CN104914098A - Method for detecting content of substance to be detected in animal manure ethanol extract - Google Patents
Method for detecting content of substance to be detected in animal manure ethanol extract Download PDFInfo
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Abstract
The invention discloses a method for detecting content of a substance to be detected in animal manure ethanol extract. The method comprises 1, adjusting a concentration of ethanol in animal manure ethanol extract to obtain animal manure ethanol extract to be detected, wherein the animal manure ethanol extract to be detected has an ethanol concentration of greater than 0 and smaller than 7% by volume and 2, detecting content of the substance to be detected in the animal manure ethanol extract by an euzymelinked immunosorbent assay method to obtain content of the substance to be detected in the animal manure ethanol extract. An experiment proves that after the ethanol volume percentage concentration of the animal manure ethanol extract is adjusted to less than 7%, the method can detect content of testosterone and other substances to be detected.
Description
Technical field
The present invention relates in biological technical field the method detecting determinand content in animal wastes ethanol extract.
Background technology
The researchist of current animal protection more and more starts the sampling method adopting non-damage; namely when not touching, to catch or even when personally not seeing animal; collect the sample that animal is multi-form; such as ight soil, urine, hair, the feather that comes off, swill, scale and chorion etc. containing mouth desquamated cells, and then abstraction and quantification is carried out to relevant physiological metabolic product in collected specimens.Widely used is at present gather the ight soil of goal in research species, and to relevant physiological metabolic product in ight soil by alcohol extract and then utilize euzymelinked immunosorbent assay (ELISA) (Enzyme-Linked Immunosorbent Assays, ELISA) to carry out quantitative measurement.But the ethanol of high concentration can impact the measurement result of ELISA.
Summary of the invention
Technical matters to be solved by this invention is the content how utilizing euzymelinked immunosorbent assay (ELISA) to detect determinand in animal wastes ethanol extract.
For solving the problems of the technologies described above, the present invention provide firstly the method detecting determinand content in animal wastes ethanol extract.
In detection animal wastes ethanol extract provided by the present invention, the method for determinand content, comprises following 1) and 2):
1) regulate the concentration of ethanol in described ight soil ethanol extract, obtain ight soil ethanol extract to be measured, in described ight soil ethanol extract to be measured, the concentration of volume percent of ethanol is greater than 0 and is less than 7%;
2) utilize euzymelinked immunosorbent assay (ELISA) (Enzyme-Linked Immunosorbent Assays, ELISA) to detect the content of determinand described in described ight soil ethanol extract to be measured, obtain the content of determinand described in described ight soil ethanol extract.
In said method, described animal can be woods musk deer (Moschusberezovskii).
In said method, in described ight soil ethanol extract to be measured, the concentration of volume percent of ethanol can be greater than 0 and be less than or equal to 6%, as 5% or 4%.
In said method, in described ight soil ethanol extract to be measured, the concentration of volume percent of ethanol can be greater than 0 and be less than or equal to 5%, as 4%.
In said method, in described ight soil ethanol extract to be measured, the concentration of volume percent of ethanol can be greater than 0 and be less than or equal to 4%.
In said method, described ight soil ethanol extract can be the leaching liquor obtained with animal wastes described in ethanol water lixiviate.
In said method, in described ethanol water, the concentration of volume percent of ethanol can be 90%.
In said method, described animal is buck; Described determinand can be testosterone.
In said method, described in described ight soil ethanol extract, the mensuration of testosterone concentration utilizes testosterone quantitative determination reagent kit to carry out.
Described testosterone quantitative determination reagent kit (euzymelinked immunosorbent assay (ELISA)) is Beijing North Institute of Biological Technology's product.
Experiment proves, the method of determinand content in detection animal wastes ethanol extract of the present invention is utilized to measure the content of testosterone in male woods musk deer ight soil ethanol extract, in male woods musk deer ight soil ethanol extract, ethanol contend percent concentration is greater than 0 when being less than 7%, and impact enzyme linked immunoassay being measured to testosterone concentration is less.Experiment proves, ethanol contend percent concentration in animal wastes ethanol extract can be adjusted to the content utilizing the method for determinand content in detection animal wastes ethanol extract of the present invention to measure testosterone and other determinands after being less than 7 ﹪.
Accompanying drawing explanation
Fig. 1 is the measured value of testosterone concentration in the male woods musk deer ight soil ethanol extract obtained according to difference ight soil to be measured ethanol extract.Wherein, ethanol percentage concentration represents ethanol contend percent concentration.
Fig. 2 is the measurement result of the testosterone concentration in the S3 liquid to be measured of different ethanol contend percent concentration.
Fig. 3 is the measurement result of the testosterone concentration in the S4 liquid to be measured of different ethanol contend percent concentration.
Fig. 4 is the measurement result of the testosterone concentration in the S5 liquid to be measured of different ethanol contend percent concentration.
Fig. 5 is the measurement result of the testosterone concentration in the S3 ' liquid to be measured of different ethanol contend percent concentration.
Fig. 6 is the measurement result of the testosterone concentration in the S4 ' liquid to be measured of different ethanol contend percent concentration.
Fig. 7 is the measurement result of the testosterone concentration in the S5 ' liquid to be measured of different ethanol contend percent concentration.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.
Experimental technique in following embodiment, if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Detect the method for determinand content in animal wastes ethanol extract, comprise following 1) and 2):
1) regulate the concentration of ethanol in described ight soil ethanol extract, obtain ight soil ethanol extract to be measured, in described ight soil ethanol extract to be measured, the concentration of volume percent of ethanol is less than 7%;
2) utilize euzymelinked immunosorbent assay (ELISA) to detect the content of determinand described in described ight soil ethanol extract to be measured, obtain the content of determinand in described ight soil ethanol extract.
Below for male woods musk deer, illustrate the assay method of testosterone concentration in animal wastes ethanol extract of the present invention.
The mensuration of testosterone concentration in embodiment 1, male woods musk deer ight soil ethanol extract
1, the preparation of male woods musk deer ight soil ethanol extract
Choose the male woods musk deer of Baoji Pien Tze Huang Lin She plant, its fresh excreta is pulverized 10min mixing in comminutor (23000rpm), obtains male woods musk deer ight soil.
Take above-mentioned male woods musk deer ight soil 0.5g in 10ml plastic centrifuge tube, add the ethanol water that 5ml ethanol contend percent concentration is 90 ﹪ in this centrifuge tube, after vortex instrument vibration 20min, at 2500rpm, (centrifugal force is 4360g; Hydro-extractor is the low speed autobalancing centrifuge of Beijing Medical Centrifugal Machine Factory, and model is LDZ 5-2) under centrifugal 15min, collect supernatant, be male woods musk deer ight soil ethanol extract.
2, the mensuration of testosterone concentration in male woods musk deer ight soil ethanol extract
By the following method, testosterone concentration in the male woods musk deer ight soil ethanol extract of determination step 1:
Get the male woods musk deer ight soil ethanol extract of 5mL step 1, (the concrete configuration method of PBS damping fluid is: take 8.5g NaCl, 2.2g Na to use sample buffer (PBS damping fluid) respectively
2hPO
4with 0.3g NaH
2pO
4be dissolved in 800ml high purity water, add 0.5ml tween, be settled to 1L) carry out gradient dilution, obtain the ight soil ethanol extract to be measured that ethanol contend percent concentration is 4 ﹪, 5 ﹪, 6 ﹪, 7 ﹪, 8 ﹪, 9 ﹪, 10 ﹪ or 15 ﹪ respectively.
Setting does not contain the ight soil ethanol extract to be measured (namely ethanol contend percent concentration is the ight soil ethanol extract to be measured of 0) of ethanol in contrast, preparation method not containing the ight soil ethanol extract to be measured of ethanol is: the male woods musk deer ight soil ethanol extract evaporate to dryness getting 5mL step 1, obtain steaming thyraden, carry out ultrasonic vibration extraction with ultrasonoscope (the desk-top instrument of KQ3200E that Kunshan Ultrasonic Instruments Co., Ltd. produces) add 5ml PBS damping fluid in steaming thyraden after, obtain not containing the ight soil ethanol extract to be measured of ethanol.
Euzymelinked immunosorbent assay (ELISA) is adopted to measure the content of testosterone in above-mentioned each ight soil ethanol extract to be measured.Enzyme micro-plate reader is ThermoMultiscan FC type, adopt testosterone quantitative determination reagent kit (euzymelinked immunosorbent assay (ELISA)) (this kit is Beijing North Institute of Biological Technology's product), this kit principal ingredient in table 1 and table 2, the important technological parameters of testosterone quantitative determination reagent kit (euzymelinked immunosorbent assay (ELISA)) is as follows:
A. sensitivity :≤0.05ng/ml
B. precision: coefficient of variation CV<15 ﹪
C. specificity: do not have cross reaction with progesterone, male glycol, DHT
The principal ingredient of table 1, testosterone quantitative determination reagent kit (euzymelinked immunosorbent assay (ELISA))
| Component/specification | 96T | Principal ingredient |
| Pre-coated plate | 12 holes/bar × 8 | Principal ingredient is for bag is by goat-anti rabbit |
| Enzyme-labelled antigen | 6ml × 1 bottle | Principal ingredient is that HRP marks T |
| Antibody | 6ml × 1 bottle | Principal ingredient is the anti-T of rabbit |
| Calibration object is a set of | 0.5ml/ bottle × 6 bottle | Principal ingredient is T, removes hormone serum |
| Developer A | 7ml × 1 bottle | Principal ingredient is urea peroxide element |
| Developer B | 7ml × 1 bottle | Principal ingredient is TMB |
| Stop buffer | 7ml × 1 bottle | Principal ingredient is dilute sulfuric acid |
| Concentrated cleaning solution | 15ml × 1 bottle | Principal ingredient is NaCl, phosphate, Tween-20 |
| Instructions | 1 part | —— |
| Viscose board | 2 | —— |
| Log-Logit coordinate paper | 1 part | —— |
| Sealing bag, drying agent | 1 cover | —— |
The concentration of table 2, testosterone quantitative determination reagent kit (euzymelinked immunosorbent assay (ELISA)) alignment product
| Unit | S0 | S1 | S2 | S3 | S4 | S5 |
| ng/ml | 0 | 0.1 | 0.4 | 1.6 | 5 | 20 |
Quality-control product concentration in testosterone quantitative determination reagent kit (euzymelinked immunosorbent assay (ELISA)): low value (L) 0.22-0.58ng/ml; High level (H): 1.75-4.13ng/ml.
In triplicate, the concrete operations at every turn repeating to test are as follows, and the reagent in following step is reagent in testosterone quantitative determination reagent kit (euzymelinked immunosorbent assay (ELISA)) in experiment:
A, the various reagent in following step are moved to room temperature (18-25 DEG C) balance half an hour; Get concentrated cleaning solution, with distilled water diluting 20 times, after mixing, obtain the cleansing solution diluted.
B, taken out from sealing bag by pre-wrapper sheet, each calibration point and Quality Control once respectively establish holes, and every hole adds respective alignment product and each 50 μ l of Quality Control; All the other each detect aperture directly add a kind of ight soil ethanol extract 50 μ l to be measured.If a blank control wells (this hole does not add any liquid).
C, every hole add enzyme-labelled antigen 50 μ L (blank control wells does not add enzyme-labelled antigen), then add antibody 50 μ L to every hole.Abundant mixing, sticks adhesive sticker mounting, puts 37 DEG C of incubations 1 hour.
Plate is washed in D, craft: discard liquid in hole, fill each hole with the cleansing solution of the dilution of steps A, leaves standstill 10 seconds and dries, pat dry after repeating 3 times.Wash trigger and wash plate: pat dry after selecting washing 3 secondary program.
E, every hole add developer A 50 μ L, developer B 50 μ L, and after vibration mixing, put 37 DEG C of lucifuges and develop the color 15 minutes, every hole adds stop buffer 50 μ L.
F, use microplate reader reading, measure each hole OD value.
G, data processing
Typical curve Y=BX+A is made by Logit-log straight-line regression according to the concentration of calibration object and the relation of OD value, A is typical curve intercept, B is slope of standard curve, after the light absorption value that ight soil ethanol extract to be measured obtains subtracts background, bring in typical curve the content X obtaining testosterone in ight soil ethanol extract to be measured into as Y.
The measured value (table 3 and Fig. 1) of testosterone concentration in the male woods musk deer ight soil ethanol extract of step 1 is obtained according to the cubage of testosterone in ight soil ethanol extract to be measured.
Table 3, obtain step 1 according to difference ight soil to be measured ethanol extract male woods musk deer ight soil ethanol extract in the measured value of testosterone concentration
Note: in table, data are the result of 3 revision tests.
In the male woods musk deer ight soil ethanol extract using SPSS16.0 software to obtain difference ight soil to be measured ethanol extract, the measured value of testosterone concentration carries out one-way analysis of variance (one way-ANOVA) and Multiple range test, and level of significance is set to P=0.05; Result is as shown in table 4 and table 5.
Table 4, one-way analysis of variance result
| Quadratic sum | df | All square | F | Conspicuousness | |
| Between group | 6181.453 | 8 | 772.682 | 14.956 | 0.00 |
| In group | 929.935 | 18 | 51.663 | ||
| Sum | 7111.388 | 26 |
Table 5, different ethanol concentration and concentration of alcohol are the significance of difference analysis in the ight soil ethanol extract to be measured of 0 between testosterone concentration measured value
Result shows, and when ethanol contend percent concentration is more than or equal to 7 ﹪, fluctuation appears in the testosterone concentration measured value of ight soil ethanol extract to be measured, and measured value significant difference when being 0% with concentration of alcohol.Show when ethanol contend percent concentration in ight soil ethanol extract is more than or equal to 7 ﹪ larger on the impact of enzyme linked immunoassay mensuration testosterone concentration, less on the impact of enzyme linked immunoassay mensuration testosterone concentration when ethanol contend percent concentration in ight soil ethanol extract is less than 7 ﹪, ethanol contend percent concentration in ight soil ethanol extract can be adjusted to the content measuring testosterone and other indexs in ight soil ethanol extract after being less than 7 ﹪ by euzymelinked immunosorbent assay (ELISA).
In embodiment 2, ight soil ethanol extract, ethanol contend percent concentration measures the checking of testosterone concentration impact on enzyme linked immunoassay
In triplicate, the concrete steps at every turn repeating to test are as follows in experiment:
(1) different ethanol contend percent concentration gradient measures testosterone concentration impact to enzyme linked immunoassay in a big way
Standard items S3, S4 and S5 of embodiment 1 are diluted according to the volume of each liquid of table 6 respectively, obtains S3, S4 and S5 liquid to be measured that ethanol contend percent concentration is respectively 0 ﹪, 2 ﹪, 5 ﹪, 7 ﹪, 10 ﹪, 15 ﹪, 20 ﹪, 30 ﹪ and 50 ﹪ respectively.
The volume of each liquid in S3, S4 and S5 liquid to be measured of table 6, different ethanol contend percent concentration
| 0﹪ | 2﹪ | 5﹪ | 7﹪ | 10﹪ | 15﹪ | 20﹪ | 30﹪ | 50﹪ | |
| PBS damping fluid (μ l) | 25 | 24 | 22.5 | 21.5 | 20 | 17.5 | 15 | 10 | 0 |
| Ethanol (μ l) | 0 | 1 | 2.5 | 3.5 | 5 | 7.5 | 10 | 15 | 25 |
| Standard items (μ l) | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 |
Measure the content of testosterone in above-mentioned each S3 liquid to be measured according to the euzymelinked immunosorbent assay (ELISA) of embodiment 1 step 2, result is as shown in table 7 and Fig. 2.Using SPSS16.0 software to carry out one-way analysis of variance (one way-ANOVA) and different ethanol concentration and concentration of alcohol to the content of testosterone in each S3 liquid to be measured is significance of difference analysis in the S3 liquid to be measured of 0 between testosterone concentration measured value, and level of significance is set to P=0.05; Result is as shown in table 8 and table 9.
The measured value of the testosterone concentration in the S3 liquid to be measured of table 7, different ethanol contend percent concentration
| Ethanol contend percent concentration | Liquid to be measured | Testosterone concentration measured value (ng/ml) |
| 0﹪ | S3 | 0.39223 |
| 2﹪ | S3 | 0.48138 |
| 5﹪ | S3 | 0.52605 |
| 7﹪ | S3 | 0.52775 |
| 10﹪ | S3 | 0.69529 |
| 15﹪ | S3 | 0.74368 |
| 20﹪ | S3 | 0.51491 |
| 30﹪ | S3 | 0.83124 |
| 50﹪ | S3 | 2.28130 |
Table 8, one-way analysis of variance
| Quadratic sum | df | All square | F | Conspicuousness | |
| Between group | 8.105 | 8 | 1.013 | 91.021 | 0.000 |
| In group | 0.200 | 18 | 0.011 | ||
| Sum | 8.305 | 26 |
Table 9, different ethanol concentration and concentration of alcohol are the significance of difference analysis in the S3 liquid to be measured of 0 between testosterone concentration measured value
| Ethanol percentage concentration (﹪) in liquid to be measured | P value |
| 2 | 0.314 |
| 5 | 0.138 |
| 7 | 0.133 |
| 10 | 0.002 |
| 15 | 0.001 |
| 20 | 0.171 |
| 30 | 0.000 |
| 50 | 0.000 |
Be that the measured value of the testosterone concentration in the S3 liquid to be measured of 0% is as standard control value using ethanol contend percent concentration, when ethanol contend percent concentration is more than or equal to 10 ﹪, there is larger fluctuation, measured value significant difference when measured value and ethanol contend percent concentration are 0% in the measured value of testosterone concentration.When ethanol contend percent concentration is 30 ﹪ and 50 ﹪, the measured value of testosterone concentration and the difference of standard value extremely remarkable.
Measure the content of testosterone in above-mentioned each S4 liquid to be measured according to the euzymelinked immunosorbent assay (ELISA) of embodiment 1 step 2, result is as shown in table 10 and Fig. 3.Using SPSS16.0 software to carry out one-way analysis of variance (one way-ANOVA) and different ethanol concentration and concentration of alcohol to the content of testosterone in each S4 liquid to be measured is significance of difference analysis in the S4 liquid to be measured of 0 between testosterone concentration measured value, and level of significance is set to P=0.05; Result is as shown in table 11 and table 12.
The measured value of the testosterone concentration in the S4 liquid to be measured of table 10, different ethanol contend percent concentration
| Ethanol contend percent concentration | Liquid to be measured | Testosterone concentration measured value (ng/ml) |
| 0﹪ | S4 | 1.11949 |
| 2﹪ | S4 | 1.27530 |
| 5﹪ | S4 | 1.18134 |
| 7﹪ | S4 | 1.25354 |
| 10﹪ | S4 | 1.01247 |
| 15﹪ | S4 | 1.52432 |
| 20﹪ | S4 | 1.14886 |
| 30﹪ | S4 | 1.24397 |
| 50﹪ | S4 | 3.18370 |
Table 11, one-way analysis of variance
| Quadratic sum | df | All square | F | Conspicuousness | |
| Between group | 10.76 | 8 | 1.345 | 96.554 | 0.000 |
| In group | 0.251 | 18 | 0.014 | ||
| Sum | 11.010 | 26 |
Table 12, different ethanol concentration and concentration of alcohol are the significance of difference analysis in the S4 liquid to be measured of 0 between testosterone concentration measured value
| Ethanol percentage concentration (﹪) in liquid to be measured | P value |
| 2 | 0.118 |
| 5 | 0.514 |
| 7 | 0.174 |
| 10 | 0.291 |
| 15 | 0.001 |
| 20 | 0.747 |
| 30 | 0.205 |
| 50 | 0.000 |
Be that the measured value of the testosterone concentration in the S4 liquid to be measured of 0% is as standard control value using ethanol contend percent concentration, when ethanol contend percent concentration is more than or equal to 15 ﹪, there is larger fluctuation, measured value significant difference when measured value and ethanol contend percent concentration are 0% in the measured value of testosterone concentration.When ethanol contend percent concentration is 50 ﹪, the measured value of testosterone concentration and the difference of standard value extremely remarkable.
Measure the content of testosterone in above-mentioned each S5 liquid to be measured according to the euzymelinked immunosorbent assay (ELISA) of embodiment 1 step 2, result is as shown in table 13 and Fig. 4.Using SPSS16.0 software to carry out one-way analysis of variance (one way-ANOVA) and different ethanol concentration and concentration of alcohol to the content of testosterone in each S5 liquid to be measured is significance of difference analysis in the S5 liquid to be measured of 0 between testosterone concentration measured value, and level of significance is set to P=0.05; Result is as shown in table 14 and table 15.
The measured value of the testosterone concentration in the S5 liquid to be measured of table 13, different ethanol contend percent concentration
| Ethanol contend percent concentration | Liquid to be measured | Testosterone concentration measured value (ng/ml) |
| 0﹪ | S5 | 5.16822 |
| 2﹪ | S5 | 4.90856 |
| 5﹪ | S5 | 5.27500 |
| 7﹪ | S5 | 4.52727 |
| 10﹪ | S5 | 5.03952 |
| 15﹪ | S5 | 4.49151 |
| 20﹪ | S5 | 3.73038 |
| 30﹪ | S5 | 4.31018 |
| 50﹪ | S5 | 6.75286 |
Table 14, one-way analysis of variance
| Quadratic sum | df | All square | F | Conspicuousness | |
| Between group | 17.057 | 8 | 2.132 | 44.463 | 0.000 |
| In group | 0.863 | 18 | 0.048 | ||
| Sum | 17.92 | 26 |
Table 15, different ethanol concentration and concentration of alcohol are the significance of difference analysis in the S5 liquid to be measured of 0 between testosterone concentration measured value
| Ethanol percentage concentration (﹪) in liquid to be measured | P value |
| 2 | 0.164 |
| 5 | 0.558 |
| 7 | 0.002 |
| 10 | 0.481 |
| 15 | 0.001 |
| 20 | 0.000 |
| 30 | 0.000 |
| 50 | 0.000 |
Be that the measured value of the testosterone concentration in the S3 liquid to be measured of 0% is as standard control value using ethanol contend percent concentration, when ethanol contend percent concentration is more than or equal to 7 ﹪, there is larger fluctuation, measured value significant difference when measured value and ethanol contend percent concentration are 0% in the measured value of testosterone concentration.When ethanol contend percent concentration is 20 ﹪, 30 ﹪ and 50 ﹪, the measured value of testosterone concentration and the difference of standard value extremely remarkable.
(2) more different ethanol contend percent concentration gradient measures testosterone concentration impact to enzyme linked immunoassay
Standard items S3, S4 and S5 of embodiment 1 are diluted according to the volume of each liquid of table 16 respectively, obtains S3 ', S4 ' and S5 ' liquid to be measured that ethanol contend percent concentration is respectively 0 ﹪, 5 ﹪, 6 ﹪, 7 ﹪, 8 ﹪, 9 ﹪, 10 ﹪, 15 ﹪ and 20 ﹪ respectively.
The volume of each liquid in S3 ', the S4 ' of table 16, different ethanol contend percent concentration and S5 ' liquid to be measured
| 0﹪ | 5﹪ | 6﹪ | 7﹪ | 8％ | 9％ | 10﹪ | 15﹪ | 20﹪ | |
| PBS damping fluid (μ l) | 25 | 22.5 | 22 | 21.5 | 21 | 20.5 | 20 | 17.5 | 15 |
| Ethanol (μ l) | 0 | 2.5 | 3 | 3.5 | 4 | 4.5 | 5 | 7.5 | 10 |
| Standard items (μ l) | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 |
Measure the content of testosterone in above-mentioned each S3 ' liquid to be measured according to the euzymelinked immunosorbent assay (ELISA) of embodiment 1 step 2, result is as shown in table 17 and Fig. 5.Using SPSS16.0 software to carry out one-way analysis of variance (one way-ANOVA) and different ethanol concentration and concentration of alcohol to the content of testosterone in each S3 ' liquid to be measured is significance of difference analysis in the S3 ' liquid to be measured of 0 between testosterone concentration measured value, and level of significance is set to P=0.05; Result is as shown in table 18 and table 19.
The measured value of the testosterone concentration in the S3 ' liquid to be measured of table 17, different ethanol contend percent concentration
| Ethanol contend percent concentration | Liquid to be measured | Testosterone concentration measured value (ng/ml) |
| 0﹪ | S3′ | 0.40104 |
| 5﹪ | S3′ | 0.54579 |
| 6﹪ | S3′ | 0.55023 |
| 7﹪ | S3′ | 0.54640 |
| 8﹪ | S3′ | 0.56917 |
| 9﹪ | S3′ | 0.61099 |
| 10﹪ | S3′ | 0.66091 |
| 15﹪ | S3′ | 0.61869 |
| 20﹪ | S3′ | 0.39075 |
Table 18, one-way analysis of variance
| Quadratic sum | df | All square | F | Conspicuousness | |
| Between group | 0.205 | 8 | 0.026 | 2.633 | 0.042 |
| In group | 0.175 | 18 | 0.01 | ||
| Sum | 0.38 | 26 |
Table 19, different ethanol concentration and concentration of alcohol are the significance of difference analysis in the S3 ' liquid to be measured of 0 between testosterone concentration measured value
| Ethanol percentage concentration (﹪) in liquid to be measured | P value |
| 5 | 0.089 |
| 6 | 0.080 |
| 7 | 0.088 |
| 8 | 0.051 |
| 9 | 0.018 |
| 10 | 0.005 |
| 15 | 0.015 |
| 20 | 0.900 |
Be that the measured value of the testosterone concentration in the S3 ' liquid to be measured of 0% is as standard control value using ethanol contend percent concentration, when ethanol contend percent concentration is more than or equal to 9 ﹪, there is larger fluctuation, measured value significant difference when measured value and ethanol contend percent concentration are 0% in the measured value of testosterone concentration.
Measure the content of testosterone in above-mentioned each S4 ' liquid to be measured according to the euzymelinked immunosorbent assay (ELISA) of embodiment 1 step 2, result is as shown in table 20 and Fig. 6.Using SPSS16.0 software to carry out one-way analysis of variance (one way-ANOVA) and different ethanol concentration and concentration of alcohol to the content of testosterone in each S4 ' liquid to be measured is significance of difference analysis in the S4 ' liquid to be measured of 0 between testosterone concentration measured value, and level of significance is set to P=0.05; Result is as shown in table 21 and table 22.
The measured value of the testosterone concentration in the S4 ' liquid to be measured of table 20, different ethanol contend percent concentration
| Ethanol contend percent concentration | Liquid to be measured | Testosterone concentration measured value (ng/ml) |
| 0﹪ | S4′ | 1.05665 |
| 5﹪ | S4′ | 1.15034 |
| 6﹪ | S4′ | 1.20404 |
| 7﹪ | S4′ | 1.30917 |
| 8﹪ | S4′ | 1.39387 |
| 9﹪ | S4′ | 1.64013 |
| 10﹪ | S4′ | 1.40464 |
| 15﹪ | S4′ | 1.52432 |
| 20﹪ | S4′ | 1.18486 |
Table 21, one-way analysis of variance
| Quadratic sum | df | All square | F | Conspicuousness | |
| Between group | 0.860 | 8 | 0.108 | 5.015 | 0.002 |
| In group | 0.386 | 18 | 0.021 | ||
| Sum | 1.246 | 26 |
Table 22, different ethanol concentration and concentration of alcohol are the significance of difference analysis in the S4 ' liquid to be measured of 0 between testosterone concentration measured value
| Ethanol percentage concentration (﹪) in liquid to be measured | P value |
| 5 | 0.443 |
| 6 | 0.234 |
| 7 | 0.049 |
| 8 | 0.011 |
| 9 | 0.000 |
| 10 | 0.009 |
| 15 | 0.001 |
| 20 | 0.298 |
Be that the measured value of the testosterone concentration in the S4 ' liquid to be measured of 0% is as standard control value using ethanol contend percent concentration, when ethanol contend percent concentration is more than or equal to 7 ﹪, there is larger fluctuation, measured value significant difference when measured value and ethanol contend percent concentration are 0% in the measured value of testosterone concentration.
Measure the content of testosterone in above-mentioned each S5 ' liquid to be measured according to the euzymelinked immunosorbent assay (ELISA) of embodiment 1 step 2, result is as shown in table 23 and Fig. 7.Using SPSS16.0 software to carry out one-way analysis of variance (one way-ANOVA) and different ethanol concentration and concentration of alcohol to the content of testosterone in each S5 ' liquid to be measured is significance of difference analysis in the S5 ' liquid to be measured of 0 between testosterone concentration measured value, and level of significance is set to P=0.05; Result is as shown in table 24 and table 25.
The measured value of the testosterone concentration in the S5 ' liquid to be measured of table 23, different ethanol contend percent concentration
| Ethanol contend percent concentration | Liquid to be measured | Testosterone concentration measured value (ng/ml) |
| 0﹪ | S5′ | 5.55619 |
| 5﹪ | S5′ | 5.27560 |
| 6﹪ | S5′ | 5.08414 |
| 7﹪ | S5′ | 4.57532 |
| 8﹪ | S5′ | 4.96171 |
| 9﹪ | S5′ | 4.71754 |
| 10﹪ | S5′ | 4.63603 |
| 15﹪ | S5′ | 4.23417 |
| 20﹪ | S5′ | 3.94385 |
Table 24, one-way analysis of variance
| Quadratic sum | df | All square | F | Conspicuousness | |
| Between group | 6.111 | 8 | 0.764 | 1.996 | 0.041 |
| In group | 6.889 | 18 | 0.383 | ||
| Sum | 13.000 | 26 |
Table 25, different ethanol concentration and concentration of alcohol are the significance of difference analysis in the S5 ' liquid to be measured of 0 between testosterone concentration measured value
| Ethanol percentage concentration (﹪) in liquid to be measured | P value |
| 5 | 0.585 |
| 6 | 0.362 |
| 7 | 0.068 |
| 8 | 0.255 |
| 9 | 0.114 |
| 10 | 0.085 |
| 15 | 0.017 |
| 20 | 0.005 |
Be that the measured value of the testosterone concentration in the S5 ' liquid to be measured of 0% is as standard control value using ethanol contend percent concentration, when ethanol contend percent concentration is more than or equal to 15 ﹪, there is larger fluctuation, measured value significant difference when measured value and ethanol contend percent concentration are 0% in the measured value of testosterone concentration.
Result shows, when in liquid to be measured, ethanol contend percent concentration is more than or equal to 7 ﹪, the determination influences of ethanol in liquid to be measured to testosterone concentration is larger, the content of testosterone and other indexs can not be detected with enzymoimmunoassay, ethanol contend percent concentration in liquid to be measured can be adjusted to the content measuring testosterone and other indexs in liquid to be measured after being less than 7 ﹪ by euzymelinked immunosorbent assay (ELISA).
Claims (8)
1. detect the method for determinand content in animal wastes ethanol extract, comprise following 1) and 2):
1) regulate the concentration of ethanol in described ight soil ethanol extract, obtain ight soil ethanol extract to be measured, in described ight soil ethanol extract to be measured, the concentration of volume percent of ethanol is greater than 0 and is less than 7%;
2) utilize euzymelinked immunosorbent assay (ELISA) to detect the content of determinand described in described ight soil ethanol extract to be measured, obtain the content of determinand described in described ight soil ethanol extract.
2. method according to claim 1, is characterized in that: described animal is woods musk deer (Moschusberezovskii).
3. method according to claim 1 and 2, is characterized in that: in described ight soil ethanol extract to be measured, the concentration of volume percent of ethanol is greater than 0 and is less than or equal to 6%.
4., according to described method arbitrary in claim 1-3, it is characterized in that: in described ight soil ethanol extract to be measured, the concentration of volume percent of ethanol is greater than 0 and is less than or equal to 5%.
5., according to described method arbitrary in claim 1-4, it is characterized in that: in described ight soil ethanol extract to be measured, the concentration of volume percent of ethanol is greater than 0 and is less than or equal to 4%.
6., according to described method arbitrary in claim 1-5, it is characterized in that: the leaching liquor of described ight soil ethanol extract for obtaining with animal wastes described in ethanol water lixiviate.
7. method according to claim 6, is characterized in that: in described ethanol water, the concentration of volume percent of ethanol is 90%.
8., according to described method arbitrary in claim 1-7, it is characterized in that: described animal is buck; Described determinand is testosterone.
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| CN110806344A (en) * | 2019-11-28 | 2020-02-18 | 北京动物园 | Method for extracting estrogen from polar bear feces |
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