CN103175960A - Enzyme immunoassay method for progestational hormone in excrement of sows and method for detecting oestrous cycle of sow - Google Patents
Enzyme immunoassay method for progestational hormone in excrement of sows and method for detecting oestrous cycle of sow Download PDFInfo
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Abstract
The invention discloses an enzyme immunoassay method for progestational hormone in excrement of sows. The method comprises the steps of: (1) sample collection: collecting the excrement of the sows, weighing and dissolving 0.5 g of excrement of the sows into 20 ml of measuring buffer liquid, carrying out centrifuging, taking and placing supernatant at 4 DEG C to be detected; and (2) measuring content of progestational hormone in a sample by utilizing competitive ELISA (enzyme-linked immuno sorbent assay): measuring according to an ELISA process. According to the enzyme immunoassay method for the progestational hormone in the excrement of the sows, the detection sensitivity of progesterone reaches 0.2 ng/ml or 8 ng/g; the recovery rate of the progesterone is 95.78%; inter-assay and intra-assay variable coefficients are respectively 6.8% (n is equal to 9) and 11.23% (n is equal to 8); the specificity of the progesterone is that the cross reaction rates of the progesterone to estradiol, estriol and oestrone are all less than 0.01%, and the cross reaction rate of the progesterone to testosterone is 0.35%; the stability is good; the required sample is easy to collect, animals are not injured, a measured result is reliable, and a method for measuring blood of the animals can be replaced to monitor a series of reproductive activities of the animals.
Description
Technical field
The present invention relates to the Enzyme immunoassay method of a broad sow ight soil progestational hormone, in mensuration ight soil, progestational hormone concentration with monitoring sow ovarian activity, belongs to biological technical field.
Background technology
The principle of enzyme-linked immunosorbent assay (ELISA): the principle of competitive ELISA be first with antibody sandwich in solid phase, add afterwards certain sample to be tested (containing determined antigen) and a certain amount of enzyme-labelled antigen bond, this moment, they can emulatively be combined with insolubilized antibody, add again the substrate colour developing after hatching washing, measure their OD
492Value calculates determinand content according to typical curve.If in sample, progesterone content is high, be coated on most of progesterone antibody on solid phase carrier and be the free progesterone combination in sample, this moment, enzyme mark progesterone was in conjunction with just relatively less, when washing, the progesterone that is combined in coated antibody is not washed, and free enzyme mark progesterone is washed, and adds the substrate colour developing, and color relation is shallow.Otherwise, if progesterone content is low, enzyme mark progesterone will with antibody knot and, what washed off is the progesterone that dissociates, enzyme mark progesterone is fixed onboard, adds the substrate colour developing, color is dark.
Use the ELISA method to measure reproductive hormone monitoring reproductive organs activity application aborning: the content of female/progestational hormone is the result that ovarian follicle on ovary, corpus luteum are grown, and is the embodiment of dam ovarian function.The essence that dam periodically oestruses is alternately the growing and the final body variation on the content of female/progestational hormone now of corpus luteum and ovary on ovary.Therefore/progestational hormone concentration female by measuring helps to understand ovarian activity state and ovarian secretion function, and the breed ability of monitor animal is to the evaluation of oestrusing, diagnosis of early gestation and ovarian function diagnosis important in inhibiting.In process of production, usually use the ELISA method animal blood sample that gathers to be carried out the mensuration of female/progestogen content, but this method of sampling can to animal produce certain stress, and implement certain difficulty.
Because steroid hormone is combined rapidly in blood in animal body, then be secreted into urine by different approach and be discharged in enteron aisle through bile, steroid hormone is by being combined with glucuronic acid or sulfate, forms the glucosiduronate of water miscible non-activity or sulfate and is excreted in ight soil and urine.In animals urine and ight soil in estrogen mainly formed by oestrone, 17 alpha-estradiols, 17 beta estradiols; The progesterone metabolin mainly contains 5 alpha-pregnanes, 5 β-pregnane in ight soil, and estrogen and progesterone kind in this and blood are basic identical.Therefore, the methods such as dam urine, ight soil of measuring are to replace measuring the activity of blood monitoring dam ovary and other reproductive organs, and the sampling of this kind detection method is simple, little, highly sensitive to the negative effect that animal produces, specificity is good, and easy operating detects, and reliable results.
Summary of the invention
For above-mentioned prior art, the objective of the invention is for the existing deficiency of method of utilizing at present blood measuring animal progestogen content, the present situation such as large in Animal stress, injury, blood sampling difficulty, utilize competitiveness enzyme to exempt from method and measure in ight soil reproductive hormone concentration with monitoring sow Monitoring Ovarian Function, the required sample easily collecting of the method, substantially without injury, the reliable results that records can replace measuring a series of reproduction activities of animal blood method monitor animal to animal.
The present invention is achieved by the following technical solutions:
The Enzyme immunoassay method of one broad sow ight soil progestational hormone, step is as follows:
1) sample collection: collect sow ight soil and also claim 0.5g to be dissolved in 20ml to measure in damping fluid, centrifugal (3000 rev/mins, 10 minutes) get supernatant, be put in 4 ℃ to be measured;
2) content of progestational hormone in the competitive ELISA working sample:
A) progesterone antibody sandwich: with the coated 96 hole ELISA Plate of the coated damping fluid that contains 1: 10000 antibody, 4 ℃ of refrigerators were placed 24 hours, and are stand-by;
B) sealing: cast out content, the sealing of sealing damping fluid is coated with the hole, and every hole 280 μ l place 2h for 37 ℃;
C) add and treat test sample: take out coated plate, cast out content, rinse three times 3 minutes/time with cleansing solution; Then every hole adds testing sample 100ul/ hole, then adds the progesterone enzyme mark thing working fluid of 1: 10000 times, and 37 ℃ of incubations are more than 2 hours;
D) colour developing: rinse three times 3 minutes/time with cleansing solution; Add substrate solution, the 200ul/ hole, 37 ℃ of lucifuges are placed more than 30 minutes;
E) stop: add stop buffer, stop enzymatic reaction;
F) read OD
492nmValue: read the optical density value in each hole at 492nm wavelength place with microplate reader;
G) the OD value is to the conversion of concentration: with the typical curve linearize: establishing the sample Concentration of Progesterone is X, (E/E0: the OD value in the OD value of sample well/zero standard hole)) for y can get equation y=a+blnx, utilizes regression equation can try to achieve the progesterone content pg/100ul working fluid of sample to be tested in conjunction with rate.
Mensuration damping fluid in described step (1) is carbonate buffer solution (the pH value is 9.6), Tris hydrochloride buffer (the pH value is 7.4), phosphate buffer (the pH value is 7.4) or distilled water (the pH value is 7.0), preferred distilled water.
The regression equation of described typical curve is: E/E0=-8.387lnx+108.7 (r=-0.9932).
A kind of method that detects the oestrus of sow phase: utilize the Concentration of Progesterone in said method mensuration sow ight soil, then judge: when nonpregnant sows excrement sample progesterone content is 50～200ng/g, can confirms as and oestrus; When the content of sow progesterone after breeding surpasses 330ng/g excrement sample, can confirm its gestation; During less than 190ng/g excrement sample, can confirm that it does not have gestation.
The inventive method progesterone detection sensitivity reaches (0.2ng/ml or 8ng/g); The recovery (95.78%); The coefficient of variation is respectively 6.8% (n=9) and 11.23% (n=8) in plate and between plate; Specificity: record progesterone antibody and estradiol, female three is groaned, the oestrone cross reacting rate is all less than 0.01% with 50% substitution method, with the testosterone cross reacting rate be 0.35%; Good stability.
The present invention systematically replaces reproductive hormone method for measurement of concentration in ight soil to measure the animal blood method and is applied to monitor Sow Genital Organs, the especially activity of ovary, and compared with prior art, its advantage shows:
1) the present invention's traditional radiommunoassay of comparing has an equipment requirement simple, without advantages such as radiating matter harm;
2) difficulty is excessive on the implementation to have avoided prior art;
3) avoid animal is damaged and injure the Blood Hormone content of bringing;
4) this invention has improved the efficient of monitoring dam physiological status greatly, has important research and production meaning;
5) to detect running program easy fast in the present invention, and from being loaded onto out result less than 3 hours, reagent chemicals is cheap, and job costs are cheap, are convenient to promote in scientific research, clinical diagnosis and herding are produced;
6) degree of accuracy of the present invention is high: the progesterone detection sensitivity reaches the 20pg/ hole, the recovery 95.78%, and the coefficient of variation is respectively 6.80% (n=9) and 11.23% (n=8) in plate and between plate; The coefficient of variation is respectively 6.8% (n=9) and 11.23% (n=8) in plate and between plate; Specificity: record progesterone antibody and estradiol, female three is groaned, the oestrone cross reacting rate is all less than 0.01% with 50% substitution method, with the testosterone cross reacting rate be 0.35%; Good stability;
7) the present invention is simple to operate, and the equipment reagent requirement is low, and is nontoxic, and production application is worth high, has good commercialization promotion prospect.
Utilize animal wastes of the present invention to measure reproductive hormone being also advantageous in that than other method:
1) progesterone and the metabolic product overwhelming majority thereof be through defecate, and in ight soil, progesterone content is blood or milk 5～10 times;
2) the ight soil protein content is far below blood or milk, and the interference of its protein background can be ignored, and method is detected impact less, and the sensitivity of kit is improved greatly;
3) in ight soil, steroid hormone is measured as a kind of non-invasive method, and monitoring wild animal reproductive endocrine state, have advantage simply accurately under state of nature;
4) collection of sample sampling is convenient, can get at any time the excrement sample by rectum, and, can be in the situation that leave animal used as test alone and take a sample, sample can regular collection within a period of time, repeat under the animal endocrinosity same individual sampling not affecting like this, can measure the short-term hormonal readiness variation of animal to particular surroundings or particular procedure in long-time;
5) in ight soil, hormonal readiness is total hormone amount in a period of time, be subjected to the influence of fluctuations of hormone secretion very little, both avoided the invasion to animal used as test, can not occur producing stress reaction because seizure makes animal yet, can reflect that animal changes in long-time internal hormone level, so fecal sample can represent the state of zoohormone very accurately.
6) the ight soil drying is easily preserved afterwards, convenient transportation, but the excrement sample of being adopted dry long preservation under 70～100 ℃ of conditions, overcome that the samples such as blood need the low temperature preservation and the transportation difficulty problem that causes.
Description of drawings
Fig. 1 is Concentration of Progesterone and typical curve schematic diagram in conjunction with rate.
Fig. 2 is the different excrement sample dilutability curve synoptic diagrams of measuring damping fluid, and the series 1,2,3,4 in figure is respectively the excrement sample dilutability curve of carbonate buffer solution, Tris hydrochloride buffer, phosphate buffer, distilled water.
Embodiment
The present invention is further illustrated below in conjunction with embodiment.
Embodiment 1: the mensuration of progesterone content in sow excrement sample
1. test method
1.1 Determination of Serum Progesterone in the excrement sample
Gather 20 sow excrement samples, utilize enzyme to exempt from method and measure progesterone content in excrement, understand that in the excrement sample, progesterone content changes, be used for judging the performance of oestrusing, diagnosis of early gestation, ovarian function diagnosis of dam etc.
1.2 test apparatus
Microplate reader is U.S. Bio-TEK product, and model is ELX800; The conventional instruments such as oscillator, precise electronic balance, sample injector, baking oven, 96 hole ELISA Plate (be purchased from Greiner company).
1.3 reagent
Antibody is purchased from Sigma company, polysorbas20,30%H
2O
2All be purchased from traditional Chinese medicines groups; OPD is base day company's product; Progesterone-11 α-hemisuccinic acid ester (available from Fitgerald company); Bovine serum albumin(BSA) (BSA), other conventional chemical reagent are to be analyzed purely, is purchased from traditional Chinese medicines groups.
1.4 enzyme mark thing (P-HRP) is synthetic
The employing mixed anhydride method is synthetic.2.2mg progesterone-11 α-hemisuccinic acid ester, 1 μ l N-methylmorpholine, be added to and contain 150 μ l N, in the reaction tube of N '-dimethyl formamide (DMF), be cooled to-15 ℃, then add 1 μ l isobutyl chlorocarbonate, then this test solution slowly is added drop-wise to its dripping quantity take each 10 μ l in the reaction tube that is cooled in advance 0 ℃ and [contains concentration in reaction tube and be the NaHCO of 0.5% (mass percent) at-15 ℃ of reaction 50min
3 Aqueous solution 100 μ l, and DMF75 μ l, wherein, NaHCO
3Be dissolved with the horseradish peroxidase (HRP) of 10.3mg in aqueous solution] ,-15 ℃ keep 1h, and jolting frequently sets to 0 a ℃ maintenance 4h.Stir dialysis 30min with distilled water, then use the 0.1M phosphate buffer (PBS, 6x1L) of pH7.5 to put 4 ℃ of dialysis 2d, then cross column chromatography by Sephadex G25 (2x33cm), eluent used is the PBS of pH7.5, concentration 0.1M.After crossing post, the 5mL eluent that contains enzyme mark thing (P-HRP) (be yellow eluent, under the 403nm wavelength, adjust optical density OD403nm to 1.0 with eluent with spectrophotometer) that obtains is carried out 50 μ l packing, freeze-drying is stored in 4 ℃ of refrigerators.
1.5 solution preparation
Coated damping fluid (200ml): Na
2CO
30.795g, NaHCO
31.465g, H
2O 500ml.
Dilution buffer liquid (500ml): Na
2HPO
412H
2O 10.925g, NaH
2PO
42H
2O 3.04g, NaCl 4.35g, BSA 0.5g, H
2O 500ml.
Sealing damping fluid (100ml): 2.0gBSA adds 0.05mol/L, the pH7.0 phosphate buffer (Na for preparing
2HPO
412H
2O10.925g, NaH
2PO
42H
2O 3.04g, NaCl 4.35g, H
2O 500ml) dissolving is quantitatively to 100ml.
Cleansing solution (500ml): 250 μ l polysorbas20s add the 0.05mol/L phosphate buffer 1 000ml for preparing.
Substrate solution: o-phenylenediamine (OPD) substrate solution;
A liquid: 0.1mol/L Na
2HPO
4 12H
24 ℃ of O save backup.(Na
2HPO
4·12H
2O，17.9g?H
2O?500ml)；
B liquid: 0.1mol/L citric acid-OPD, OPD 0.6 gram are dissolved in 500 milliliters of 0.1mol/L citric acid solutions and are namely, 0 ℃ of freezing preservation of following lucifuge, and the term of validity is 1 month (citric acid 10.5g, H
2O 500ml, OPD 0.6g is dissolved in citric acid solution);
Before use, A liquid and B liquid are made into working fluid with 2: 1 ratios of volume ratio, every 100 milliliters of working fluids add 3.0% (percent by volume) H
2O
21ml.
Stop buffer (30ml): the H of 2mol/L
2SO
4, by 98% concentrated sulphuric acid: water=1: 9 (volume ratio) is formulated.
1.6 determining of progesterone antibody and progesterone enzyme mark bond solubility
Adopt the chessboard titrimetry:
1) coated antibody: become different diluent (as 1: 5000 with coated damping fluid dilution progesterone antiserum, 1: 10000,1: 20000 etc.), in numbered carrier board each hole, the 1st row adds coated damping fluid, the progesterone antiserum dilution 200 μ l that the 2nd row begins to add variable concentrations add a cover standing 24h in 4 ℃ of refrigerators.
3) sealing: cast out content, the sealing of sealing damping fluid is coated with the hole, and every hole 280 μ l place 2h for 37 ℃.
4) washing: take out reaction plate, discard the sealing damping fluid, with cleansing solution washing 3 times, blot with thieving paper.
5) add the progesterone enzyme mark thing dilution of variable concentrations: 1.4 synthetic enzyme mark thing eluents are diluted to variable concentrations (as 1: 5000 with dilution buffer liquid, 1: 10000,1: 20000 etc.), take out reaction plate, add progesterone enzyme mark thing dilution 100 μ l in each hole, repeat two row, that is: the every hole of 1-2 row adds 1: 5000, the every hole of 3-4 row adds 1: 10000, and 5-6 is listed as every hole and adds 1: 20000, Separately add dilution buffer liquid 100 μ l, 37 ℃ were reacted 2 hours.
6) washing: take out reaction plate, discard reactant liquor, with cleansing solution washing 3 times, blot with thieving paper.
7) add substrate solution: the preparation substrate solution, add substrate solution 200 μ l in each hole, room temperature lucifuge reaction 30 minutes, this moment, reactant liquor was orange-yellow.
8) add stop buffer: every hole adds stop buffer 50 μ l, and this moment, reactant liquor was brown color.
9) read the OD492 value: read the optical density value in each hole with microplate reader at 492nm wavelength place.
10) choosing progesterone antiserum dilution and the progesterone enzyme mark thing dilution of OD492 value in 1.0 left and right is working fluid.
Under this experiment condition, progesterone antiserum dilution and progesterone enzyme mark thing dilution are 1: 10000.
1.7 operating process
1) the excrement sample gathers: collect sow ight soil and also claim 0.5g to be dissolved in 20ml to measure in damping fluid, centrifugal (3000 rev/mins, 10 minutes) get supernatant, be put in 4 ℃ to be measured.The mensuration damping fluid is selected: carbonate buffer solution (the pH value is 9.6), Tris hydrochloride buffer (the pH value is 7.4), phosphate buffer (the pH value is 7.4), distilled water (the pH value is 7.0).
2) coated antibody: antiprogestin IgG dilution (1: 10000) is become coated working fluid with coated damping fluid dilution, (the 12nd is listed as except F, G, H row's 3 holes) adds coated working fluid 200 μ l in numbered carrier board each hole, add a cover standing 24h in 4 ℃ of refrigerators.
3) sealing: cast out content, the sealing of sealing damping fluid is coated with the hole, and every hole 280 μ l place 2h for 37 ℃.
4) washing: take out reaction plate, discard the sealing damping fluid, with cleansing solution washing 3 times, blot with thieving paper.
5) application of sample: arrange 1-12 row and F at coated plate A and arrange 1-11 and be listed as and add successively sample to be tested (being the resulting supernatant of step 1) 100 μ l in each hole.The multiple A row of B and C re-scheduling application of sample, the multiple F row of G and H re-scheduling application of sample.D and E row adds standard items progesterone 100 μ l, 5 gradients successively according to the low paramount order of concentration: 0.1,0.2,0.5,1.0,2.0ng/ hole, each gradient repeats 3 times.
6) add progesterone enzyme mark thing working fluid: after application of sample, plate is placed in 4 ℃ of refrigerators, prepares simultaneously progesterone enzyme mark thing working fluid (1.4 synthetic enzyme mark thing eluents are diluted to 1: 10000 with dilution buffer liquid); Take out carrier board, add progesterone enzyme mark thing working fluid 100 μ l in each hole, 37 ℃ were reacted 2 hours.
7) washing: take out reaction plate, discard reactant liquor, with cleansing solution washing 3 times, blot with thieving paper.
8) add substrate solution: the preparation substrate solution, add substrate solution 200 μ l in each hole, room temperature lucifuge reaction 30 minutes, this moment, reactant liquor was orange-yellow.
9) add stop buffer: every hole adds stop buffer 50 μ l, and this moment, reactant liquor was brown color.
10) read the OD value: read the optical density value in each hole with microplate reader at 492nm wavelength place.
11) the OD value is to the conversion of concentration: with the typical curve linearize: establishing Concentration of Progesterone is x, in conjunction with rate (E/E
0: sample well OD/ zero standard hole OD
0) for y can get equation y=a+blnP, namely available regression equation is tried to achieve the content pg/100 μ l working fluid (ng/g excrement sample) of progesterone in sample (P).
Described typical curve is made by conventional method: a certain amount of progesterone standard is carried out gradient dilution, and measure it in the optical density value at 492nm wavelength place.The representative standard curve that the present embodiment is set up as shown in Figure 1, as seen from the figure, in the general operation situation, in conjunction with the typical linear regression equation between the natural logarithm lnP of rate (E/E0) and Concentration of Progesterone be: E/E0=-8.387lnx+108.7 (r=-0.9932), E/E0 refers to the optical density value at 492nm wavelength place, x refers to Concentration of Progesterone, the pg/ of unit hole.
The method of setting up is carried out methodology identify, sensitivity and the recovery: the sensitivity of progesterone ELISA kit: 20pg/ hole, the recovery: 95.78%; Repeatability: (Concentration of Progesterone of high, medium and low three levels is respectively 50 to high, medium and low three horizontal samples of replication, 100,500ng/g excrement sample) progesterone content in, the coefficient of variation is respectively 6.8% (n=9) and 11.23% (n=8) in plate and between plate; Specificity: record progesterone antibody and estradiol, estriol, oestrone cross reacting rate all less than 0.01% with 50% substitution method, with the testosterone cross reacting rate be 0.35%; Good stability.
2 measurement results
2.1 the different excrement sample dilutability curves of measuring damping fluid
use respectively carbonate buffer solution (the pH value is 9.6), Tris hydrochloride buffer (the pH value is 7.4), phosphate buffer (the pH value is 7.4), distilled water (the pH value is 7.0) is made excrement sample dilution, press the operation of Salmonella step, measurement result as shown in Figure 2, series 1 in figure, 2, 3, 4 are respectively carbonate buffer solution, the Tris hydrochloride buffer, phosphate buffer, the excrement sample dilutability curve of distilled water, as seen from the figure, the Tris hydrochloride buffer, phosphate buffer, distilled water measurement result consistance is higher, carbonate buffer solution and other three kinds of damping fluid measurement results are lower.It is neutral that its reason is that the PH of Tris-hydrochloride buffer, phosphate buffer, distilled water approaches, and carbonate buffer solution is strong basicity (9.6).
ELISA has two kinds of fundamental reaction systems, i.e. immune response system and enzymatic reaction system, and these two kinds of reaction systems all are subjected to the impact of pH value and damping fluid composition.Reaction system requires pH closely neutral, too high or too low equal can the reaction by Immunosuppression of pH.Because the pH of Tris-hydrochloride buffer, phosphate buffer, distilled water is approaching neutral, be fit to immune response and enzymatic reaction, and carbonate buffer solution pH is too high, Immunosuppression reaction and enzymatic reaction.Therefore, this test findings confirmation pH value is exempted from method mensuration excrement sample Concentration of Progesterone to enzyme and is had a significant impact, and neutrality is more suitable in dilution excrement sample.Consider from user's angle, with distilled water diluting excrement sample more economically.
2.2 20 sows on certain pig farm have been carried out the mensuration of ight soil progestogen content, wherein ten gilt and ten multiparity sows, by measuring progesterone content in excrement with the competitive ELISA method, understand sow ovarian activity and diagnosis of early gestation, measurement result is as shown in table 1, table 2:
Progesterone content in table 1 gilt excrement sample
The content of the excrement of dam or the estradiol in blood, progesterone is the result that ovarian follicle on ovary, corpus luteum are grown, and is the embodiment of dam ovarian function.The essence that dam periodically oestruses is alternately the growing of ovarian follicle and corpus luteum on ovary, being reflected in ight soil is the cyclical variation of estradiol and progesterone content, in oestrus, due to luteolysis on ovary, follicular development, in excrement, progesterone content is very low, and estradiol content is higher; If dam oestruses after semen deposition the fertilization, gestation, the cycle corpus luteum of rear formation of oestrusing is no longer dissolved, degenerates by the prostaglandin that endometrium is secreted, terminations of periodically oestrusing, formation corpus luteum graviditatis, within the time that arrives next oestrus, the progesterone content in excrement maintains higher level.Therefore can be used for judging oestrus performance, the early stage pregnant diagnosis etc. of dam by the progesterone content in mensuration gilt and multiparity sow excrement.
As shown in Table 1, gilt comes into play at 60d left and right ovary.Sow before puberty growth course can be divided into 0-2 monthly age, 2-5 monthly age and repose period three phases.0-2 monthly age sow ovary and fallopian tubal poor growth only have ovarian follicle before the chamber on ovary, but developing womb are very fast.All reproductive organs of 2-5 monthly age section are Fast Growth all, and are especially outstanding with ovary; There is the chamber ovarian follicle to begin to project to the ovary surface during 3 monthly age.Hence one can see that, measured before puberty the fecal hormone of sow extremely important to the early stage seed selection replacement gilt of breeding station.
Progesterone content in table 2 multiparity sow excrement sample
As shown in Table 2, subtract two standard deviations as benchmark (n=5) with the average of Concentration of Progesterone in in-pig excrement sample, when nonpregnant sows excrement sample progesterone content is 50～200ng/g, can confirms as and oestrus; When the content of sow progesterone after breeding surpasses 330ng/g excrement sample, just can confirm its gestation; The average of Concentration of Progesterone deducts two standard deviations as benchmark (n=5) in the nonpregnant pig manure sample, during less than 190ng/g excrement sample, can confirm that it does not have gestation, and result is substantially similar to the result of study of Masaharu M etc.
3 conclusions
This method is by measuring the progesterone content in different phase sow ight soil, and result shows, in sow ight soil, the content of progesterone is consistent with theoretical analysis.Compare with progesterone method for measuring in blood, excrement sample sampling method has convenience, simple to operate, need not to use the advantages such as micro liquid sampler, more is conducive to promotion and application in pig farm, self-employed pig raiser, self-employed worker.The present invention utilizes ELISA to carry out measurement operation in addition, and the radiommunoassay of comparing has an equipment requirement simple, easy and simple to handle, without advantages such as radiating matter harm.At present oestrus of sow is identified manyly by observation and sow teasing method, but observation needs certain experience, and examination feelings method is also more time-consuming, and accuracy is not high, often occurs false heat pig false diagnosis in production, and then medication or semen deposition cause the situation of miscarriage.Measure progesterone level in excrement by enzyme immunoassay technique, just can reflect very exactly the functional status of ovary, can correctly diagnose true and false oestrus with oestrus not obvious, thereby avoid well the generation of false diagnosis, so this method also can be used for the evaluation of oestrusing.Equally, current sow gestation diagnosis clinical examination method and the hormone methods of adopting more, the clinical examination method needs certain experience, and false diagnosis also easily occurs; The hormone method does not slightly note also easily causing miscarriage; Simultaneously can not accomplish early diagnosis.Use B ultrasound for animals to diagnose though have at present, generally also need just can carry out more than 35 days after semen deposition.And use enzyme immunity hormone determination technology just can be after semen deposition a feelings phase (19-23 days) carry out cyesiognosis accurately.Therefore use the method to measure progesterone content in the excrement sample, can carry out oestrus evaluation, the early pregnancy diagnosis of pig, business promotion prospect of the present invention is better.
Claims (10)
1. the Enzyme immunoassay method of a broad sow ight soil progestational hormone, it is characterized in that: step is as follows:
1) sample collection: collect sow ight soil and also claim 0.5g to be dissolved in 20ml to measure in damping fluid, centrifugal, get supernatant, be put in 4 ℃ to be measured;
2) content of progestational hormone in the competitive ELISA working sample:
A) progesterone antibody sandwich: with the coated 96 hole ELISA Plate of the coated damping fluid that contains 1: 10000 antibody, 4 ℃ of refrigerators were placed 24～48 hours, and are stand-by;
B) sealing: cast out content, the sealing of sealing damping fluid is coated with the hole, and every hole 280 μ l place 2h for 37 ℃;
C) add and treat test sample: take out coated plate, cast out content, rinse three times 3 minutes/time with cleansing solution; Then every hole adds testing sample 100ul, then adds the progesterone enzyme mark thing working fluid of 1: 10000 times, and 37 ℃ of incubations are more than 2 hours;
D) colour developing: rinse three times 3 minutes/time with cleansing solution; Add substrate solution, the 200ul/ hole, 37 ℃ of lucifuges are placed more than 30 minutes;
E) stop: add stop buffer, stop enzymatic reaction;
F) read OD
492nmValue: read the optical density value in each hole at 492nm wavelength place with microplate reader;
G) the OD value is to the conversion of concentration: with the typical curve linearize: establishing the sample Concentration of Progesterone is x, be y in conjunction with rate (E/E0: sample well OD value/zero standard hole OD value), can get equation y=a+blnx, utilize regression equation can try to achieve the progesterone content pg/100ul working fluid of sample to be tested.
2. the Enzyme immunoassay method of sow ight soil progestational hormone according to claim 1 is characterized in that: the mensuration damping fluid in described step (1) is that the pH value is that 9.6 carbonate buffer solution, pH value are that 7.4 Tris hydrochloride buffer, pH value are 7.4 phosphate buffer or distilled water.
3. the Enzyme immunoassay method of sow ight soil progestational hormone according to claim 1 is characterized in that: the consisting of of described coated damping fluid: Na
2CO
30.795g, NaHCO
31.465g, H
2O 500ml.
4. the Enzyme immunoassay method of sow ight soil progestational hormone according to claim 1 is characterized in that: the consisting of of described sealing damping fluid: 2.0gBSA adds 0.05mol/L, the pH7.0 phosphate buffer dissolves quantitatively to 100ml; Consisting of of described phosphate buffer: Na
2HPO
412H
2O 10.925g, NaH
2PO
42H
2O 3.04g, NaCl 4.35g, H
2O 500ml.
5. the Enzyme immunoassay method of sow ight soil progestational hormone according to claim 1 is characterized in that: the consisting of of described cleansing solution: 250 μ l polysorbas20s add 0.05mol/L phosphate buffer 1 000ml.
6. the Enzyme immunoassay method of sow ight soil progestational hormone according to claim 1 is characterized in that: described progesterone enzyme mark thing working fluid is that enzyme mark thing eluent is diluted to 1: 10000 with dilution buffer liquid and obtains;
Described enzyme mark thing eluent prepares by the following method: 2.2mg progesterone-11 α-hemisuccinic acid ester, 1 μ l N-methylmorpholine, be added to and contain 150 μ l N, in the reaction tube of N '-dimethyl formamide, be cooled to-15 ℃, then add 1 μ l isobutyl chlorocarbonate, this test solution is at-15 ℃ of reaction 50min, then its dripping quantity with each 10 μ l slowly is added drop-wise in the reaction tube that is cooled in advance 0 ℃, contains concentration in reaction tube and be 0.5% NaHCO
3Aqueous solution 100 μ l, and DMF75 μ l, wherein, NaHCO
3Be dissolved with the horseradish peroxidase (HRP) of 10.3mg in aqueous solution ,-15 ℃ keep 1h, set to 0 a ℃ maintenance 4h; Stir dialysis 30min with distilled water, then put 4 ℃ of dialysis 2d with the 0.1M phosphate buffer of pH7.5, then cross column chromatography by Sephadex G25, eluent used is the PBS of pH7.5, concentration 0.1M; After crossing post, obtain yellow eluent, adjust optical density OD403nm to 1.0 with eluent, namely get enzyme mark thing eluent;
Consisting of of described dilution buffer liquid: Na
2HPO
412H
2O 10.925g, NaH
2PO
42H
2O 3.04g, NaCl 4.35g, BSA 0.5g, H
2O 500ml.
7. the Enzyme immunoassay method of sow ight soil progestational hormone according to claim 1 is characterized in that: the consisting of of described substrate solution:
A liquid: 0.1mol/L Na
2HPO
412H
2O, 4 ℃ save backup;
B liquid: 0.1mol/L citric acid-OPD:OPD 0.6 gram is dissolved in 500 milliliters of 0.1mol/L citric acid solutions and is namely, 0 ℃ of freezing preservation of following lucifuge, and the term of validity is 1 month;
Before use, A liquid and B liquid are made into working fluid with 2: 1 ratios of volume ratio, every 100 milliliters of working fluids add 3.0%H
2O
21ml.
8. the Enzyme immunoassay method of sow ight soil progestational hormone according to claim 1, it is characterized in that: described stop buffer is the H of 2mol/L
2SO
4
9. the Enzyme immunoassay method of sow ight soil progestational hormone according to claim 1, it is characterized in that: the regression equation of described typical curve is: E/E0=-8.387lnx+108.7, and r=-0.9932, E/E0 refer to the optical density value at 492nm wavelength place, x refers to Concentration of Progesterone, the pg/ of unit hole.
10. method that detects the oestrus of sow phase, it is characterized in that: utilize the Enzyme immunoassay method of the described sow ight soil of any one progestational hormone in claim 1～9 to measure Concentration of Progesterone in sow ight soil, then judge: when nonpregnant sows excrement sample progesterone content is 50～200ng/g, can confirms as and oestrus; When the content of sow progesterone after breeding surpasses 330ng/g excrement sample, can confirm its gestation; During less than 190ng/g excrement sample, can confirm that it does not have gestation.
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| CN114814241A (en) * | 2022-05-31 | 2022-07-29 | 广东省农业科学院动物科学研究所 | Use of protein and screening method |
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