CN112014340A - Method for detecting ghrelin of nonhuman primate - Google Patents
Method for detecting ghrelin of nonhuman primate Download PDFInfo
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- 102000012004 Ghrelin Human genes 0.000 title claims abstract description 26
- 101800001586 Ghrelin Proteins 0.000 title claims abstract description 26
- GNKDKYIHGQKHHM-RJKLHVOGSA-N ghrelin Chemical compound C([C@H](NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)CN)COC(=O)CCCCCCC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C1=CC=CC=C1 GNKDKYIHGQKHHM-RJKLHVOGSA-N 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 14
- 241000288906 Primates Species 0.000 title claims description 6
- 210000004369 blood Anatomy 0.000 claims abstract description 21
- 239000008280 blood Substances 0.000 claims abstract description 21
- 238000001514 detection method Methods 0.000 claims abstract description 17
- 239000003112 inhibitor Substances 0.000 claims abstract description 11
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims abstract description 7
- 229910052708 sodium Inorganic materials 0.000 claims abstract description 7
- 239000011734 sodium Substances 0.000 claims abstract description 7
- 238000003118 sandwich ELISA Methods 0.000 claims abstract description 5
- 239000000126 substance Substances 0.000 claims description 16
- 241001465754 Metazoa Species 0.000 claims description 15
- 238000011534 incubation Methods 0.000 claims description 15
- 238000005406 washing Methods 0.000 claims description 13
- 238000002965 ELISA Methods 0.000 claims description 10
- 238000012549 training Methods 0.000 claims description 8
- 238000007789 sealing Methods 0.000 claims description 6
- 238000003908 quality control method Methods 0.000 claims description 5
- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 239000002250 absorbent Substances 0.000 claims description 3
- 230000002745 absorbent Effects 0.000 claims description 3
- 235000021152 breakfast Nutrition 0.000 claims description 3
- 238000011161 development Methods 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 239000003550 marker Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims description 2
- 230000015556 catabolic process Effects 0.000 abstract description 4
- 238000006731 degradation reaction Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 2
- 230000002401 inhibitory effect Effects 0.000 abstract description 2
- 210000002381 plasma Anatomy 0.000 abstract 1
- 210000002784 stomach Anatomy 0.000 abstract 1
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 6
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 4
- 108010039627 Aprotinin Proteins 0.000 description 3
- 241000282567 Macaca fascicularis Species 0.000 description 3
- 229960004405 aprotinin Drugs 0.000 description 3
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 3
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 description 3
- WRDABNWSWOHGMS-UHFFFAOYSA-N AEBSF hydrochloride Chemical compound Cl.NCCC1=CC=C(S(F)(=O)=O)C=C1 WRDABNWSWOHGMS-UHFFFAOYSA-N 0.000 description 2
- 238000012286 ELISA Assay Methods 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- MGSKVZWGBWPBTF-UHFFFAOYSA-N aebsf Chemical compound NCCC1=CC=C(S(F)(=O)=O)C=C1 MGSKVZWGBWPBTF-UHFFFAOYSA-N 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000700 radioactive tracer Substances 0.000 description 2
- 235000010234 sodium benzoate Nutrition 0.000 description 2
- 239000004299 sodium benzoate Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 239000005711 Benzoic acid Substances 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 244000141359 Malus pumila Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 208000031964 Other metabolic disease Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 235000021016 apples Nutrition 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 235000012631 food intake Nutrition 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- YFZOUMNUDGGHIW-UHFFFAOYSA-M p-chloromercuribenzoic acid Chemical compound OC(=O)C1=CC=C([Hg]Cl)C=C1 YFZOUMNUDGGHIW-UHFFFAOYSA-M 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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Abstract
The invention discloses a method for detecting ghrelin in stomach of non-human primate, which comprises the steps of treating collected blood samples within 30s by using sodium p-hydroxy-mercuric benzoate as an inhibitor, centrifuging for 10min at the temperature of more than 3500g and 4 ℃ to obtain blood plasma to be detected, and establishing operation steps according to the double-antibody sandwich ELISA principle. The invention has the advantages and effects that: the method is simple to operate, relatively stable and strong in specificity, and the final result is approved by engineers of kit manufacturers (Bertin Pharma). Since ghrelin is easily degraded, especially acy-ghrelin, and the half-life is 9-13 minutes, the invention obtains a detection method for inhibiting the degradation of ghrelin by determining the inhibitor.
Description
Technical Field
The invention relates to a method for detecting ghrelin of a nonhuman primate.
Background
Ghrelin is an endogenous Ghrelin secreted from the gastrointestinal tract and composed of 28 amino acids, and has various physiological functions including stimulating appetite and increasing food intake.
At present, a method for detecting ghrelin of animals such as mice and sheep is disclosed, but non-human primates are incomparable with other rodent and non-rodent experimental animals due to the fact that the non-human primates have close advantages with humans in terms of phylogeny and medical research. Therefore, the method for detecting ghrelin suitable for non-human primates is explored, and a very precious NHPs experimental basis is provided for the research and development of human obesity, diabetes and other metabolic diseases and new drugs thereof.
Disclosure of Invention
In order to solve the problems, the invention provides a method for detecting the non-human primate gastrohunger, so as to realize accurate, stable and simple detection of the non-human primate gastrohunger.
The invention is realized by the following technical scheme: a method for detecting ghrelin of nonhuman primate comprises the following steps:
(1) the non-human primates after animal training are fasted overnight, and blood samples are collected before breakfast by routine;
(2) treating the blood sample collected in the step (1) by using sodium p-hydroxy-mercuric benzoate as an inhibitor within 30s, namely adding sodium p-hydroxy-mercuric benzoate and 10ul/ml of the blood sample into the blood sample by using a liquid-transferring gun, then reversing the blood-collecting tube for several times, and uniformly mixing;
(3) centrifuging the blood sample treated in the step (2) for 10min at the temperature of more than 3500g and 4 ℃ to obtain plasma to be detected, subpackaging the plasma into a plurality of tubes, and freezing and storing at the temperature of minus 80 ℃ until detection;
(4) establishing operation steps according to a double-antibody sandwich ELISA principle:
a. standing the kit and the plasma to be detected for 30 minutes at room temperature;
b. washing the holes of the enzyme-labeled plate in the kit;
c. sample adding: distributing a standard substance, a quality control substance and a plasma sample to be detected; wherein the standard substance and the quality control substance are carried by the kit, the plasma sample to be detected is the plasma frozen in the step (3), and the blank control hole is a hole without any substance;
d. and (3) incubation: add AChE marker per well except for blank control wells; sealing the plate with a sealing plate film, placing the plate in a shaking table for soft oscillation to uniformly mix the sample, and then placing the sample at room temperature for incubation;
e. washing: washing the ELISA plate for several times by using a plate washing machine, and then patting the ELISA plate on absorbent paper;
f. color development: adding Ellman reagent into each well, placing on a shaker (keeping out of the light), and incubating at room temperature;
g. reading a plate: reading an OD value by using a microplate reader;
(5) and drawing a standard curve according to the OD value of the standard substance and the known concentration, and finally measuring and calculating the concentration of the ghrelin according to the OD value of the plasma sample to be measured.
The room-temperature standing incubation of the step d is carried out at the incubation temperature of 25 ℃ for 3 hours.
The detection principle of the double-antibody sandwich ELISA is as follows:
the detection method is based on a double-antibody sandwich technology. Monoclonal antibody specifically combined with the C end of the Ghrelin is coated in the hole of the ELISA plate. The antibody binds to any Ghrelin (including standards or samples) added to the well. The tracer added to the well recognizes the N-terminus of the Ghrein added to the well. The two antibodies form a sandwich by binding to different sites of Ghrelin. The sandwich is immobilized on an microplate and excess reagent is washed away. The color developer is added and binds to the tracer to form a yellow complex. The concentration of Ghrelin is proportional to the shade of yellow. And reading the absorbance of each hole on a microplate reader, establishing a standard curve according to the absorbance and the concentration of the standard substance, and finally calculating to obtain the concentration of the Ghrelin.
The invention has the advantages and effects that: the method is simple to operate, relatively stable and strong in specificity, and the final result is approved by engineers of kit manufacturers (Bertin Pharma). Since ghrelin is easily degraded, especially acy-ghrelin, and the half-life is 9-13 minutes, the invention obtains a detection method for inhibiting the degradation of ghrelin by determining the inhibitor.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
Acy-ghrelin ELISA assay kit and Unacy-ghrelin ELISA assay kit, procurement manufacturers: BertinPharma, France.
Ghrelin degradation inhibitor: including p-hydroxymercuric benzoate (PHMB), p-hydroxymercuric benzoate (4- (hydroxyymecure) benzoic acid sodium salt, PCMB), Phenylmethylsulfonylfluoride (PMSF), 4- (2-Aminoethyl) benzenesulfonylfluoride hydrochloride (4- (2-Aminoethyl) benzenesulfonylfluoride, AEBSF), aprotinin (aprotinin).
(1) Animal training:
a. selecting 200 male cynomolgus monkeys: 150 obese animals, 50 healthy control animals. The selection criteria are as follows: 1) matching ages; 2) CRL (crown hip length) match; 3) obese animal BMI (weight/crown hip length) > 45 kg/m, healthy control animal BMI < 33 kg/m.
b. And (3) food taking training: feeding granulated feed at 8:00AM per day for 100 g/animal, and removing residual feed at 10:00 AM; 14:00PM is fed to apples, and 100g of the feed is fed to animals; 17:00PM feed pellet, 100g per animal.
c. Training by hand pulling: the animal's hands were gently pulled out of the single cage, fitted with a gentle utterance. The animal's hands are grasped until the animal is quiet. The time from hand grasping to animal quiescence was recorded. Training frequency is 3 times a day, 7:30-8:30 AM; 10:00-11:00 AM; 16:00-17:00 PM.
d. Simulating blood sampling training: in hand-pulled training, the skin around the blood vessel is punctured with an injection needle, but the skin is not really punctured.
(2) Blood sample collection:
after dinner, the cynomolgus monkey trained in the step (1) is fasted all night (18: 00PM-8:00 AM), blood samples are respectively collected before breakfast, the collected blood samples are treated by using sodium p-hydroxy-mercuric benzoate as an inhibitor within 30s, namely, the sodium p-hydroxy-mercuric benzoate is added into the blood samples by using a liquid-transferring gun, the blood samples are 10ul/ml, then the blood-taking tube is reversed for a plurality of times, and the blood-taking tube is evenly mixed;
(3) centrifuging the blood sample treated in the step (2) for 10min at the temperature of more than 3500g and 4 ℃ to obtain plasma to be detected, then respectively subpackaging the plasma into a plurality of tubes, and freezing and storing the tubes in a refrigerator at the temperature of-80 ℃ until detection is carried out (the longest storage time is 30 days);
(4) establishing operation steps according to a double-antibody sandwich ELISA principle:
a. standing the kit (namely Acy-ghrelin ELISA detection kit and Unacy-ghrelin ELISA detection kit) and the plasma to be detected for 30 minutes at room temperature;
b. washing the holes of the enzyme-labeled plate in the kit;
c. sample adding: distributing a standard substance, a quality control substance and a plasma sample to be detected; wherein the standard substance and the quality control substance are carried by the kit, the plasma sample to be detected is the plasma frozen in the step (3), and the blank control hole is a hole without any substance;
d. and (3) incubation: add AChE marker per well except for blank control wells; sealing the plate with a sealing plate membrane, placing the plate in a shaking table to gently oscillate so as to uniformly mix the sample, and then placing the sample in a room temperature for standing and incubating (namely, the incubation temperature is 25 ℃, and the incubation time is 3 hours);
e. washing: washing the ELISA plate for several times by using a plate washing machine, and then patting the ELISA plate on absorbent paper;
f. color development: adding Ellman reagent into each well, placing on a shaking table (keeping out of the light), and incubating at room temperature (namely, the incubation temperature is 25 ℃, and the incubation time is 3 hours);
g. reading a plate: OD values were read using a microplate reader. And the observed OD value is in direct proportion to the concentration of the sample, and finally, the standard curve is established repeatedly for a plurality of times, the repeatability is good, and the preliminary establishment of the detection step is confirmed.
And (3) verification:
1. selection of fitting curves
The OD value is in proportional relation with the concentration of the sample, and a standard curve is drawn according to the OD value of the standard substance and the known concentration. And finally, measuring specific concentration according to the OD value of the sample.
And fitting by respectively adopting a linear curve and a nonlinear curve, and comparing the final results to find that the fitting degree of the four-parameter logic fitting curve is highest. And selecting a logic four-parameter fitting curve for fitting.
2. Inhibitor screening
Experiments are designed, blood samples treated by different inhibitors are respectively used for detection, and the following inhibitors are respectively used in 30s after blood collection: and (3) treating the blood sample by using p-hydrargyrum benzoic acid, p-hydrargyrum sodium benzoate, phenylmethylsulfonyl fluoride, 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride and aprotinin, and finally selecting the p-hydrargyrum sodium benzoate as a Ghrelin degradation inhibitor according to the comparison result.
3. Optimization of detection conditions and flow
Aiming at factors which are easy to influence the detection result, such as detection environment temperature, incubation time, sample adding operation, plate washing mode and times, sample dilution times, sample preservation time and the like, corresponding tests are respectively designed to find out the optimal conditions and procedures of the experiment.
Finally, the optimal environment temperature is 25 ℃, the incubation time is 3 hours, the dilution time of the sample is 5 or 10 times (the sample is diluted by 5 or 10 times before the sample is added to prevent the sample concentration from being too large and influence the accuracy), and the storage time of the sample is one month (-80 ℃).
4. Stability of the assay
3 samples were taken and assayed separately in the same kit to determine intra-batch differences and in 2 different kits to determine inter-batch differences. Intra-and inter-batch differences are used to illustrate the stability and reproducibility of the assay. The Acy-ghrelin and Unacy-ghrelin had both an inter-batch coefficient of variation of 9.8% and intra-batch coefficients of variation of 6.3% and 6.2%, respectively.
5. Establishment and application of nonhuman primate (cynomolgus monkey) plasma Ghrelin ELISA detection method
Through the verification, the establishment of the Ghrelin detection method is successful.
The method is simple to operate, relatively stable and strong in specificity.
Claims (2)
1. A method for detecting ghrelin in nonhuman primates is characterized by comprising the following steps:
(1) the non-human primates after animal training are fasted overnight, and blood samples are collected before breakfast by routine;
(2) treating the blood sample collected in the step (1) by using sodium p-hydroxy-mercuric benzoate as an inhibitor within 30s, namely adding sodium p-hydroxy-mercuric benzoate and 10ul/ml of the blood sample into the blood sample, then reversing the blood collecting tube for several times, and uniformly mixing;
(3) centrifuging the blood sample treated in the step (2) for 10min at the temperature of more than 3500g and 4 ℃ to obtain plasma to be detected, subpackaging the plasma into a plurality of tubes, and freezing and storing at the temperature of minus 80 ℃ until detection;
(4) establishing operation steps according to a double-antibody sandwich ELISA principle:
a. standing the kit and the plasma to be detected for 30 minutes at room temperature;
b. washing the holes of the enzyme-labeled plate in the kit;
c. sample adding: distributing a standard substance, a quality control substance and a plasma sample to be detected;
d. and (3) incubation: add AChE marker per well except for blank control wells; sealing the plate with a sealing plate film, placing the plate in a shaking table for soft oscillation to uniformly mix the sample, and then placing the sample at room temperature for incubation;
e. washing: washing the ELISA plate for several times by using a plate washing machine, and then patting the ELISA plate on absorbent paper;
f. color development: adding Ellman reagent into each well, placing on a shaker (keeping out of the light), and incubating at room temperature;
g. reading a plate: reading an OD value by using a microplate reader;
(5) and drawing a standard curve according to the OD value of the standard substance and the known concentration, and finally measuring and calculating the concentration of the ghrelin according to the OD value of the plasma sample to be measured.
2. The method for detecting ghrelin in a non-human primate according to claim 1, wherein: the room-temperature standing incubation of the step d is carried out at the incubation temperature of 25 ℃ for 3 hours.
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