaker：200mg17- ketosteroids derivative, 3.5ml diformazans Base formamide, 3.5ml ethanol, 7.0ml 10mM, pH 5.0 kaliumphosphate buffer, 200mg1- ethyls -3- (- 3- diformazans ammonia third Base) carbodiimide, 50mg N- hydroxy thiosuccinimides, by these chemicals be stirred at room temperature dissolving reaction 30min；
3. the solution dissolved is added dropwise in BSA solution, and it is stirred overnight at 2~8 DEG C, obtains antigen；Will synthesis Good antigen is purified by dialysis, obtains 17-KS immunogene.
Embodiment two：The preparation of anti-17-KS specific antibody
The 17-KS immunogene that embodiment one is prepared uses conventional method inoculation experiments animal rabbit, strengthens Antiserum is taken after immune, is comprised the following steps that：
1. the 17-KS immunogene of above-mentioned synthesis is diluted into 1.0mg/ml with PBS, antigenic solution is obtained, then Mixed with 1.0ml antigenic solutions with equivalent Freund's complete adjuvant, experimental animal rabbit is injected.
After 2.2~3 weeks, then with 1.0ml identicals antigenic solution and equivalent incomplete Freund's adjuvant to above-mentioned experimental animal Rabbit is injected once, afterwards every surrounding injection once, is injected 4 times altogether.
3. the experimental animal rabbit of pair step 2 takes blood, isolate and purify to obtain potency as 1:30000~1:50000 anti-17- ketone Steroids specific antibody.
Embodiment three：The ELISA of 17-KS is examined
The foundation of the ELISA examination criteria curves of 1.17- ketosteroids
(1) preparation of standard items
17-KS powder (being purchased from Sigma companies) is dissolved in methanol solution, is prepared into 1mg/ml storing liquid. Storing liquid is diluted to 20.00mg/L, 10.00mg/L, 5.00mg/L, 2.50mg/L, 1.25mg/L successively with ELISA buffer solutions With 0.00mg/L standard liquid.Wherein, ELISA buffer solutions contain 50.0mM Tris, 145mM NaCl and 0.25% BSA.
(2) standard curve is prepared using the ELISA methods of inspection of 17-KS
Anti- 17-KS specific antibody prepared in embodiment two is diluted to 1 with PBS:8000 final concentration Solution, 100 μ L/ holes are coated on 96 hole elisa plates, 4 DEG C of placement 12-24h；With PBS anti-17-KS is coated with by above-mentioned After 96 hole elisa plates of antibody wash 3 times, the 0.5% BSA solution in 200 μ L/ holes is added, 8-16h is placed in 4 DEG C of closings.Then Washed 3 times with PBS, add the standard items in 20 μ L/ holes.Add the HRP-17- ketosteroids coupling of 100 μ L/ holes working concentrations Thing；PBS board-washings 5 times after incubation 30min at room temperature；Then 100 μ L tmb substrates are added per hole, are incubated at room temperature 30min.Again per hole Add 100 μ L terminate liquids (2M sulfuric acid).Determine 450nm light absorption value.The light absorption value of 450nm according to corresponding to each standard items is determined Mark, standard curve is made, as a 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 companies) is dissolved in methanol solution and is made 1000mg/L's Storing liquid, and this storing liquid is diluted in blank diaper, it is respectively 0.00,1.50,8.00,20.00mg/L to final concentration, shape Into blank, the urine specimen of basic, normal, high concentration.The blank diaper is the Healthy People urine without 17-KS.
(2) method of testing
Using the ELISA methods of inspection of above-mentioned 17-KS, by above-mentioned blank, the urine specimen of basic, normal, high concentration Instead of standard items, test above-mentioned blank, the urine specimen of basic, normal, high concentration 450nm light absorption value.
(3) test result
The standard curve that the ELISA of 17-KS shown in compares figure 1 is examined, calculates 17- ketones in each sample Sterol content, and 3 multiple holes measure are carried out to each sample, calculated according to the actual content of 17-KS in above-mentioned sample The rate of recovery, as a result as shown in table 1.
The ELISA detection recovery experiments of table 117- ketosteroids
Urine sample Blank It is low In It is 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
Average 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：Various concentrations sample is determined using the ELISA detection reagents of 17-KS of the present invention In the 17-KS rate of recovery it is all higher, equal ＞ 90%, illustrate anti-17-KS specific antibody of the present invention It can be used for the detection of 17-KS in sample, and result precision is high.
Example IV：The preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing
The preparation of glucose-6-phosphate dehydrogenase (G6PD) 1. (G6PDH) solution：
(1) accurate to weigh the G6PDH that 15mg specifications are 100KU, room-temperature dissolution contains 72.6mg (0.05M) in 12mL Tris、8mg MgCl2In (3.3mM) and 100mg NaCl solution, pH value of solution=9.0, this step is carried out in beaker C.
(2) NADH (NADH) of 225mg reduction-states, 135mg grapes are added in above-mentioned beaker C Sugar -6- phosphoric acid (G-6-P) and 0.75mL carbitols (Carbitol).
(3) 2mL dimethyl sulfoxide (DMSO)s (dimethy sulfoxide, DMSO) are added dropwise again in above-mentioned beaker C.
The activation of 2.17- ketosteroid derivatives：
(1) the above-mentioned 17-KS derivatives of 10mg are weighed under anhydrous conditions, are dissolved in 600 μ L DMF.
(2) above-mentioned solution temperature is made to drop to -2~-8 DEG C.
(3) 3 μ L tri-n-butylamines (tributylamine) are added.
(4) 1.5 μ L isobutyl chlorocarbonates (isobutylchloroformate) are added.
(5) -2~-8 DEG C are stirred 30 minutes.
3.G6PDH and 17-KS derivative connection：
(1) the 17-KS derivative solution of above-mentioned activation is added dropwise in the G6PDH solution of above-mentioned dissolving.
(2) 2-8 DEG C is stirred overnight.
4. purified product：
By the solution in G-25 gel chromatographies column purification step 3, the final product of acquisition is G-6-P dehydrogenation Enzyme-hapten conjugation thing, is stored at 2-8 DEG C.
Embodiment five：The preparation of 17-KS homogeneous enzyme immunoassay detection reagent
1. the preparation of reagent A：By the NADH (NAD) of 4.036g (11.25mM) oxidation state, 1.711g (11.25mM) G-6-P (G-6-P) is placed in beaker D, with 1L 55mM, pH=8.0 Tris buffer solutions Homogeneous zymolyte is made in dissolving；The anti-17-KS specific antibody of above-mentioned preparation is added in above-mentioned homogeneous zymolyte, resisted The volume ratio of body and homogeneous zymolyte is 1:1000.
2. reagent B preparation：Glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing prepared by example IV is added to In 120mM, pH=8.2 Tris buffer solutions, the volume ratio of above-mentioned conjugate and Tris buffer solutions is 1:3000.
Embodiment six：17-KS homogeneous enzyme immunoassay is examined and result
1. obtain standard curve：
(1) set and step auspicious BS-480 automatic clinical chemistry analyzers response parameter (being shown in Table 2).
(2) operating procedure is：First reagent adding A, adds standard items, is eventually adding reagent B.After adding reagent B, measure is not With the OD at time point340Light absorption value, reaction rate during various criterion product concentration is calculated, needs constantly to adjust in actual mechanical process The volume ratio of reagent A and reagent B, while light-metering point is adjusted, comparatively ideal reaction normal curve map is finally drawn, such as Fig. 3 institutes Show.
Table 2 steps auspicious BS-480 automatic clinical chemistry analyzers response parameter
2. pattern detection：By the obtained standard curve of homogeneous enzyme immunoassay detection reagent of the present invention, replication is low, Middle and high concentration Quality Control sample 10 times, above-mentioned Quality Control sample are：17-KS standard items are dissolved in human urine, to concentration Respectively 1.50,8.00,20.00mg/L.Detection data and data analysis are shown in Table 3.
The sample of table 3 determines and precision and rate of recovery assessment
Urine sample It is low In It is 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
Average 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
Testing result：The degree of accuracy of the homogeneous enzyme immunoassay detection reagent measure of the present invention is high, and the rate of recovery reaches 95%- 105%, precision is high, and CV is below 5%.
Embodiment seven：Interfering effects of drug is tested
Choose 62 kinds of Common drugs and carry out Interference Detection, adjustment concentration is to 1.00mg/L, using the homogeneous enzyme of embodiment six Immunization method is measured：
1. reagent A haptoreaction prepared by interference medicament to be measured and embodiment five, adds reagent B；
2. the OD of the above-mentioned mixed solution of detection340Light absorption value, the dense of respective substance is obtained according to the standard curve of embodiment six Degree.
62 kinds of common medicine names and measurement result are referring specifically to table 4.
The common interference drug monitoring result of table 4
Measurement result is shown：The concentration that above-mentioned 62 kinds of Common drugs are equivalent to 17-KS is respectively less than 0.01mg/L.By This is visible, and antibody of the invention is the specific antibody of anti-17-KS, with other medicines no cross reaction.
Embodiment eight：Correlation analysis
Spain's biosystem (chromatogram-AAS) and of the invention homogeneous is used respectively to 100 clinical samples Enzyme immunoreagent carries out correlation analysis, and the data of measure are referring to table 5.
The clinical sample measured value of table 5
Above-mentioned data are mapped, referring to Fig. 3, obtained linear equation is：Y=1.0113x-0.1401, coefficient R2 =0.9907, show that the degree of accuracy of the detection reagent measure 17-KS clinical samples of the present invention is high.
It should be noted that the foregoing is only embodiments of the invention, it is not intended to limit the scope of the invention, Every equivalent structure done using description of the invention and accompanying drawing content or equivalent flow conversion, or be directly or indirectly used in Other correlative technology fields, are included within the scope of the present invention.

Claims (8)

  1. A kind of 1. 17-KS immunogene, shown in its structural formula such as formula (I)：
    Carrier is the bovine serum albumin(BSA) with immunogenicity.
  2. 2. a kind of preparation method of 17-KS immunogene as claimed in claim 1, it is characterised in that include following step Suddenly：
    (1) 100~300mg of carrier protein is dissolved in 25~75ml 0.2M, in pH 8.5 phosphate buffer；
    (2) following chemicals is added to stirring and dissolving in small beaker：100~300mg17- ketosteroids derivative, 1.75~ 5.25ml dimethylformamides, 1.75~5.25ml ethanol, 3.5~10.5ml 10mM, pH 5.0 kaliumphosphate buffer, 100 ~300mg 1- ethyls -3- (- 3- dimethylaminopropyls) carbodiimide, 25~75mg N- hydroxy thiosuccinimides, will be above-mentioned 30~60min of dissolving reaction is stirred at room temperature in chemicals；
    (3) solution dissolved is added dropwise in carrier protein solution, and be stirred overnight at 2~8 DEG C, obtain antigen；It will close Purified into good antigen by dialysis, obtain 17-KS immunogene；Described 17-KS derivant structure Shown in formula such as formula (II)：
  3. 3. a kind of anti-17-KS specific antibody, it is the 17-KS immunogen immune experiment described in claim 1 Caused complete antibody molecule after animal.
  4. A kind of 4. anti-17-KS specific antibody according to claim 3, to use single 17-KS The polyclonal antibody that immunogene is obtained to animal booster immunization, or be that the monoclonal obtained after being immunized through somatic hybridization resists Body；Described experimental animal is one kind of rabbit, goat, mouse, sheep, cavy or horse.
  5. A kind of 5. preparation method of anti-17-KS specific antibody as described in claim 3 or 4, it is characterised in that bag Containing following steps：
    (1) 17-KS immunogene is diluted to 0.1~3.0mg/ml with PBS, obtains antigenic solution, then with 0.5~ 5.0ml antigenic solutions are mixed with equivalent Freund's complete adjuvant, and experimental animal is injected；
    After (2) 2~3 weeks, then with 0.5~5.0ml identicals antigenic solution above-mentioned experiment is moved with equivalent incomplete Freund's adjuvant Thing is injected once, afterwards every surrounding injection once, is injected 3~6 times altogether；
    (3) blood is taken to the experimental animal of step (2), isolates and purifies to obtain potency as 1:30000~1:50000 anti-17- ketones Sterol specific antibody.
  6. 6. a kind of 17-KS detection reagent, containing the anti-17-KS specific antibody described in claim 3 or 4 and Indicator, described indicator are enzymatic reagent；Described enzymatic reagent is by 17-KS enzyme mark conjugate and the substrate of enzyme Composition, enzyme mark conjugate is glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate, and the substrate of enzyme is glucose -6- phosphorus Acid.
  7. 7. a kind of preparation method of 17-KS detection reagent as claimed in claim 6, it is characterised in that include following step Suddenly：
    (1) reagent A：By 2.018~8.072g, 5.625~22.50mM oxidation state NADH and 0.856~3.422g, 5.625~22.50mM G-6-Ps, 0.5~2L 55mM, pH=8.0 Tris buffer solutions are molten Homogeneous zymolyte is made in solution；Anti- 17-KS specific antibody described in claim 3 or 4 is added to above-mentioned homogeneous enzyme bottom In thing, the volume ratio of anti-17-KS specific antibody and homogeneous zymolyte is 1:1000；
    (2) reagent B：17-KS enzyme mark conjugate is added in 120mM, pH=8.2 Tris buffer solutions, 17- ketones are consolidated The volume ratio of alcoholase mark conjugate and Tris buffer solutions is 1:3000.
  8. 8. the preparation method of 17-KS detection reagent according to claim 7, it is characterised in that described 17- ketone The preparation method of steroids enzyme mark conjugate comprises the steps of：
    (1) preparation of glucose-6-phosphate dehydrogenase (G6PD) solution：Weigh the glucose -6- phosphorus that 7.5~22.5mg specifications are 100KU Acidohydrogenase, room-temperature dissolution contain 72.6mg 0.05M Tris, 8mg 3.3mM MgCl in 6~18mL2With 100mg NaCl's In solution, pH=9.0；In the solution add 112.5~337.5mg reduction-states NADH, 67.5~ 202.5mg G-6-Ps and 0.375~1.125mL carbitols；1~3mL dimethyl sulfoxide (DMSO)s are added dropwise again；
    (2) activation of 17-KS derivative：5~15mg17- ketosteroid derivatives are weighed under anhydrous conditions, are dissolved In 300~900 μ L dimethylformamides；Above-mentioned solution temperature is set to drop to -2~-8 DEG C；Add 1.5~4.5 μ L tri-n-butylamines；Add Enter 0.75~2.25 μ L isobutyl chlorocarbonates；- 2~-8 DEG C are stirred 30~60 minutes；Described 17-KS derivant structure Shown in formula such as formula (II)：
    (3) connection of glucose-6-phosphate dehydrogenase (G6PD) and 17-KS derivative：The 17- ketones that step (2) activates are consolidated 01 derivatives solution is added dropwise in the glucose-6-phosphate dehydrogenase (G6PD) solution of step (1) dissolving；2-8 DEG C is stirred overnight；
    (4) purified product：Connection product is purified by G-25 gel chromatography columns, the final product of acquisition is G-6-P Dehydrogenase-hapten conjugation thing, is stored at 2-8 DEG C.
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CN102617729A (en) * 2012-04-06 2012-08-01 苏州博源医疗科技有限公司 Tacrolimus immunogen, anti-tacrolimus specific antigen and tacrolimus detection reagent
CN102998464A (en) * 2011-09-16 2013-03-27 武汉优尔生科技股份有限公司 Dehydroepiandrosterone enzyme-linked immunosorbent assay kit development method

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CN101538307A (en) * 2009-04-27 2009-09-23 华南农业大学 Dehydroepiandrosterone hapten, artificial antigen and antibody as well as preparation method and application thereof
CN102998464A (en) * 2011-09-16 2013-03-27 武汉优尔生科技股份有限公司 Dehydroepiandrosterone enzyme-linked immunosorbent assay kit development method
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