CN110187094A - A kind of 5-HIAA measurement reagent - Google Patents
A kind of 5-HIAA measurement reagent Download PDFInfo
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- CN110187094A CN110187094A CN201910567947.3A CN201910567947A CN110187094A CN 110187094 A CN110187094 A CN 110187094A CN 201910567947 A CN201910567947 A CN 201910567947A CN 110187094 A CN110187094 A CN 110187094A
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
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Abstract
The present invention provides a kind of 5-HIAAs to measure reagent；Contain 5-HIAA specific antibody and indicator；The 5-HIAA specific antibody is the complete antibody molecule that generates after 5-HIAA immunogen immune experimental animal；5-HIAA immunogens of the invention are strong, immunogenicity is high, anti-5-HIAA specific antibody high specificity, the potency height prepared, and with 62 kinds of common drugs without any cross reaction；The present invention can measure multiple samples simultaneously on automatic clinical chemistry analyzer, realize the rapid measurement of high throughput of 5-HIAA, accuracy is high, high specificity, accuracy is all enhanced before comparing with detection efficiency, the full-automation for realizing detection process simultaneously, to the of less demanding of testing staff, it is easy to accomplish and promote the use of.
Description
Technical field
The present invention relates to a kind of biological reagent field；More particularly to a kind of 5-HIAA measures reagent.
Background technique
Metabolite of the 5-HIAA (5-HIAA) as serotonin (5-HT), is catalyzed by monoamine oxidase
Change through 5-OHi acetaldehyde.5-HIAA is metabolized in liver, and is drained with urine to external.5-HT level is at noon
Time-division highest, midnight is minimum, therefore the content daytime of its metabolite 5-HIAA is high compared with night, therefore measuring urine for 24 hours more can be quasi-
The contents level of true reaction 5-HIAA.Clinically 5-HIAA content raising is common in carcinoid tumor, ceylon sore mouth, branch
The diseases such as tracheae oat-cell carcinoma；And the reduction of its content is common in nephrosis, depression, obsessive-compulsive neurosis.Therefore, measurement urine
5-HIAA content can provide important evidence for the research of the diagnosis of a variety of diseases, treatment and pathogenesis in liquid.
Method currently used for 5-HIAA content detection mainly has: high performance liquid chromatography, chromatography of ions etc..But
Time-consuming for these methods, at high cost, detection small scale, is not particularly suited for the detection of 5-HIAA content in clinical patients urine.Mesh
Preceding deficient in stability in the market is good, high sensitivity, the 5-HIAA detection reagent of high specificity, especially high-quality automation inspection
Test reagent.Therefore, it researches and develops a kind of quality and reaches that clinical examination requirement, practical, cost performance is high, can be applied to full-automatic biochemical
The 5-HIAA detection reagent of analyzer is imperative.
Summary of the invention
The object of the present invention is to provide regulate and control a kind of 5-HIAA measurement reagent.
In a first aspect, the present invention is achieved by the following technical solutions:
The present invention relates to a kind of 5-HIAAs to measure reagent, contains 5-HIAA specific antibody and instruction
Reagent；
The 5-HIAA specific antibody is complete to generate after 5-HIAA immunogen immune experimental animal
Whole antibody molecule；
Any two kinds in enzymatic reagent, radioactive isotope reagent, fluorescent reagent or luminescence reagent of the indicator
It is mixed in mass ratio for 1:1；Wherein, the enzymatic reagent by 5-HIAA enzyme mark conjugate and enzyme substrate group
At enzyme mark conjugate is glucose -6- phosphate dehydrogenase-haptens enzyme mark conjugate, and the substrate of enzyme is glucose -6-
Phosphoric acid.
Preferably, the complete antibody molecule is to be reinforced using single 5-HIAA immunogene animal
Polyclonal antibody obtained is immunized.
Preferably, the preparation method of the 5-HIAA immunogene, includes the following steps:
Step 1, carrier protein is dissolved in phosphate buffer；
Step 2, by 5-HIAA, dimethylformamide, ethyl alcohol, kaliumphosphate buffer 1- ethyl -3- (- 3- diformazan ammonia
Propyl) carbodiimide, N- hydroxy thiosuccinimide, it is added to stirring and dissolving in small beaker according to certain proportion, in room temperature
Lower stirring and dissolving reaction, obtains solution A；
Step 3, the solution A dissolved is added dropwise in carrier protein solution, is stirred overnight, obtain antigen；Synthetic is resisted
Original is purified by dialysis, obtains 5-HIAA immunogene.
Preferably, in step 1, the quality of the albumen is 100~300g.
Preferably, in step 1, the phosphate buffer is 25~75ml 0.2M, pH 8.5.
Preferably, in step 2, certain proportion is as follows: 50-100mg 5- hydroxyindoleacetic acid, 0.5-1.5ml bis-
Methylformamide, 0.5-1.5ml ethyl alcohol, 2.5-7.5ml 10mM, pH5.0 kaliumphosphate buffer 50-100mg 1- ethyl-
3- (- 3- dimethylaminopropyl) carbodiimide, 15-20mg N- hydroxy thiosuccinimide.
Preferably, in step 2, the stirring and dissolving reacts 30~60min.
Preferably, in step 3, the whipping temp is 2~8 DEG C.
The 5-HIAA specific antibody the preparation method is as follows:
5-HIAA immunogene is diluted with PBS, obtains antigenic solution by step 1, then uses 0.1ml antigen
Solution is mixed with equivalent Freund's complete adjuvant, is injected to experimental animal；
Step 2 after 1 week, then with the identical antigenic solution of 0.1ml and equivalent incomplete Freund's adjuvant is infused above-mentioned experimental animal
It penetrates once, it is primary every injection in 2 weeks later；
Step 3 takes blood to the experimental animal of step 2, isolates and purifies to obtain the anti-5- oxyindole that potency is 1:20000
Acetic acid specific antibody.
Preferably, the 5-HIAA immunogene is diluted to 0.1~3.0mg/ml.
The present invention says the preparation method for the 5-HIAA detection reagent being related to comprising the steps of:
(1) reagent A: by the nicotinamide adenine dinucleotide and 0.6-2.1g, 3.5- of 2-3g, 4-9mM oxidation state
Homogeneous enzyme bottom is made with the Tris buffer solution of 0.1-0.5L 30mM, pH=8.0 in 15.8mM G-6-P
Object；The anti-5-HIAA specific antibody is added in above-mentioned homogeneous zymolyte, anti-5- oxyindole second
The volume ratio of sour specific antibody and homogeneous zymolyte is 1:500；
(2) 5-HIAA enzyme mark conjugate reagent B: is added to the Tris of 0.1-0.5L 30mM, pH=8.0
In buffer, the volume ratio of 5-HIAA enzyme mark conjugate and Tris buffer is 1:1000.
It is had the advantage that in method of the invention
(1) 5-HIAA immunogens of the invention are strong, immunogenicity is high, the anti-5-OHi second prepared
Sour specific antibody high specificity, potency are high, and with 62 kinds of common drugs without any cross reaction；
(2) present invention containing above-mentioned anti-5-HIAA specific antibody homogeneous enzyme immunoassay detection reagent can be convenient,
It quickly and accurately determines the 5-HIAA content in the biological samples such as urine, and can be analyzed in full-automatic biochemical
Multiple samples are measured simultaneously on instrument, realize the rapid measurement of high throughput of 5-HIAA, accuracy is high, high specificity,
Accuracy is all enhanced before comparing with detection efficiency, while realizing the full-automation of detection process, to detection
Personnel's is of less demanding, it is easy to accomplish and promote the use of.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.It should be pointed out that embodiment below is only
It is to further explanation of the invention, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
The present invention relates to a kind of 5-HIAAs to measure reagent, contains 5-HIAA specific antibody and instruction
Reagent；
The 5-HIAA specific antibody is complete to generate after 5-HIAA immunogen immune experimental animal
Whole antibody molecule；
The indicator is selected from enzymatic reagent, radioactive isotope reagent mixes in mass ratio for 1:1；Wherein, described
Enzymatic reagent be made of the substrate of 5-HIAA enzyme mark conjugate and enzyme, enzyme mark conjugate be glucose -6- phosphoric acid
Dehydrogenase-haptens enzyme mark conjugate, the substrate of enzyme are glucose -6- phosphoric acid.
The complete antibody molecule is is obtained using single 5-HIAA immunogene to animal booster immunization
The polyclonal antibody obtained.
The preparation method of the 5-HIAA immunogene, includes the following steps:
Step 1, carrier protein is dissolved in phosphate buffer；
Step 2, by 5- hydroxyindoleacetic acid, dimethylformamide, ethyl alcohol, kaliumphosphate buffer 1- ethyl -3- (- 3- diformazan ammonia
Propyl) carbodiimide, N- hydroxy thiosuccinimide, it is added to stirring and dissolving in small beaker according to certain proportion, in room temperature
Lower stirring and dissolving reaction, obtains solution A；
Step 3, the solution A dissolved is added dropwise in carrier protein solution, is stirred overnight, obtain antigen；Synthetic is resisted
Original is purified by dialysis, obtains 5-HIAA immunogene.
In step 1, the quality of the albumen is 100g, and the phosphate buffer is 75ml 0.2M, pH 8.5.
In step 2, certain proportion is as follows: 50mg 5-HIAA, 0.5ml dimethylformamide, 0.5ml
Ethyl alcohol, 2.5ml 10mM, pH5.0 kaliumphosphate buffer 50mg 1- ethyl -3- (- 3- dimethylaminopropyl) carbodiimide,
15mg N- hydroxy thiosuccinimide, in step 2, the stirring and dissolving reacts 30min.
In step 3, the whipping temp is 2 DEG C.
The 5-HIAA specific antibody the preparation method is as follows:
5-HIAA immunogene is diluted with PBS, obtains antigenic solution by step 1, then uses 0.1ml antigen
Solution is mixed with equivalent Freund's complete adjuvant, is injected to experimental animal；
Step 2 after 1 week, then with the identical antigenic solution of 0.1ml and equivalent incomplete Freund's adjuvant is infused above-mentioned experimental animal
It penetrates once, it is primary every injection in 2 weeks later；
Step 3 takes blood to the experimental animal of step 2, isolates and purifies to obtain the anti-5- oxyindole that potency is 1:20000
Acetic acid specific antibody.
The 5-HIAA immunogene is diluted to 0.1mg/ml.
The present invention says the preparation method for the 5-HIAA detection reagent being related to comprising the steps of:
(1) reagent A: by the nicotinamide adenine dinucleotide of 2g, 4mM oxidation state and 0.6g, 3.5mM glucose-
Homogeneous zymolyte is made with the Tris buffer solution of 0.1L 30mM, pH=8.0 in 6- phosphoric acid；By the anti-5- hydroxyl
Base heteroauxin specific antibody is added in above-mentioned homogeneous zymolyte, anti-5-HIAA specific antibody and homogeneous enzyme
The volume ratio of substrate is 1:500；
(2) 5-HIAA enzyme mark conjugate reagent B: is added to the Tris of 0.1L 30mM, pH=8.0 buffering
In liquid, the volume ratio of 5-HIAA enzyme mark conjugate and Tris buffer is 1:1000.
Embodiment 2
The present invention relates to a kind of 5-HIAAs to measure reagent, contains 5-HIAA specific antibody and instruction
Reagent；
The 5-HIAA specific antibody is complete to generate after 5-HIAA immunogen immune experimental animal
Whole antibody molecule；
The indicator is selected from fluorescent reagent, luminescence reagent mixes in mass ratio for 1:1；Wherein, enzyme examination
Agent is made of the substrate of 5-HIAA enzyme mark conjugate and enzyme, and enzyme mark conjugate is glucose -6- phosphate dehydrogenase
Haptens enzyme mark conjugate, the substrate of enzyme are glucose -6- phosphoric acid.
The complete antibody molecule is is obtained using single 5-HIAA immunogene to animal booster immunization
The polyclonal antibody obtained.
The preparation method of the 5-HIAA immunogene, includes the following steps:
Step 1, carrier protein is dissolved in phosphate buffer；
Step 2, by 5-HIAA, dimethylformamide, ethyl alcohol, kaliumphosphate buffer 1- ethyl -3- (- 3- diformazan ammonia
Propyl) carbodiimide, N- hydroxy thiosuccinimide, it is added to stirring and dissolving in small beaker according to certain proportion, in room temperature
Lower stirring and dissolving reaction, obtains solution A；
Step 3, the solution A dissolved is added dropwise in carrier protein solution, is stirred overnight, obtain antigen；Synthetic is resisted
Original is purified by dialysis, obtains 5-HIAA immunogene.
In step 1, the quality of the albumen is 300g, and the phosphate buffer is 75ml 0.2M, pH 8.5.
In step 2, it is described it is certain proportion it is as follows: 100mg 5-HIAA, 1.5ml dimethylformamide,
Kaliumphosphate buffer 100mg 1- ethyl -3- (- 3- dimethylaminopropyl) carbon two of 1.5ml ethyl alcohol, 7.5ml 10mM, pH5.0
Imines, 20mg N- hydroxy thiosuccinimide, in step 2, the stirring and dissolving reacts 60min.
In step 3, the whipping temp is 8 DEG C.
The 5-HIAA specific antibody the preparation method is as follows:
5-HIAA immunogene is diluted with PBS, obtains antigenic solution by step 1, then uses 0.1ml antigen
Solution is mixed with equivalent Freund's complete adjuvant, is injected to experimental animal；
Step 2 after 1 week, then with the identical antigenic solution of 0.1ml and equivalent incomplete Freund's adjuvant is infused above-mentioned experimental animal
It penetrates once, it is primary every injection in 2 weeks later；
Step 3 takes blood to the experimental animal of step 2, isolates and purifies to obtain the anti-5-OHi second that potency is 1:20000
Sour specific antibody.
The 5-HIAA immunogene is diluted to 3.0mg/ml.
The present invention says the preparation method for the 5-HIAA detection reagent being related to comprising the steps of:
(1) reagent A: by the nicotinamide adenine dinucleotide of 3g, 9mM oxidation state and the Portugal 2.1g, 3.5-15.8mM
Homogeneous zymolyte is made with the Tris buffer solution of 0.5L 30mM, pH=8.0 in grape sugar -6- phosphoric acid；It will be described
Anti- 5-HIAA specific antibody is added in above-mentioned homogeneous zymolyte, anti-5-HIAA specific antibody with
The volume ratio of homogeneous zymolyte is 1:500；
(2) 5-HIAA enzyme mark conjugate reagent B: is added to the Tris of 0.5L 30mM, pH=8.0 buffering
In liquid, the volume ratio of 5- hydroxyindoleacetic acid enzyme mark conjugate and Tris buffer is 1:1000.
Embodiment 3
The present invention relates to a kind of 5-HIAAs to measure reagent, contains 5-HIAA specific antibody and instruction
Reagent；
The 5-HIAA specific antibody is complete to generate after 5-HIAA immunogen immune experimental animal
Whole antibody molecule；
The indicator is selected from enzymatic reagent, fluorescent reagent mixes in mass ratio for 1:1；Wherein, the enzymatic reagent
It is made of the substrate of 5-HIAA enzyme mark conjugate and enzyme, enzyme mark conjugate is glucose -6- phosphate dehydrogenase -
Haptens enzyme mark conjugate, the substrate of enzyme are glucose -6- phosphoric acid.
The complete antibody molecule is is obtained using single 5-HIAA immunogene to animal booster immunization
The polyclonal antibody obtained.
The preparation method of the 5-HIAA immunogene, includes the following steps:
Step 1, carrier protein is dissolved in phosphate buffer；
Step 2, by 5-HIAA, dimethylformamide, ethyl alcohol, kaliumphosphate buffer 1- ethyl -3- (- 3- diformazan ammonia
Propyl) carbodiimide, N- hydroxy thiosuccinimide, it is added to stirring and dissolving in small beaker according to certain proportion, in room temperature
Lower stirring and dissolving reaction, obtains solution A；
Step 3, the solution A dissolved is added dropwise in carrier protein solution, is stirred overnight, obtain antigen；Synthetic is resisted
Original is purified by dialysis, obtains 5-HIAA immunogene.
In step 1, the quality of the albumen is 200g, and the phosphate buffer is 50ml 0.2M, pH 8.5.
In step 2, certain proportion is as follows: 60mg 5-HIAA, 0.8ml dimethylformamide, 0.8ml
Kaliumphosphate buffer 60mg 1- ethyl -3- (- 3- dimethylaminopropyl) carbodiimide, 16mg of ethyl alcohol, 3ml 10mM, pH5.0
N- hydroxy thiosuccinimide, in step 2, the stirring and dissolving reacts 40min.
In step 3, the whipping temp is 3 DEG C.
The 5-HIAA specific antibody the preparation method is as follows:
5-HIAA immunogene is diluted with PBS, obtains antigenic solution by step 1, then uses 0.1ml antigen
Solution is mixed with equivalent Freund's complete adjuvant, is injected to experimental animal；
Step 2 after 1 week, then with the identical antigenic solution of 0.1ml and equivalent incomplete Freund's adjuvant is infused above-mentioned experimental animal
It penetrates once, it is primary every injection in 2 weeks later；
Step 3 takes blood to the experimental animal of step 2, isolates and purifies to obtain the anti-5-OHi second that potency is 1:20000
Sour specific antibody.
The 5-HIAA immunogene is diluted to 0.5mg/ml.
The present invention says the preparation method for the 5-HIAA detection reagent being related to comprising the steps of:
(1) reagent A: by the nicotinamide adenine dinucleotide of 2.5g, 5mM oxidation state and 0.8g, 4.5mM grape
Homogeneous zymolyte is made with the Tris buffer solution of 0.45L 30mM, pH=8.0 in sugar -6- phosphoric acid；Described is resisted
5-HIAA specific antibody is added in above-mentioned homogeneous zymolyte, anti-5-HIAA specific antibody and
The volume ratio of phase zymolyte is 1:500；
(2) 5-HIAA enzyme mark conjugate reagent B: is added to the Tris of 0.2L 30mM, pH=8.0 buffering
In liquid, the volume ratio of 5-HIAA enzyme mark conjugate and Tris buffer is 1:1000.
Embodiment 4
The present invention relates to a kind of 5-HIAAs to measure reagent, contains 5-HIAA specific antibody and instruction
Reagent；
The 5-HIAA specific antibody is complete to generate after 5-HIAA immunogen immune experimental animal
Whole antibody molecule；
The indicator is selected from enzymatic reagent, luminescence reagent mixes in mass ratio for 1:1；Wherein, the enzymatic reagent
It is made of the substrate of 5-HIAA enzyme mark conjugate and enzyme, enzyme mark conjugate is glucose -6- phosphate dehydrogenase -
Haptens enzyme mark conjugate, the substrate of enzyme are glucose -6- phosphoric acid.
The complete antibody molecule is is obtained using single 5-HIAA immunogene to animal booster immunization
The polyclonal antibody obtained.
The preparation method of the 5-HIAA immunogene, includes the following steps:
Step 1, carrier protein is dissolved in phosphate buffer；
Step 2, by 5-HIAA, dimethylformamide, ethyl alcohol, kaliumphosphate buffer 1- ethyl -3- (- 3- diformazan ammonia
Propyl) carbodiimide, N- hydroxy thiosuccinimide, it is added to stirring and dissolving in small beaker according to certain proportion, in room temperature
Lower stirring and dissolving reaction, obtains solution A；
Step 3, the solution A dissolved is added dropwise in carrier protein solution, is stirred overnight, obtain antigen；Synthetic is resisted
Original is purified by dialysis, obtains 5-HIAA immunogene.
In step 1, the quality of the albumen is 100~300g, and the phosphate buffer is 25~75ml 0.2M,
pH 8.5。
In step 2, certain proportion is as follows: 70mg 5-HIAA, 0.7ml dimethylformamide, 0.7ml
Ethyl alcohol, 3.5ml 10mM, pH5.0 kaliumphosphate buffer 80mg 1- ethyl -3- (- 3- dimethylaminopropyl) carbodiimide,
17mg N- hydroxy thiosuccinimide, in step 2, the stirring and dissolving reacts 50min.
In step 3, the whipping temp is 5 DEG C.
The 5-HIAA specific antibody the preparation method is as follows:
5-HIAA immunogene is diluted with PBS, obtains antigenic solution by step 1, then uses 0.1ml antigen
Solution is mixed with equivalent Freund's complete adjuvant, is injected to experimental animal；
Step 2 after 1 week, then with the identical antigenic solution of 0.1ml and equivalent incomplete Freund's adjuvant is infused above-mentioned experimental animal
It penetrates once, it is primary every injection in 2 weeks later；
Step 3 takes blood to the experimental animal of step 2, isolates and purifies to obtain the anti-5-OHi second that potency is 1:20000
Sour specific antibody.
The 5-HIAA immunogene is diluted to 0.1mg/ml.
The present invention says the preparation method for the 5-HIAA detection reagent being related to comprising the steps of:
(1) reagent A: by the nicotinamide adenine dinucleotide of 2g, 6mM oxidation state and 1.6g, 5.5mM glucose-
Homogeneous zymolyte is made with the Tris buffer solution of 0.45L 30mM, pH=8.0 in 6- phosphoric acid；By the anti-5-
Hydroxyindoleacetic acid specific antibody is added in above-mentioned homogeneous zymolyte, anti-5-HIAA specific antibody and homogeneous
The volume ratio of zymolyte is 1:500；
(2) 5-HIAA enzyme mark conjugate reagent B: is added to the Tris of 0.45L 30mM, pH=8.0 buffering
In liquid, the volume ratio of 5-HIAA enzyme mark conjugate and Tris buffer is 1:1000.
Embodiment 5
The present invention relates to a kind of 5-HIAAs to measure reagent, contains 5-HIAA specific antibody and instruction
Reagent；
The 5-HIAA specific antibody is complete to generate after 5-HIAA immunogen immune experimental animal
Whole antibody molecule；
The indicator is selected from enzymatic reagent, fluorescent reagent mixes in mass ratio for 1:1；Wherein, the enzymatic reagent
It is made of the substrate of 5-HIAA enzyme mark conjugate and enzyme, enzyme mark conjugate is glucose -6- phosphate dehydrogenase -
Haptens enzyme mark conjugate, the substrate of enzyme are glucose -6- phosphoric acid.
The complete antibody molecule is is obtained using single 5-HIAA immunogene to animal booster immunization
The polyclonal antibody obtained.
The preparation method of the 5-HIAA immunogene, includes the following steps:
Step 1, carrier protein is dissolved in phosphate buffer；
Step 2, by 5-HIAA, dimethylformamide, ethyl alcohol, kaliumphosphate buffer 1- ethyl -3- (- 3- diformazan ammonia
Propyl) carbodiimide, N- hydroxy thiosuccinimide, it is added to stirring and dissolving in small beaker according to certain proportion, in room temperature
Lower stirring and dissolving reaction, obtains solution A；
Step 3, the solution A dissolved is added dropwise in carrier protein solution, is stirred overnight, obtain antigen；Synthetic is resisted
Original is purified by dialysis, obtains 5-HIAA immunogene.
In step 1, the quality of the albumen is 200g, and the phosphate buffer is 60ml 0.2M, pH 8.5.
In step 2, certain proportion is as follows: 85mg 5-HIAA, 0.5ml dimethylformamide, 0.5ml
Ethyl alcohol, 7.5ml 10mM, pH5.0 kaliumphosphate buffer 85mg 1- ethyl -3- (- 3- dimethylaminopropyl) carbodiimide,
16mg N- hydroxy thiosuccinimide, in step 2, the stirring and dissolving reacts 30min.
In step 3, the whipping temp is 2 DEG C.
The 5-HIAA specific antibody the preparation method is as follows:
5-HIAA immunogene is diluted with PBS, obtains antigenic solution by step 1, then uses 0.1ml antigen
Solution is mixed with equivalent Freund's complete adjuvant, is injected to experimental animal；
Step 2 after 1 week, then with the identical antigenic solution of 0.1ml and equivalent incomplete Freund's adjuvant is infused above-mentioned experimental animal
It penetrates once, it is primary every injection in 2 weeks later；
Step 3 takes blood to the experimental animal of step 2, isolates and purifies to obtain the anti-5-OHi second that potency is 1:20000
Sour specific antibody.
The 5-HIAA immunogene is diluted to 3.0mg/ml.
The present invention says the preparation method for the 5-HIAA detection reagent being related to comprising the steps of:
(1) reagent A: by the nicotinamide adenine dinucleotide of 3g, 9mM oxidation state and 2.1g, 15.8mM glucose-
Homogeneous zymolyte is made with the Tris buffer solution of 0.1L 30mM, pH=8.0 in 6- phosphoric acid；By the anti-5-
Hydroxyindoleacetic acid specific antibody is added in above-mentioned homogeneous zymolyte, anti-5-HIAA specific antibody and homogeneous
The volume ratio of zymolyte is 1:500；
(2) 5-HIAA enzyme mark conjugate reagent B: is added to the Tris of 0.1L 30mM, pH=8.0 buffering
In liquid, the volume ratio of 5-HIAA enzyme mark conjugate and Tris buffer is 1:1000.
Embodiment 6
The present invention relates to a kind of 5-HIAAs to measure reagent, contains 5-HIAA specific antibody and instruction
Reagent；
The 5-HIAA specific antibody is complete to generate after 5-HIAA immunogen immune experimental animal
Whole antibody molecule；
The indicator is selected from fluorescent reagent, luminescence reagent mixes in mass ratio for 1:1；Wherein, enzyme examination
Agent is made of the substrate of 5-HIAA enzyme mark conjugate and enzyme, and enzyme mark conjugate is glucose -6- phosphate dehydrogenase
Haptens enzyme mark conjugate, the substrate of enzyme are glucose -6- phosphoric acid.
The complete antibody molecule is is obtained using single 5-HIAA immunogene to animal booster immunization
The polyclonal antibody obtained.
The preparation method of the 5-HIAA immunogene, includes the following steps:
Step 1, carrier protein is dissolved in phosphate buffer；
Step 2, by 5-HIAA, dimethylformamide, ethyl alcohol, kaliumphosphate buffer 1- ethyl -3- (- 3- diformazan ammonia
Propyl) carbodiimide, N- hydroxy thiosuccinimide, it is added to stirring and dissolving in small beaker according to certain proportion, in room temperature
Lower stirring and dissolving reaction, obtains solution A；
Step 3, the solution A dissolved is added dropwise in carrier protein solution, is stirred overnight, obtain antigen；Synthetic is resisted
Original is purified by dialysis, obtains 5-HIAA immunogene.
In step 1, the quality of the albumen is 250g, and the phosphate buffer is 70ml 0.2M, pH 8.5.
In step 2, certain proportion is as follows: 90mg 5-HIAA, 1.4ml dimethylformamide, 0.2ml
Kaliumphosphate buffer 90mg 1- ethyl -3- (- 3- dimethylaminopropyl) carbodiimide, 15- of ethyl alcohol, 6.5ml 10mM, pH5.0
20mg N- hydroxy thiosuccinimide, in step 2, the stirring and dissolving reacts 30min.
In step 3, the whipping temp is 7 DEG C.
The 5-HIAA specific antibody the preparation method is as follows:
5-HIAA immunogene is diluted with PBS, obtains antigenic solution by step 1, then uses 0.1ml antigen
Solution is mixed with equivalent Freund's complete adjuvant, is injected to experimental animal；
Step 2 after 1 week, then with the identical antigenic solution of 0.1ml and equivalent incomplete Freund's adjuvant is infused above-mentioned experimental animal
It penetrates once, it is primary every injection in 2 weeks later；
Step 3 takes blood to the experimental animal of step 2, isolates and purifies to obtain the anti-5-OHi second that potency is 1:20000
Sour specific antibody.
The 5-HIAA immunogene is diluted to 0.1~3.0mg/ml.
The present invention says the preparation method for the 5-HIAA detection reagent being related to comprising the steps of:
(1) reagent A: by the nicotinamide adenine dinucleotide of 2-3g, 4-9mM oxidation state and the Portugal 1.9g, 14.6mM
Homogeneous zymolyte is made with the Tris buffer solution of 0.5L 30mM, pH=8.0 in grape sugar -6- phosphoric acid；It will be described
Anti- 5-HIAA specific antibody is added in above-mentioned homogeneous zymolyte, anti-5-HIAA specific antibody with
The volume ratio of homogeneous zymolyte is 1:500；
(2) 5-HIAA enzyme mark conjugate reagent B: is added to the Tris of 0.5L 30mM, pH=8.0 buffering
In liquid, the volume ratio of 5-HIAA enzyme mark conjugate and Tris buffer is 1:1000.
Embodiment 7
The present invention relates to a kind of 5-HIAAs to measure reagent, contains 5-HIAA specific antibody and instruction
Reagent；
The 5-HIAA specific antibody is complete to generate after 5-HIAA immunogen immune experimental animal
Whole antibody molecule；
The indicator is selected from radioactive isotope reagent, fluorescent reagent mixes in mass ratio for 1:1；Wherein, institute
The enzymatic reagent stated is made of the substrate of 5-HIAA enzyme mark conjugate and enzyme, and enzyme mark conjugate is glucose -6- phosphorus
Acidohydrogenase-haptens enzyme mark conjugate, the substrate of enzyme are glucose -6- phosphoric acid.
The complete antibody molecule is is obtained using single 5-HIAA immunogene to animal booster immunization
The polyclonal antibody obtained.
The preparation method of the 5-HIAA immunogene, includes the following steps:
Step 1, carrier protein is dissolved in phosphate buffer；
Step 2, by 5- hydroxyindoleacetic acid, dimethylformamide, ethyl alcohol, kaliumphosphate buffer 1- ethyl -3- (- 3- diformazan ammonia
Propyl) carbodiimide, N- hydroxy thiosuccinimide, it is added to stirring and dissolving in small beaker according to certain proportion, in room temperature
Lower stirring and dissolving reaction, obtains solution A；
Step 3, the solution A dissolved is added dropwise in carrier protein solution, is stirred overnight, obtain antigen；Synthetic is resisted
Original is purified by dialysis, obtains 5-HIAA immunogene.
In step 1, the quality of the albumen is 300g, and the phosphate buffer is 75ml 0.2M, pH 8.5.
In step 2, certain proportion is as follows: 50mg 5-HIAA, 0.5ml dimethylformamide, 0.5ml
Kaliumphosphate buffer 50mg 1- ethyl -3- (- 3- dimethylaminopropyl) carbodiimide, 15- of ethyl alcohol, 2.5ml 10mM, pH5.0
20mg N- hydroxy thiosuccinimide, in step 2, the stirring and dissolving reacts 60min.
In step 3, the whipping temp is 2 DEG C.
The 5-HIAA specific antibody the preparation method is as follows:
5-HIAA immunogene is diluted with PBS, obtains antigenic solution by step 1, then uses 0.1ml antigen
Solution is mixed with equivalent Freund's complete adjuvant, is injected to experimental animal；
Step 2 after 1 week, then with the identical antigenic solution of 0.1ml and equivalent incomplete Freund's adjuvant is infused above-mentioned experimental animal
It penetrates once, it is primary every injection in 2 weeks later；
Step 3 takes blood to the experimental animal of step 2, isolates and purifies to obtain the anti-5-OHi second that potency is 1:20000
Sour specific antibody.
The 5-HIAA immunogene is diluted to 3.0mg/ml.
The present invention says the preparation method for the 5-HIAA detection reagent being related to comprising the steps of:
(1) reagent A: by the nicotinamide adenine dinucleotide and 0.6-2.1g, 15.8mM of 2-3g, 4-9mM oxidation state
Homogeneous zymolyte is made with the Tris buffer solution of 0.5L 30mM, pH=8.0 in G-6-P；It will be described
Anti- 5-HIAA specific antibody is added in above-mentioned homogeneous zymolyte, anti-5-HIAA specific antibody with
The volume ratio of homogeneous zymolyte is 1:500；
(2) 5-HIAA enzyme mark conjugate reagent B: is added to the Tris of 0.5L 30mM, pH=8.0 buffering
In liquid, the volume ratio of 5-HIAA enzyme mark conjugate and Tris buffer is 1:1000.
Embodiment 8
The present invention relates to a kind of 5-HIAAs to measure reagent, contains 5-HIAA specific antibody and instruction
Reagent；
The 5-HIAA specific antibody is complete to generate after 5-HIAA immunogen immune experimental animal
Whole antibody molecule；
The indicator is selected from enzymatic reagent, luminescence reagent mixes in mass ratio for 1:1；Wherein, the enzymatic reagent
It is made of the substrate of 5-HIAA enzyme mark conjugate and enzyme, enzyme mark conjugate is glucose -6- phosphate dehydrogenase -
Haptens enzyme mark conjugate, the substrate of enzyme are glucose -6- phosphoric acid.
The complete antibody molecule is is obtained using single 5-HIAA immunogene to animal booster immunization
The polyclonal antibody obtained.
The preparation method of the 5-HIAA immunogene, includes the following steps:
Step 1, carrier protein is dissolved in phosphate buffer；
Step 2, by 5-HIAA, dimethylformamide, ethyl alcohol, kaliumphosphate buffer 1- ethyl -3- (- 3- diformazan ammonia
Propyl) carbodiimide, N- hydroxy thiosuccinimide, it is added to stirring and dissolving in small beaker according to certain proportion, in room temperature
Lower stirring and dissolving reaction, obtains solution A；
Step 3, the solution A dissolved is added dropwise in carrier protein solution, is stirred overnight, obtain antigen；Synthetic is resisted
Original is purified by dialysis, obtains 5-HIAA immunogene.
In step 1, the quality of the albumen is 300g, and the phosphate buffer is 25ml 0.2M, pH 8.5.
In step 2, certain proportion is as follows: 50mg 5-HIAA, 0.5ml dimethylformamide, 1.5ml
Ethyl alcohol, kaliumphosphate buffer 50-100mg 1- ethyl -3- (- 3- dimethylaminopropyl) carbon two of 2.5ml 10mM, pH5.0 are sub-
Amine, 15-20mg N- hydroxy thiosuccinimide, in step 2, the stirring and dissolving reacts 60min.
In step 3, the whipping temp is 8 DEG C.
The 5-HIAA specific antibody the preparation method is as follows:
5-HIAA immunogene is diluted with PBS, obtains antigenic solution by step 1, then uses 0.1ml antigen
Solution is mixed with equivalent Freund's complete adjuvant, is injected to experimental animal；
Step 2 after 1 week, then with the identical antigenic solution of 0.1ml and equivalent incomplete Freund's adjuvant is infused above-mentioned experimental animal
It penetrates once, it is primary every injection in 2 weeks later；
Step 3 takes blood to the experimental animal of step 2, isolates and purifies to obtain the anti-5-OHi second that potency is 1:20000
Sour specific antibody.
The 5-HIAA immunogene is diluted to 3.0mg/ml.
The present invention says the preparation method for the 5-HIAA detection reagent being related to comprising the steps of:
(1) reagent A: by the nicotinamide adenine dinucleotide of 2g, 9mM oxidation state and 2.1g, 15.8mM glucose-
Homogeneous zymolyte is made with the Tris buffer solution of 0.5L 30mM, pH=8.0 in 6- phosphoric acid；By the anti-5- hydroxyl
Base heteroauxin specific antibody is added in above-mentioned homogeneous zymolyte, anti-5-HIAA specific antibody and homogeneous enzyme
The volume ratio of substrate is 1:500；
(2) 5-HIAA enzyme mark conjugate reagent B: is added to the Tris of 0.5L 30mM, pH=8.0 buffering
In liquid, the volume ratio of 5-HIAA enzyme mark conjugate and Tris buffer is 1:1000.
Embodiment 9
The present invention relates to a kind of 5-HIAAs to measure reagent, contains 5-HIAA specific antibody and instruction
Reagent；
The 5-HIAA specific antibody is complete to generate after 5-HIAA immunogen immune experimental animal
Whole antibody molecule；
The indicator is selected from enzymatic reagent, fluorescent reagent mixes in mass ratio for 1:1；Wherein, the enzymatic reagent
It is made of the substrate of 5-HIAA enzyme mark conjugate and enzyme, enzyme mark conjugate is glucose -6- phosphate dehydrogenase -
Haptens enzyme mark conjugate, the substrate of enzyme are glucose -6- phosphoric acid.
Preferably, the complete antibody molecule is to be reinforced using single 5-HIAA immunogene animal
Polyclonal antibody obtained is immunized.
Preferably, the preparation method of the 5-HIAA immunogene, includes the following steps:
Step 1, carrier protein is dissolved in phosphate buffer；
Step 2, by 5-HIAA, dimethylformamide, ethyl alcohol, kaliumphosphate buffer 1- ethyl -3- (- 3- diformazan ammonia
Propyl) carbodiimide, N- hydroxy thiosuccinimide, it is added to stirring and dissolving in small beaker according to certain proportion, in room temperature
Lower stirring and dissolving reaction, obtains solution A；
Step 3, the solution A dissolved is added dropwise in carrier protein solution, is stirred overnight, obtain antigen；Synthetic is resisted
Original is purified by dialysis, obtains 5-HIAA immunogene.
In step 1, the quality of the albumen is 150g, and the phosphate buffer is 45ml 0.2M, pH 8.5.
In step 2, certain proportion is as follows: 75mg 5-HIAA, 1.0ml dimethylformamide, 0.8ml
Ethyl alcohol, 4.5ml 10mM, pH5.0 kaliumphosphate buffer 80mg 1- ethyl -3- (- 3- dimethylaminopropyl) carbodiimide,
15mg N- hydroxy thiosuccinimide, in step 2, the stirring and dissolving reacts 30min.
In step 3, the whipping temp is 8 DEG C.
The 5-HIAA specific antibody the preparation method is as follows:
5-HIAA immunogene is diluted with PBS, obtains antigenic solution by step 1, then uses 0.1ml antigen
Solution is mixed with equivalent Freund's complete adjuvant, is injected to experimental animal；
Step 2 after 1 week, then with the identical antigenic solution of 0.1ml and equivalent incomplete Freund's adjuvant is infused above-mentioned experimental animal
It penetrates once, it is primary every injection in 2 weeks later；
Step 3 takes blood to the experimental animal of step 2, isolates and purifies to obtain the anti-5-OHi second that potency is 1:20000
Sour specific antibody.
The 5-HIAA immunogene is diluted to 3.0mg/ml.
The present invention says the preparation method for the 5-HIAA detection reagent being related to comprising the steps of:
(1) reagent A: by the nicotinamide adenine dinucleotide of 3g, 9mM oxidation state and 2.1g, 3.5mM glucose -6-
Homogeneous zymolyte is made with the Tris buffer solution of 0.5L 30mM, pH=8.0 in phosphoric acid；By the anti-5- hydroxyl
Base heteroauxin specific antibody is added in above-mentioned homogeneous zymolyte, anti-5-HIAA specific antibody and homogeneous enzyme
The volume ratio of substrate is 1:500；
(2) 5-HIAA enzyme mark conjugate reagent B: is added to the Tris of 0.5L 30mM, pH=8.0 buffering
In liquid, the volume ratio of 5-HIAA enzyme mark conjugate and Tris buffer is 1:1000.
Embodiment 10
The present invention relates to a kind of 5-HIAAs to measure reagent, contains 5-HIAA specific antibody and instruction
Reagent；
The 5-HIAA specific antibody is complete to generate after 5-HIAA immunogen immune experimental animal
Whole antibody molecule；
The indicator is selected from enzymatic reagent, luminescence reagent mixes in mass ratio for 1:1；Wherein, the enzymatic reagent
It is made of the substrate of 5-HIAA enzyme mark conjugate and enzyme, enzyme mark conjugate is glucose -6- phosphate dehydrogenase -
Haptens enzyme mark conjugate, the substrate of enzyme are glucose -6- phosphoric acid.
The complete antibody molecule is is obtained using single 5-HIAA immunogene to animal booster immunization
The polyclonal antibody obtained.
The preparation method of the 5-HIAA immunogene, includes the following steps:
Step 1, carrier protein is dissolved in phosphate buffer；
Step 2, by 5-HIAA, dimethylformamide, ethyl alcohol, kaliumphosphate buffer 1- ethyl -3- (- 3- diformazan ammonia
Propyl) carbodiimide, N- hydroxy thiosuccinimide, it is added to stirring and dissolving in small beaker according to certain proportion, in room temperature
Lower stirring and dissolving reaction, obtains solution A；
Step 3, the solution A dissolved is added dropwise in carrier protein solution, is stirred overnight, obtain antigen；Synthetic is resisted
Original is purified by dialysis, obtains 5-HIAA immunogene.
In step 1, the quality of the albumen is 300g, and the phosphate buffer is 75ml 0.2M, pH 8.5.
In step 2, it is described it is certain proportion it is as follows: 100mg 5-HIAA, 1.5ml dimethylformamide,
Kaliumphosphate buffer 100mg 1- ethyl -3- (- 3- dimethylaminopropyl) carbon two of 1.5ml ethyl alcohol, 7.5ml 10mM, pH5.0
Imines, 20mg N- hydroxy thiosuccinimide, in step 2, the stirring and dissolving reacts 60min.
In step 3, the whipping temp is 8 DEG C.
The 5-HIAA specific antibody the preparation method is as follows:
5-HIAA immunogene is diluted with PBS, obtains antigenic solution by step 1, then uses 0.1ml antigen
Solution is mixed with equivalent Freund's complete adjuvant, is injected to experimental animal；
Step 2 after 1 week, then with the identical antigenic solution of 0.1ml and equivalent incomplete Freund's adjuvant is infused above-mentioned experimental animal
It penetrates once, it is primary every injection in 2 weeks later；
Step 3 takes blood to the experimental animal of step 2, isolates and purifies to obtain the anti-5-OHi second that potency is 1:20000
Sour specific antibody.
The 5-HIAA immunogene is diluted to 3.0mg/ml.
The present invention says the preparation method for the 5-HIAA detection reagent being related to comprising the steps of:
(1) reagent A: by the nicotinamide adenine dinucleotide of 3g, 8mM oxidation state and 0.85g, 15.8mM glucose-
Homogeneous zymolyte is made with the Tris buffer solution of 0.5L 30mM, pH=8.0 in 6- phosphoric acid；By the anti-5-
Hydroxyindoleacetic acid specific antibody is added in above-mentioned homogeneous zymolyte, anti-5-HIAA specific antibody and homogeneous
The volume ratio of zymolyte is 1:500；
(2) 5-HIAA enzyme mark conjugate reagent B: is added to the Tris of 0.5L 30mM, pH=8.0 buffering
In liquid, the volume ratio of 5-HIAA enzyme mark conjugate and Tris buffer is 1:1000.
Embodiment 11
The present invention relates to a kind of 5-HIAAs to measure reagent, contains 5-HIAA specific antibody and instruction
Reagent；
The 5-HIAA specific antibody is complete to generate after 5-HIAA immunogen immune experimental animal
Whole antibody molecule；
The indicator is selected from enzymatic reagent；Wherein, the enzymatic reagent is by 5-HIAA enzyme mark conjugate and enzyme
Substrate composition, enzyme mark conjugate is glucose -6- phosphate dehydrogenase-haptens enzyme mark conjugate, and the substrate of enzyme is Portugal
Grape sugar -6- phosphoric acid.
In summary: 5-HIAA immunogens of the invention are strong, immunogenicity is high, the anti-5- prepared
Hydroxyindoleacetic acid specific antibody high specificity, potency are high, and with 62 kinds of common drugs without any cross reaction；
The homogeneous enzyme immunoassay detection reagent that the present invention contains above-mentioned anti-5-HIAA specific antibody can be convenient, quickly, it is quasi-
Really determine the 5-HIAA content in the biological samples such as urine, and can be same on automatic clinical chemistry analyzer
When measure multiple samples, realize the rapid measurement of high throughput of 5-HIAA, accuracy is high, high specificity, accuracy
It is all enhanced before being compared with detection efficiency, while realizing the full-automation of detection process, to testing staff's
It is of less demanding, it is easy to accomplish and promote the use of.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring essence of the invention.
Claims (10)
1. a kind of 5-HIAA measures reagent, which is characterized in that containing 5-HIAA specific antibody and refer to
Show reagent；
The 5-HIAA specific antibody is complete to generate after 5-HIAA immunogen immune experimental animal
Whole antibody molecule；
Any two kinds in enzymatic reagent, radioactive isotope reagent, fluorescent reagent or luminescence reagent of the indicator
It is mixed in mass ratio for 1:1；Wherein, the enzymatic reagent by 5-HIAA enzyme mark conjugate and enzyme substrate group
At enzyme mark conjugate is glucose -6- phosphate dehydrogenase-haptens enzyme mark conjugate, and the substrate of enzyme is glucose -6-
Phosphoric acid.
2. 5-HIAA as described in claim 1 measures reagent, which is characterized in that the complete antibody molecule
To use single 5-HIAA immunogene to animal booster immunization polyclonal antibody obtained.
3. 5-HIAA as described in claim 1 measures reagent, which is characterized in that the 5-HIAA
The preparation method of immunogene, includes the following steps:
Step 1, carrier protein is dissolved in phosphate buffer；
Step 2, by 5-HIAA, dimethylformamide, ethyl alcohol, kaliumphosphate buffer 1- ethyl -3- (- 3- diformazan ammonia
Propyl) carbodiimide, N- hydroxy thiosuccinimide, it is added to stirring and dissolving in small beaker according to certain proportion, in room temperature
Lower stirring and dissolving reaction, obtains solution A；
Step 3, the solution A dissolved is added dropwise in carrier protein solution, is stirred overnight, obtain antigen；Synthetic is resisted
Original is purified by dialysis, obtains 5-HIAA immunogene.
4. 5-HIAA as claimed in claim 3 measures reagent, which is characterized in that in step 1, the albumen
Quality is 100~300g.
5. 5-HIAA as claimed in claim 3 measures reagent, which is characterized in that in step 1, the phosphoric acid is slow
Fliud flushing is 25~75ml 0.2M, pH 8.5.
6. 5-HIAA as claimed in claim 3 measures reagent, which is characterized in that described centainly to match in step 2
Than as follows: 50-100mg 5-HIAA, 0.5-1.5ml dimethylformamide, 0.5-1.5ml ethyl alcohol, 2.5-
Kaliumphosphate buffer 50-100mg 1- ethyl -3- (- 3- dimethylaminopropyl) carbodiimide, 15- of 7.5ml 10mM, pH5.0
20mg N- hydroxy thiosuccinimide.
7. 5-HIAA as claimed in claim 3 measures reagent, which is characterized in that in step 2, the stirring is molten
30~60min of solution reaction.
8. 5-HIAA as claimed in claim 3 measures reagent, which is characterized in that in step 3, the stirring temperature
Degree is 2~8 DEG C.
9. 5-HIAA as described in claim 1 measures reagent, which is characterized in that the 5- hydroxyindoleacetic acid
Specific antibody the preparation method is as follows:
5-HIAA immunogene is diluted with PBS, obtains antigenic solution by step 1, then uses 0.1ml antigen
Solution is mixed with equivalent Freund's complete adjuvant, is injected to experimental animal；
Step 2 after 1 week, then with the identical antigenic solution of 0.1ml and equivalent incomplete Freund's adjuvant is infused above-mentioned experimental animal
It penetrates once, it is primary every injection in 2 weeks later；
Step 3 takes blood to the experimental animal of step 2, isolates and purifies to obtain the anti-5- oxyindole that potency is 1:20000
Acetic acid specific antibody.
10. 5-HIAA as claimed in claim 9 measures reagent, which is characterized in that the 5- oxyindole second
Sour immunogene is diluted to 0.1~3.0mg/ml.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1321466A1 (en) * | 2001-12-20 | 2003-06-25 | Randox Laboratories Ltd. | Haptens, immunogens, antibodies and conjugates for 2-oxo-3-hydroxy LSD |
| CN105131106A (en) * | 2015-07-27 | 2015-12-09 | 苏州博源医疗科技有限公司 | 5-hydroxyindoleacetic acid immunogen, antibody and detection reagent, and preparation methods thereof |
| CN105175531A (en) * | 2015-08-14 | 2015-12-23 | 苏州博源医疗科技有限公司 | Hydroxyproline immunogen, specific antibody and detection reagent, and preparation methods thereof |
-
2019
- 2019-06-27 CN CN201910567947.3A patent/CN110187094A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1321466A1 (en) * | 2001-12-20 | 2003-06-25 | Randox Laboratories Ltd. | Haptens, immunogens, antibodies and conjugates for 2-oxo-3-hydroxy LSD |
| CN105131106A (en) * | 2015-07-27 | 2015-12-09 | 苏州博源医疗科技有限公司 | 5-hydroxyindoleacetic acid immunogen, antibody and detection reagent, and preparation methods thereof |
| CN105175531A (en) * | 2015-08-14 | 2015-12-23 | 苏州博源医疗科技有限公司 | Hydroxyproline immunogen, specific antibody and detection reagent, and preparation methods thereof |
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Application publication date: 20190830 |