CN1156701C - Enzyme immunoanalysis test kit for fast in-situ diagnosis of milk cow's early pregnancy and oestrus - Google Patents
Enzyme immunoanalysis test kit for fast in-situ diagnosis of milk cow's early pregnancy and oestrus Download PDFInfo
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- CN1156701C CN1156701C CNB021132631A CN02113263A CN1156701C CN 1156701 C CN1156701 C CN 1156701C CN B021132631 A CNB021132631 A CN B021132631A CN 02113263 A CN02113263 A CN 02113263A CN 1156701 C CN1156701 C CN 1156701C
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Abstract
The present invention relates to an enzyme immunoassay test kit for fast diagnosing the early pregnancy and the oestrus of milk cows in site, which comprises a solid phase carrier and a reagent for detecting. The reagent for detecting comprises a progesterone resisting antibody as a first antibody and a second antibody for resisting the first antibody. The reagent for detecting comprises the following components of enzyme marking progesterone dissolved by phosphate buffer before being used, a progesterone standard prepared by the phosphate buffer, an antibody for resisting progesterone, the second antibody for resisting the first antibody, hydrogen peroxide, first developer, second developer, phosphate buffer of which the pH value is from 7 to 7.4 and phosphate buffer of which the pH value is from 9 to 9.8, wherein the antibody for resisting progesterone is diluted by the phosphate buffer before being used; the second antibody for resisting the first antibody is diluted by the buffer bicarbonate before being used; and the first developer and the second developer contain tetramethylbenzidine buffer. The enzyme immunoassay test kit can fast and accurately judge whether the milk cows impregnate or are in heat by analyzing and detecting the results of milk samples of the milk cows in site.
Description
Technical field
What the present invention relates to is a kind ofly to be used to detect and to judge whether milk cow is in the EIA enzyme immunoassay testing cassete in early pregnancy or oestrus, specifically being a kind of for using at the scene, is that the sample fast detecting is judged the early pregnancy of milk cow and the EIA enzyme immunoassay testing cassete of the situation of oestrusing with the milk of milk cow.
Background technology
The output of milk of milk cow is in close relations with its residing different cyclostages.Infertile after the milk cow breeding be cause the calving interval to prolong, breeding potential reduces, and makes the output of milk reduce the major reason that feeding cost increases.Have now found that the progesterone concentration in the dairy cow milk can present growth and decline clocklike and change in the oestrous cycle of milk cow, can accurately reflect the situation of its cyclostage of living in.Thereby, can understand and judge whether milk cow becomes pregnant, whether oestruses, and the disease of whether suffering from reproductive systems such as some ovary by measuring progesterone levels in the dairy cow milk.
Detect the assay method of progesterone content in the dairy cow milk at present, radio immunoassay and enzyme immunoassay (EIA) two classes generally can be arranged.Radio immunoassay can not use at the scene owing to can only carry out in the laboratory, and the sense cycle time is long, and cowboying person obtains testing result generally needs 2-7 days, and easily causes radioactive contamination.Enzyme immunoassay (EIA) is a kind of method of testing of not having injury, does not have alpha-contamination problem, its sensitivity not only with radio immunoassay quite, even can be higher, and equipment needed thereby is all relative with reagent, material more simple and convenient and inexpensive.But present needed cycle detection time of general enzyme immunoassay (EIA) used in medical science and animal science is still longer, and the mensuration of finishing batch to be tested generally needs 3-16 hour, equally also is difficult to use at the scene.On the other hand, detect the enzyme immunoassay (EIA) of milk progesterone of cow content at present, what adopted all is principle and the method that monospecific antibody detects.As, at Can.J.Anim.Sci., 70:997-1003, the Target testing cassete of being reported in 1990 is first with the little panel surface of antiprogestin antibody sandwich in a kind of porous, adds titer and milk sample respectively, add enzyme mark progesterone liquid and colour developing liquid after the reaction again, carry out colorimetric and observe evaluation.J.DairySci., 69, Suppl.1, the method for report is that monoclonal antibody is coated on the plastics spillikin in 1986, during mensuration spillikin is placed tubule, makes itself and other test solution carry out immune response.At J.Dairy Sci., 69:1115-1121, in 1986 report be with the antiprogestin antibody sandwich on paper slip, be placed on during mensuration in the plastic tube, successively add standard, sample and enzyme mark progesterone, add the method that colour developing liquid is observed evaluation after the reaction again.The report of the detection method of same test principle also has therewith.This basic process that detects the principle method is: earlier a certain amount of hormone antibody is coated on the surface of solid phase carriers such as plastic tube, sticking plaster, plastic bead, titer plate or paper slip before detecting, seals with bovine serum albumin(BSA).To after wrapping quilt and the solid phase carrier water handled of sealing and cleaning, add the immune response of being at war with property of testing sample and enzyme mark hormone during mensuration, again through washing with add the also placement of substrate that contains developer, with instrument or visual inspection change in color result.Though these testing cassetes can use at the scene, and have fast, advantage such as easy, accuracy rate of diagnosis to calver can reach 70%-85%, the 80%-95% that can be to calver not, but except that required expense is higher, because what adopt all is the EIA enzyme immunoassay of monospecific antibody form, practice result shows, solid phase carrier with this hormone antibody bag by after, easy prolongation because of the holding time, processing such as the cleaning in the mensuration and operation and former this hormone antibody that is attached on the solid phase carrier that wrapped is lost, the amount that has caused participating in immunoreactive antibody produces certain difference between Kong Yukong, be the accuracy that influences the immune response result, a sensitivity and a reproducible major reason.
Summary of the invention
According to above-mentioned situation, the present invention will provide a kind of and can use at the scene, and can carry out the EIA enzyme immunoassay testing cassete that fast detecting is judged to early pregnancy of milk cow and the situation of oestrusing simply and easily.
The EIA enzyme immunoassay testing cassete of the on-the-spot quick diagnosis milk cow's early pregnancy of the present invention and the usefulness of oestrusing comprises solid phase carrier and detection reagent two parts, detects with enzyme mark progesterone being arranged, progesterone standard items, antiprogestin antibody, chromogenic reagent and buffering reagent in the reagent.Be first antibody with antiprogestin antibody in the detectable, also include the second antibody of anti-first antibody, detect consisting of with reagent:
A) enzyme mark progesterone dissolves with phosphate buffer before using,
B) progesterone standard items, with the phosphate buffer preparation,
C) antiprogestin antibody is first antibody, dilute with phosphate buffer before using,
D) second antibody of anti-first antibody is diluted with carbonate buffer solution before using,
E) chromogenic reagent:
First developer is that volume content is 0.004% superoxol,
Second developer be contain the 0.05 mole of phosphoric acid salt that weight/volume is 0.01% tetramethyl benzidine-
Citrate buffer, pH5.0,
F) phosphate buffer contains 0.04 mole of phosphoric acid disodium hydrogen, sodium dihydrogen phosphate and 0.14 mole nacl, pH7-7.4,
G) carbonate buffer solution of forming by sodium carbonate-sodium bicarbonate, pH9-9.8.
Clearly, a distinguishing feature of the above-mentioned EIA enzyme immunoassay testing cassete of the present invention is that what to adopt is the principle of double antibody EIA enzyme immunoassay, promptly, detecting with in the composition of reagent, except that containing antibody with usually used antiprogestin hormone, also has the second antibody of anti-first antibody simultaneously as the first antibody composition.
Generally speaking, said various antibody all is a kind of immunoglobulin G (IgG) usually.The first antibody of above-mentioned said this antiprogestin hormone of the present invention also is a kind of immunoglobulin G that can method of operating routinely prepares.For example: to contain said progesterone hormone is the immunogen immune rabbit, separates this immunoglobulin G that makes as first antibody by its serum then.On structure, as a kind of embodiment, the antiprogestin antibody of said this first antibody can be that connection thing with progesterone-6 beta-hydroxies-hemisuccinic acid salt and bovine serum albumin(BSA) is as antigen, rabbit is carried out immune prepared specific antibody serum, and make its titre remain at least 1: 210,000 is good.
The second antibody of above-mentioned said anti-first antibody generally is after employing is the another kind of animal of immunogen immune, to separate the another kind of immunoglobulin G that makes from its serum again with the first antibody.For example, as a kind of example, can be immunogen immune goat or sheep, and obtain the second antibody of the anti-rabbit immunoglobulin G of goat or sheep thus as anti-first antibody with first antibody such as immunoglobulin G of above-mentioned rabbit.
Above-mentioned said enzyme mark progesterone can adopt the form of the progesterone-6 beta-hydroxies-hemisuccinic acid salt that connects with horseradish peroxidase usually, can adopt conventional amine condensation method, for example prepares with reference to S.Liebermann institute method of reporting.
For ease of preserving, in above-mentioned detectable, can also contain the antiseptic of a small amount of appropriate format in said antiprogestin antibody and the enzyme mark progesterone respectively in the usual way again.As a kind of embodiment, it is the formalin of 1%-3% that said antiseptic can be adopted as thimerosal and the volume/volume that weight/volume is 5%-20%.
In addition, for more helping cryopreservation, can also add the freeze-point depressant commonly used that volume ratio is 15-30% in antiprogestin antibody in above-mentioned detectable and the enzyme mark progesterone.As a kind of embodiment, said freeze-point depressant can adopt the glycerine of 10%-20%.
General and have no special requirements to said solid phase carrier in the above-mentioned analytical test box.For example, except that the titer plate that can use the form that can not break the most frequently used when carrying out immune response, the solid phase carrier that special recommendation is used is to be good according to the required plate hole quantity of the testing sample detachable titer plate that declines that uses of can selecting easily to break.
The above-mentioned analytical test box of the present invention in use, earlier with the second antibody bag by the detection immune response zone in the solid phase carrier, the plate hole inwall of required use in for example above-mentioned said detachable form titer plate that maybe can not break, second antibody is attached to carries out immunoreactive regional position, and then do sealing with bovine serum albumin(BSA) and handle.During detection, in the plate hole that has added milk sample to be measured or progesterone standard items, add enzyme mark progesterone and first antibody respectively, it is carried out competitive reaction respectively.First antibody can combine with the second antibody on the solid phase carrier therebetween, and the progesterone in the milk sample then can be competed the binding site on the first antibody simultaneously with enzyme mark progesterone and combine with it.The result of competition, the amount with the enzyme of first antibody combination mark progesterone on the solid phase carrier will be different and different with progesterone levels in the milk.Progesterone content in the milk is many, and the amount that then is combined in the enzyme mark progesterone on the solid phase carrier is less relatively, and the amount of enzyme is corresponding at least also more shallow by the shown color of developer; Otherwise if the progesterone content in the milk sample is few, the enzyme mark progesterone that then is combined on the solid phase carrier will be more, and is corresponding darker by the color relation that developer demonstrates.Change because the progesterone content in the dairy cow milk is growth and decline clocklike in its oestrous cycle, the progesterone content in the back dairy cow milk of becoming pregnant can obviously raise, do not become pregnant or the dairy cow milk when being in heat in progesterone content then obviously lower.Therefore by developer to the shown shade of milk sample in the above-mentioned testing process and progesterone standard items can reflect in the milk sample progesterone content just, thereby can judge exactly that milk cow is in period of pregnancy or oestrus, so that take measures areput.
By the Principle of Process of above-mentioned using method and detection as can be seen, when using the EIA enzyme immunoassay testing cassete of the above-mentioned form of the present invention, before detecting essential earlier with the second antibody bag by the detection immune response zone of used solid phase carrier---as the plate hole inwall in the titer plate, make second antibody be attached to this position, zone, and then seal processing with bovine serum albumin(BSA).Therefore, EIA enzyme immunoassay testing cassete of the present invention is except that above-mentioned form, the form that can also further adopt is: make to using above-mentioned detection method to provide and store titration orifice plate in testing cassete etc. and detect and use solid phase carrier, when production is dispatched from the factory, become its detect in the conversion zone of immunity in advance with above-mentioned said second antibody wrap by, also seal the backup form of having handled well with bovine serum albumin(BSA).The solid phase carrier of this kind form is detecting the processing operation that need not temporarily to wrap again quilt and sealing when using, and for making on-the-spot the detection use the convenient of operation and improve detection efficiency, obviously is very favourable.When adopting the testing cassete of this kind form, generally also can save this second antibody reagent for detecting in the detectable that needs to provide and be equipped with, thereby make the composition of testing cassete also further be able to corresponding simplification.
As above-mentioned, when adopting monospecific antibody enzyme immunity principle analyzing and testing at present, since solid phase carrier with this monospecific antibody bag by after can be because of the prolongation of holding time, factors such as the cleaning treatment in the mensuration and this antibody is lost, thereby there is certain difference in the amount that causes participating in immunoreactive antibody between Kong Yukong, has influenced immune response result's accuracy, sensitivity and reappearance.And adopt double antibody method of the present invention to carry out check and analysis, because solid phase carrier has carried out bag quilt and sealing with excessive second antibody in advance, during mensuration be simultaneously with the first antibody of amount respectively with milk sample to be measured in contained progesterone, progesterone standard items and enzyme mark progesterone react, avoided the quantitative difference of the first antibody between different plate holes before the immune response; Simultaneously, even excessive second antibody is lost in preservation, washing process, also still can also have enough sites to combine, and it also can not disturb the immune response of other reagent with the first antibody that is added.This just the above-mentioned testing cassete of the present invention can significantly improve and guarantee accuracy, stability and the reproducible basic reason place of testing result.Test findings shows, when adopting the above-mentioned analytical test box of the present invention that the milk sample of milk cow is carried out analyzing and testing, generally can reach 85%-90% and 95%-100% respectively to pregnant and the judging nicety rate unpregnancy milk cow, and can judge, and can do sth. in advance 21-68 days in 21-24 days after the milk cow breeding than traditional rectal palpation method.Simultaneously, when checked oestrus,, or some signs but when still being difficult to determine, using the above-mentioned testing cassete of the present invention also can make things convenient for exactly and is differentiated of oestrusing are arranged, help to find that recessiveness is oestrused and the false heat ox even the milk cow that has has the recessiveness phenomenon of oestrusing; After the milk cow calving, can also promptly and accurately judge and when recover oestrus, be convenient to breed early, prevent that the nonpregnant phase from prolonging.Test findings shows, adopts the above-mentioned analytical test box of the present invention, and the time of finishing one-time detection generally only needs 16-20 minute, and wherein the Guan Jian immune response time generally only needs 5-7 minute.In addition, because only need get 1 milk sample when detecting can check, milk cow there is not any injury fully, need not to use special instrument and complex instrument again, test result can be judged by color comparison of naked eye, so simple to operation, and can be that common dexterity is operated, fully possess in the cattle farm or condition and the feasibility that promote the use of at the scene of man of peasant household complicated and technical very strong work simplification originally.Test findings also shows, test material in the above-mentioned analytical test box of the present invention and detection also have long effective holding time with reagent, effective storage life under the condition of 4 ℃ and-20 ℃ can reach respectively more than 5 months and 6 months, to be applicable under various conditions and occasion promote the use of more favourable.
Except that can be used in the early pregnancy of fast detecting milk cow and oestrusing, above-mentioned testing cassete of the present invention can also help some disease of ovary, the inspection and the diagnosis of diseases such as, ovarian cyst static, retained corpus luteum as ovary.These also all have great importance and practical value in producing for actual milk cow.
Below will again foregoing of the present invention be described in further detail, but this should be interpreted as that the scope of the above-mentioned theme of the present invention only is confined to following embodiment by the form of embodiment.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Embodiment
The EIA enzyme immunoassay testing cassete of this routine on-the-spot quick diagnosis milk cow's early pregnancy and the usefulness of oestrusing comprises solid phase carrier and detection reagent two parts.Solid phase carrier can be conventional detachable form 48 holes of using or the titer plate in 96 holes in the immune response test.Detect with there being enzyme to mark progesterone in the reagent, the progesterone standard items, as the antiprogestin antibody of first antibody, the second antibody of anti-first antibody, and developer and buffering agent etc.Its concrete corresponding preparation method of forming and can be for reference is as follows:
A) enzyme mark progesterone: can adopt during preparation 20 milligrams of horseradish peroxidases (RZ＞3.0) are dissolved in 500 microlitre distilled water, add 300 microlitre dimethyl formamides; In addition 5 milligrams of progesterone-6 beta-hydroxies-hemisuccinic acid salt is dissolved in the 300 microlitre dimethyl formamides, add 6 microlitre methyl morpholines and 3 microlitre isobutyl chlorocarbonates, mix with above-mentioned enzyme liquid then, stirred 1 hour-15 ℃ of conditions, under 4 ℃ of conditions, stirred 2 hours again, in distilled water, dialysed at least 15 hours then.After making chromatographic purifying and handle with sephadex G-25, the freeze-drying cryopreservation is in-20 ℃.Be dissolved in the phosphate buffer before the use.
B) progesterone standard items: analytically pure progesterone is formulated in the following phosphate buffer, under-20 ℃ of conditions, preserves.
C) antiprogestin antibody: can bovine serum albumin(BSA) and progesterone-6 beta-hydroxies-hemisuccinic acid salt connection be made antigen with carbodiimides during preparation, it is injected in the rabbit body, in 5-7 month, can successively inject 7-10 time, the acquisition titre is 1:210,000 or higher specific corrosioning anteserum, the freeze-drying cryopreservation.Be dissolved in before the use in the following phosphate buffer, and to add weight/volume be that the thimerosal of 5%-20% and formalin that volume/volume is 1%-3% are as antiseptic, and volume/volume be the glycerine of 10-20% as freeze-point depressant, under the condition of 4 ℃ or-20 ℃, preserve.
D) goat anti-rabbit igg (second antibody): can adopt centrifugal from rabbit anteserum, to isolate immunoglobulin G (IgG), be injected in goat or the sheep body as immunogene with this, in 4-5 month, successively inject 4-6 time, collect serum after the conventional method check titre, immunosorbent with routine, for example rabbit igg-agarose coagulation-CL-6B etc. carries out conventional purification process and makes, and freeze-drying is preserved standby under-20 ℃～-70 ℃ conditions.Dissolve with carbonate buffer solution before using.
E) chromogenic reagent: form by two kinds of test solutions.One is to contain 0.004% superoxol; It is two for being dissolved with the tetramethyl benzidine of 0.01% (w/v) in 0.05 mole phosphate-citrate salts damping fluid molten (pH5.0).
F) phosphate buffer: contain 0.04 mole of phosphoric acid disodium hydrogen, sodium dihydrogen phosphate and 0.14 mole nacl, pH7-7.4.
G) carbonate buffer solution: form pH9-9.8 by sodium carbonate-sodium bicarbonate.
Adopt the above-mentioned testing cassete of the present invention, the milk sample to be measured of taking from milk cow can be detected by following mode of operation:
1) plate hole is prepared, promptly with above-mentioned second antibody bag by the microtitre plate hole: after this second antibody is diluted to the concentration of 10-20 mcg/ml with said carbonate buffer solution, add 100 microlitres, discard after at least 15 hours 0 ℃～4 ℃ placements to every hole.Add 0.1% bovine serum albumin(BSA) liquid, room temperature is placed after 1 hour and is discarded; Add phosphate buffer 1 80 microlitres/hole of containing 15-25 milligram/100 milliliter thimerosal antiseptic, seal up for safekeeping in-20 ℃ standby.
The milk sample size of surveying as required during detection retracts the plate hole of respective numbers.For example: generally need to use two holes when only detecting a milk sample, wherein a hole is as sample well, and another is as gauge orifice; If detect a plurality of milk samples simultaneously, for example during 2-5 sample,, then can be shared one for the gauge orifice of progesterone standard items use except that each milk sample needs each with the hole; If the milk sample size that detects simultaneously is more, when above, can take the circumstances into consideration to be provided with the gauge orifice more than 2 as 6.Use clear water hole flushing three times before the detection according to a conventional method.
2) add phosphate buffer: in the plate hole of each use, add phosphate buffer 3-4 and drip.
3) drip sample: drips 1 in the new fresh milk sample that detects the same day with transfer pipet in sample well respectively, dropping progesterone standard items are 1 in gauge orifice.
4) in sample well and gauge orifice, add the first antibody of equivalent and the enzyme mark progesterone reagent of 10-20 microlitre respectively simultaneously, make that liquid fully mixes in the hole in, lucifuge placement 5-7 minute.
5) add developer: get rid of the liquid of abandoning in the plate hole, with clean water hole flushing 5 times, add each two of two kinds of developers respectively in each plate hole, and it is mixed, lucifuge is placed.
6) observe judgement: after～5 minutes, liquid color is deeper than or is same as gauge orifice in the blue shade that liquid showed in each hole of comparison that detects by an unaided eye---the sample well, then can be judged as to belong to unpregnancy; Otherwise then can be judged as milk cow and become pregnant if the color of sample well is shallower than gauge orifice.
For making things convenient for practical operation, according to the activity index experimental result of every batch of detection being used reagent, all ingredients that can each hole at every turn detecting is required quantitatively is packaged in same tubule or other the suitable packing material in advance, only needs during use in a manner described its disposable adding plate hole to be got final product.
Claims (7)
1. the EIA enzyme immunoassay testing cassete of an on-the-spot quick diagnosis milk cow's early pregnancy and the usefulness of oestrusing, comprise solid phase carrier and detection reagent two parts, detect with enzyme mark progesterone is arranged in the reagent, the progesterone standard items, antiprogestin antibody, chromogenic reagent and buffering reagent is characterized in that in the detectable with antiprogestin antibody being first antibody, also include the second antibody of anti-first antibody simultaneously, detect consisting of with reagent:
A) enzyme mark progesterone, and to contain thimerosal and the volume/volume that weight/volume is 5%-20% be the antiseptic of the formalin of 1%-3%, before using with the phosphate buffer dissolving,
B) progesterone standard items, with the phosphate buffer preparation,
C) antiprogestin antibody is first antibody, for the connection thing with progesterone-6 beta-hydroxies-hemisuccinic acid salt and bovine serum albumin(BSA) is the antibody that the antigen immune rabbit is obtained, and to contain thimerosal and the volume/volume that weight/volume is 5%-20% be the antiseptic of the formalin of 1%-3%, dilute with phosphate buffer before using
D) second antibody of anti-first antibody is the anti-rabbit immunoglobulin G of goat or sheep, dilutes with carbonate buffer solution before using,
E) chromogenic reagent:
First developer is that volume content is 0.004% superoxol,
Second developer is to contain 0.05 mole of phosphoric acid salt-citrate buffer that weight/volume is 0.01% tetramethyl benzidine, pH5.0,
F) phosphate buffer contains 0.04 mole of phosphoric acid disodium hydrogen, sodium dihydrogen phosphate and 0.14 mole nacl, pH7-7.4,
G) carbonate buffer solution of forming by sodium carbonate-sodium bicarbonate, pH9-9.8
H) freeze-point depressant is the glycerine of 15-30% for volume ratio.
2. the EIA enzyme immunoassay testing cassete of the on-the-spot quick diagnosis milk cow's early pregnancy as claimed in claim 1 and the usefulness of oestrusing is characterized in that said freeze-point depressant is the glycerine of 10%-20%.
3. the EIA enzyme immunoassay testing cassete of the on-the-spot quick diagnosis milk cow's early pregnancy as claimed in claim 1 or 2 and the usefulness of oestrusing is characterized in that said antiprogestin antibody is that titre was at least 1: 210,000 specific antibody.
4. the EIA enzyme immunoassay testing cassete of the on-the-spot quick diagnosis milk cow's early pregnancy as claimed in claim 1 or 2 and the usefulness of oestrusing is characterized in that said enzyme mark progesterone is the connection thing of horseradish peroxidase and progesterone-6 beta-hydroxies-hemisuccinic acid salt.
5. the EIA enzyme immunoassay testing cassete of the on-the-spot quick diagnosis milk cow's early pregnancy as claimed in claim 1 or 2 and the usefulness of oestrusing is characterized in that said solid phase carrier is detachable titer plate.
6. the EIA enzyme immunoassay testing cassete of an on-the-spot quick diagnosis milk cow's early pregnancy and the usefulness of oestrusing, comprise solid phase carrier and detection reagent two parts, detect with enzyme mark progesterone is arranged in the reagent, the progesterone standard items, antiprogestin antibody, chromogenic reagent and buffering reagent, it is characterized in that in the detectable with antiprogestin antibody being first antibody, at the first second antibody bag quilt of the plate hole inwall of solid phase carrier with anti-first antibody, then doing sealing with bovine serum albumin(BSA) handles, the last phosphate buffer 1 80 microlitres/hole of containing 15-25 milligram/100 milliliter thimerosal that adds in plate hole is sealed up for safekeeping standbyly, and said detection consists of with reagent:
A) enzyme mark progesterone, and to contain thimerosal and the volume/volume that weight/volume is 5%-20% be the antiseptic of the formalin of 1%-3%, with the phosphate buffer dissolving,
B) progesterone standard items, with the phosphate buffer preparation,
C) antiprogestin antibody is first antibody, for the connection thing with progesterone-6 beta-hydroxies-hemisuccinic acid salt and bovine serum albumin(BSA) is the antibody that the antigen immune rabbit is obtained, and to contain thimerosal and the volume/volume that weight/volume is 5%-20% be the antiseptic of the formalin of 1%-3%
D) second antibody of anti-first antibody is the anti-rabbit immunoglobulin G of goat or sheep, dilutes with carbonate buffer solution before using,
E) chromogenic reagent:
First developer is that volume content is 0.004% superoxol,
Second developer is to contain 0.05 mole of phosphoric acid salt-citrate buffer that weight/volume is 0.01% tetramethyl benzidine, pH5.0,
F) phosphate buffer contains 0.04 mole of phosphoric acid disodium hydrogen, sodium dihydrogen phosphate and 0.14 mole nacl, pH7-7.4,
G) carbonate buffer solution of forming by sodium carbonate-sodium bicarbonate, pH9-9.8
H) freeze-point depressant is the glycerine of 15-30% for volume ratio.
7. the EIA enzyme immunoassay testing cassete of the on-the-spot quick diagnosis milk cow's early pregnancy as claimed in claim 6 and the usefulness of oestrusing is characterized in that said freeze-point depressant is the glycerine of 10%-20%.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB021132631A CN1156701C (en) | 2002-01-23 | 2002-01-23 | Enzyme immunoanalysis test kit for fast in-situ diagnosis of milk cow's early pregnancy and oestrus |
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| CNB021132631A CN1156701C (en) | 2002-01-23 | 2002-01-23 | Enzyme immunoanalysis test kit for fast in-situ diagnosis of milk cow's early pregnancy and oestrus |
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| CN101598735B (en) * | 2009-07-28 | 2012-10-24 | 南京农业大学 | Method for measuring paper milk sample reproductive hormones concentration by enzyme-immunoassay method |
| CN101915847A (en) * | 2010-06-13 | 2010-12-15 | 黑龙江八一农垦大学 | Biosensor for detecting milk progesterone of cow |
| CN103134716A (en) * | 2011-11-30 | 2013-06-05 | 内蒙古蒙牛乳业（集团）股份有限公司 | Pretreatment method and detection method for detecting progesterone content in lactobacillis beverage |
| CN103134715A (en) * | 2011-11-30 | 2013-06-05 | 内蒙古蒙牛乳业（集团）股份有限公司 | Pretreatment method and detection method for detecting prolactin content in lactobacillis beverage |
| CN103163291A (en) * | 2013-03-09 | 2013-06-19 | 黑龙江八一农垦大学 | Kit and operation method thereof |
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