CN105675890B - A kind of device and method of test experience mouse pregnancy - Google Patents
A kind of device and method of test experience mouse pregnancy Download PDFInfo
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- CN105675890B CN105675890B CN201511028901.2A CN201511028901A CN105675890B CN 105675890 B CN105675890 B CN 105675890B CN 201511028901 A CN201511028901 A CN 201511028901A CN 105675890 B CN105675890 B CN 105675890B
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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Abstract
The invention discloses a kind of device and method of test experience mouse pregnancy, including hollow detection box, detector bar, slide switch, colloidal gold strip and observation window, detector bar is accommodated in detection box, detector bar outlet is provided with detection box, sliding groove is provided with the wall of detection box side, slide switch outside the wall of detection box side and is fixed on detector bar through sliding groove, detector bar is controlled to pass in and out by slide switch, colloidal gold strip is fixed in detection box, the sample contact tip of colloidal gold strip foremost is extend out to outside detection box, the invention is main by using female mice vaginal anatomy feature, male female mice mating behavior feature and male mouse sperm biological property, with reference to a variety of detection techniques, reach the purpose of test experience mouse pregnancy.The present invention have it is easy to detect, stress not, the degree of accuracy height, the advantages of data reliability is high.
Description
Technical field
The invention belongs to experimental animal cyesiognosis technology and immunological technique field, and in particular to a kind of test experience mouse
The device and method of pregnancy.
Background technology
Experimental animal is the animal for being engaged in scientific research, teaching, production, detection and its scientific experiment, is widely used in
In terms of biomedical, medical research and development, teaching experiment, biological detection, very important effect is played in life science.
Because the gene similarity of mouse and people have 99%, and the breeding and raising of mouse and rat are relatively inexpensive, and breeding is rapid.Mesh
Before, it is mouse or rat to have about 9 in every 10 experimental animals, and application field includes immunology, oncology, physiology.Disease
Of science, embryology and toxicology etc., and application in Neuscience is also being on the increase, and experimental mouse is also into experimental animal
Synonym.
The breeding of experimental mouse is the key of experimental animal production and experimentation.In experimental mouse reproductive process, be pregnant day
Number and pregnant time are to determine experimental mouse point cage feeding time and test deciding factor prepared by pregnant mouse.Accurate cyesiognosis
Experimental animal manufacturer and researcher can be made to grasp accurate pregnant time, it is more rationally effective to arrange cultured space,
And the utilization rate of missing inspection raising experimental animal is further reduced, and reduce feeding cost.Conventional experimental mouse is pregnant at present examines
Disconnected method has cloudy bolt range estimation detection method and vagina sperm plate coating checking method.
(1) cloudy bolt detection method：Cloudy bolt is a kind of white serosity material that mouse is formed after mating 4-5min, is blocked in
Vagina is to the intracavitary of cervix, and cloudy bolt is typically in post-coitum 12-24h Automatic-fallings.Cloudy bolt detection method is exactly after mouse mates
M seq is main at present to be determined by estimating and detecting using whether cloudy bolt is checked as its mating and the mark of gestation.
Range estimation is exactly directly to use eye observation, and detection, which needs to hold detector bar, touches mouse introitus, contradicts sense if any obvious and then shows have
Cloudy bolt, without cloudy bolt if no conflict sense and detector bar unresisted can be inserted.This method is simple and convenient, but because the cloudy bolt of mouse is big
It is small to differ, introitus is then protruded from greatly is easy to observation, and it is small, gone through in cervix, it is necessary to open introitus, therefore
It is difficult to make a definite diagnosis only by range estimation, and the probability of missing inspection is also very big so that feeding cost is improved, and is caused production and is wasted.Detection is checked
Detector bar is mostly made by oneself, easily produces larger stimulate in detection process to female mice.Pregnant mouse caused by missing inspection and nascent
Mouse is because that can not determine index of correlation, it is also necessary to handle, and there is also negative influence in terms of animal welfare.
(2) vagina sperm plate coating checking method：M seq softly revolves the cotton swab for speckling with physiological saline after mouse mates
Enter the vagina of female mice, rotation is taken out after turning around, then by cotton swab smear, pap staining microscopy.Although this method accuracy is higher,
Program is cumbersome.In actually large-scale production and experimentation, because size of animal is more, it need to take considerable time, and it is impracticable.
The content of the invention
In order to solve the deficiency that can not quickly and easily and be accurately detected under existence conditions, present invention design provides a set of comprehensive
Quick detection method, with it is easy to carry, detect quick and precisely, it is simple to operate the characteristics of.
Realizing the technical scheme of the object of the invention is：
A kind of device of test experience mouse pregnancy, including hollow detection box, detector bar, slide switch, colloid gold test paper
Bar and observation window, described detector bar are accommodated in detection box, are provided with detector bar outlet, detection box side wall and are provided with detection box
Sliding groove, slide switch outside the wall of detection box side and is fixed on detector bar through sliding groove, and detector bar is by slide switch control
System turnover, described colloidal gold strip is fixed in detection box, and the sample contact tip of colloidal gold strip foremost stretches out
To outside detection box, colloidal gold strip includes absorption pad, nitrocellulose filter, colloidal gold pad, sample pad, pH test paper layer and PVC
Bottom plate, the nitrocellulose filter is provided with detection line and nature controlling line, and described sample pad sample is located at as sample contact tip
Foremost, colloidal gold pad, nitrocellulose filter and absorption pad are pasted onto PVC bottom plates to colloidal gold strip successively from front to back again
On, described pH test paper layer is connected on absorption pad stage casing, detection box and is provided with observation window, and described observation window is opened in collaurum
The top of the detection lines of test strips, nature controlling line and pH test paper layer, described colloidal gold pad is coated with recognizing for colloid gold label
The mouse AsAb of mouse sperm antigen, the detection line is coated with mouse AsAb, and the nature controlling line is coated with goat and resisted
Mouse IgG.
A kind of device of described test experience mouse pregnancy, the described mouse AsAb that can recognize mouse sperm antigen is
It is extracted from immune infertility mouse seminal fluid.
A kind of device of described test experience mouse pregnancy, in addition to test paper protection cap, test paper protection cap are covered in detection box
On, to protect the colloidal gold strip extending out to outside detection box, and test paper protection cap is opposite in detection box with detector bar outlet
Two ends.
A kind of device of described test experience mouse pregnancy, in addition to test paper fixed projection, described test paper fixed projection
For hollow prismoid, test paper fixed projection, which is fixed on, to be extend out at the colloidal gold strip outside detection box, colloidal gold strip
Stretched out from the upper bottom surface of test paper fixed projection.
A kind of method of test experience mouse pregnancy, utilizes the device as described in claim 1-4 is any, including following step
Suddenly：
Step 1：First look at dams private parts to be measured whether there is macroscopic cloudy bolt, have, complete to detect and be judged as bosom
Pregnant, otherwise the detector bar on extending apparatus, to being detected inside dams private parts to be measured, is judged as there is the moon if having obstruction to feel
Bolt, completes to detect and be judged as pregnancy, otherwise performs step 2；
Step 2：Test paper is inserted toward female mice intravaginal to be measured, and test paper is detected into contact tip and dams vagina to be measured
Internal secretion is fully contacted, and then reads the result shown on test paper, if detection pH value is in neutrality, PH test paper is brown, and
Generation AsAb immune response is that two red instruction lines are presented in immunity test strip, then is strong positive, is judged as pregnancy；If
It is in faintly acid to detect pH value, and PH test paper is red or orange, and sends AsAb immune response immunity test strip presentation two
Bar red instruction line is then the positive, is judged as pregnancy；If detecting pH value in neutrality, PH test paper is brown, and immune response does not occur
It is then false positive, is judged as not being pregnant；If it is in faintly acid to detect pH value, PH test paper is red or orange, and immune response does not occur
It is then feminine gender, is judged as not being pregnant.
The technical effects of the invention are that, the present invention is using probe method, three kinds of sides of colloid gold test paper method and pH value determination method
The appraisement system that method is combined.And develop a kind of new colloid gold test paper and detection coupling apparatus.
Colloidal gold immunochromatographimethod technology is novel immune determination techniques, mainly for detection of various diseases.The technology can be with
Reduce analysis time and sample collection amount, improve detection sensitivity and the degree of accuracy.Mouse AsAb (AsAb) be immunity not
A kind of pathologic antibody that the male mouse educated produces, it can be combined with sperm antigen.Damxung mouse is mated with female mice after ejaculation, and meeting exists
Female mice intravaginal, which is remained, contains a large amount of sperm antigens in a large amount of seminal fluid, seminal fluid.The present invention is proposed a kind of using anti-by the principle
The qualitative checking method that sperm antibody is combined with sperm antigen carries out cyesiognosis to experimental mouse.
The seminal fluid pH range of normal hero mouse is in 6.4-7.5, in neutrality, but in oestrus, female mice intravaginal lactobacillus
Content table makes the reduction of vagina environment pH value, scope is in 5.6-4.5, in faintly acid up to increasing.Therefore, Damxung mouse enters with dams
After row mating ejaculation, seminal fluid has the effect of pH value neutralization with vagina environment liquid, and pH value can change.The present invention passes through
This principle, is detected to pH value simultaneously on Test paper.Further verify the accuracy of female mice gestation.
During detection, if intravaginal remains sperm antigen, detection line will not be formed on red precipitate line, nature controlling line, formed
Red precipitate line；If intravaginal residual is respectively formed on red precipitate line without sperm antigen, detection line and nature controlling line.
Brief description of the drawings
Fig. 1 is the structural representation of apparatus of the present invention；
Fig. 2 is the structural representation of test strips；
Wherein 1 is detector bar；2 be slide switch；3 be test paper protection cap；4 be colloidal gold strip；5 be that test paper fixation is prominent
Rise；6 be reading display screen；7 be test paper contact tip；8 be sliding groove；9 be absorption pad；10 be PVC bottom plates；11 be collaurum
Pad；12 be detection line；13 be nature controlling line；14 be nitrocellulose filter；15 be PH test paper layer；16 be absorption pad.
Embodiment
Illustrate the present invention with reference to embodiment, the scheme of embodiment described here does not limit application of the present invention.
The device of the present embodiment includes hollow detection box, detector bar, slide switch, colloidal gold strip and observation window,
Detector bar is accommodated in detection box, is provided with detection box on detector bar outlet, detection box side wall and is provided with sliding groove, slide switch is set
In detecting outside the wall of box side and being fixed on through sliding groove on detector bar, detector bar is controlled to pass in and out by slide switch, colloid gold test paper
Bar is fixed in detection box, and the sample contact tip of colloidal gold strip foremost is extend out to outside detection box, colloidal gold strip
Including absorption pad, nitrocellulose filter, colloidal gold pad, sample pad, pH test paper layer and PVC bottom plates, on the nitrocellulose filter
Provided with detection line and nature controlling line, sample pad sample is located at colloidal gold strip foremost as sample contact tip, colloidal gold pad, nitre
Acid cellulose film and absorption pad are pasted onto on PVC bottom plates successively from front to back again, and pH test paper layer is connected to absorption pad stage casing, detects
Observation window is provided with box, observation window is opened in the top of detection line, nature controlling line and the pH test paper layer of colloidal gold strip, collaurum
What pad was coated with colloid gold label can recognize the mouse AsAb of mouse sperm antigen, and detection line is coated with mouse AsAb,
Nature controlling line is coated with mountain sheep anti-mouse igg.
The present embodiment can recognize that the mouse AsAb of mouse sperm antigen is to be extracted from immune infertility mouse seminal fluid.Below
Provide specific mouse AsAb preparation process：
The preparation and purifying of AsAb：With reference to the method in the patent No. 200710190243.6：Collect immunity not
The testicular cell of mouse is educated, membrane antigen is extracted with NP-40, then with ultrasonic grind, supernatant is removed after ultracentrifugation, as
Crude antigen A liquid.With the testicular cell immunization experiment animal of immune infertility mouse, antiserum is taken, is exempted from through salting out method runic γ
Epidemic disease globulin, claims immune serum IgG；Take simultaneously not by the pooled serum of this kind of immune experimental animal, through salting out method runic γ
Immunoglobulin, claims non-immune serum IgG；With the testicular cell immunization experiment animal of normal mouse, antiserum is taken, through saltouing
Method runic γ immunoglobulins, claim anti-mouse whole serum IgG.With Sepharose-4B Ago-Gels respectively with above-mentioned immune blood
Clear IgG, non-immune serum IgG and anti-mouse whole serum IgG are coupled into immunoaffinity chromatography A posts, B posts and C posts.Remove Crude antigen A
Liquid crosses affinity chromatography A posts, collection and the antigenic substance that IgG is combined on A posts, is Crude antigen B liquid after concentration；The latter is excessively affine layer
Analyse B posts, collect not with the antigenic substance combined on B posts, after concentration be antigens c liquid；C liquid after affinity chromatography C posts, collect not with
The antigenic substance being combined on C posts, refines antigen liquid after concentration.
The mice serum of immune infertility is collected, it is immune with salting out method and DEAE52 cellulose column IgG purification types and IgA types
Globulin, and respectively rough IgG types antibody D liquid and rough IgA types antibody D liquid.The serum solution of normal mouse is collected, is collected not
The testicular cell of mouse containing purpose antigen, and it is prepared into testicular cell lysate.With Sepharose-4B Ago-Gels point
Testicular Cells lysate coupling not with refined antigen liquid, the mixed semen without purpose antigen and without purpose antigen
Into affinity chromatography D posts, E posts and F posts.Take rough IgG types or IgA type antibody D liquid to cross affinity chromatography D posts, collect with D posts itself
It is rough IgG types or IgA type antibody E liquid after the antibody materials that antigen is combined, concentration；The latter crosses affinity chromatography E posts, collects not
With being rough IgG or IgA types antibody F liquid after the antibody materials that albumen is combined on E posts, concentration；F liquid after affinity chromatography F posts,
Collect not with obtaining refined mouse AsAb after the antibody materials that albumen is combined on F posts, concentration, vacuum freezedrying into
Saved backup after powder.
The present embodiment device also includes test paper protection cap, and test paper protection cap is covered on detection box, and detection is extend out to protect
Colloidal gold strip outside box, and test paper protection cap and detector bar exported in the opposite two ends of detection box.
In order to fix test paper, the present embodiment also includes test paper fixed projection, and test paper fixed projection is hollow prismoid, examination
Paper fixed projection, which is fixed on, to be extend out at the colloidal gold strip outside detection box, and colloidal gold strip is upper from test paper fixed projection
Bottom surface is stretched out.
The present embodiment is used for the method that test experience mouse is pregnant, and using above-mentioned device, comprises the following steps：
Step 1：First look at dams private parts to be measured whether there is macroscopic cloudy bolt, have, complete to detect and be judged as bosom
Pregnant, otherwise the detector bar on extending apparatus, to being detected inside dams private parts to be measured, is judged as there is the moon if having obstruction to feel
Bolt, completes to detect and be judged as pregnancy, otherwise performs step 2；
Step 2：Test paper is inserted toward female mice intravaginal to be measured, and test paper is detected into contact tip and dams vagina to be measured
Internal secretion is fully contacted, and then reads the result shown on test paper, if detection pH value is in neutrality, PH test paper is brown, and
Generation AsAb immune response is that two red instruction lines are presented in immunity test strip, then is strong positive, is judged as pregnancy；If
It is in faintly acid to detect pH value, and PH test paper is red or orange, and sends AsAb immune response immunity test strip presentation two
Bar red instruction line is then the positive, is judged as pregnancy；If detecting pH value in neutrality, PH test paper is brown, and immune response does not occur
It is then false positive, is judged as not being pregnant；If it is in faintly acid to detect pH value, PH test paper is red or orange, and immune response does not occur
It is then feminine gender, is judged as not being pregnant.
The present invention is compared with cloudy bolt ocular estimate and vagina sperm plate coating checking method
Having looked into bolt has ear tag number but CD-1 female mices not of uniform size 30, and the female mice that do not mate 15 is randomly divided into 3 groups, i.e.,
Invention group, range estimation group and smear group, every group has bolt female mice 10, and do not mate mouse 5.Every group of experience quite tests people
Member is checked.Three groups of experimenter's inspection results are such as shown in Table 1.
Table 1 detects contrast table
| Group | Look into bolt number (only) | Time (second) | Accuracy rate (%) |
| Range estimation group | 6 | 147 | 60% |
| Smear group | 9 | 732 | 90% |
| Invention group | 9 | 321 | 90% |
As known from Table 1, present invention group accuracy rate is apparently higher than range estimation group, and the review time is considerably less than smear group.From reality
Show in production practices, of the present invention group accurately can carry out cyesiognosis in efficient quick to experimental mouse.
Claims (4)
1. a kind of device of test experience mouse pregnancy, it is characterised in that including hollow detection box, detector bar, slide switch, glue
Body gold test paper strip and observation window, described detector bar are accommodated in detection box, and detector bar outlet, detection box side are provided with detection box
Sliding groove is provided with wall, slide switch outside the wall of detection box side and is fixed on detector bar through sliding groove, and detector bar is by sliding
Dynamic switch control turnover, described colloidal gold strip is fixed in detection box, the sample contact of colloidal gold strip foremost
Termination is extend out to outside detection box, and colloidal gold strip includes absorption pad, nitrocellulose filter, colloidal gold pad, sample pad, pH test paper
Layer and PVC bottom plates, the nitrocellulose filter are provided with detection line and nature controlling line, and described sample pad sample is used as sample contact jaw
Head is located at colloidal gold strip foremost, and colloidal gold pad, nitrocellulose filter and absorption pad are pasted onto PVC successively from front to back again
On bottom plate, described pH test paper layer is connected on absorption pad stage casing, detection box and is provided with observation window, and described observation window is opened in glue
The top of the detection line of body gold test paper strip, nature controlling line and pH test paper layer, described colloidal gold pad is coated with the energy of colloid gold label
The mouse AsAb of mouse sperm antigen is recognized, the detection line is coated with mouse AsAb, and the nature controlling line is coated with mountain
Sheep anti-mouse igg, the described mouse AsAb that can recognize mouse sperm antigen is to be extracted from immune infertility mouse seminal fluid.
2. the device of a kind of test experience mouse pregnancy according to claim 1, it is characterised in that also protected including test paper
Lid, test paper protection cap is covered on detection box, and to protect the colloidal gold strip extending out to outside detection box, and test paper protection cap is with visiting
Rod outlet is surveyed in the opposite two ends of detection box.
3. the device of a kind of test experience mouse pregnancy according to claim 1, it is characterised in that also fix prominent including test paper
Rise, described test paper fixed projection is hollow prismoid, test paper fixed projection is fixed on the collaurum extending out to outside detection box
At test strips, colloidal gold strip stretches out from the upper bottom surface of test paper fixed projection.
4. a kind of method of test experience mouse pregnancy, it is characterised in that utilize the device as described in claim 1-3 is any, bag
Include following steps：
Step 1：First look at dams private parts to be measured whether there is macroscopic cloudy bolt, have, complete to detect and be judged as pregnancy, it is no
Then the detector bar on extending apparatus, to being detected inside dams private parts to be measured, is judged as there is cloudy bolt if having obstruction to feel, completes
Detect and be judged as pregnancy, otherwise perform step 2；
Step 2：Test paper is inserted toward female mice intravaginal to be measured, and test paper is detected inside contact tip and dams vagina to be measured
Secretion is fully contacted, and then reads the result shown on test paper, if detection pH value is in neutrality, PH test paper is brown, and is occurred
AsAb immune response is that two red instruction lines are presented in immunity test strip, then is strong positive, is judged as pregnancy；If detection
PH value is in faintly acid, and PH test paper is red or orange, and it is red to send AsAb immune response immunity test strip presentation two
Color instruction line is then the positive, is judged as pregnancy；If detecting pH value in neutrality, PH test paper is brown, and immune response does not occur is then
False positive, is judged as not being pregnant；If it is in faintly acid to detect pH value, PH test paper is red or orange, and immune response does not occur is then
Feminine gender, is judged as not being pregnant.
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| DD295246A5 (en) * | 1990-04-12 | 1991-10-24 | Zi Fuer Molekularbiologie,Ag Patent- Neuererwesen,De | ENZYME IMMUNOASSAY FOR THE IDENTIFICATION OF SPERMASPURES THROUGH THE DETECTION OF SPERM-SPECIFIC 30 KDA ANTIGEN |
| US5480799A (en) * | 1993-12-10 | 1996-01-02 | The University Of North Carolina At Chapel Hill | Sperm antigen corresponding to a sperm zona binding protein autoantigenic epitope |
| JP2915369B2 (en) * | 1997-01-14 | 1999-07-05 | 日本農産工業株式会社 | Rat sperm fertility test method |
| CN101158681A (en) * | 2007-11-23 | 2008-04-09 | 东南大学 | Complementary type affinity chromatography purification method of autoantibody |
| CN203083999U (en) * | 2013-01-25 | 2013-07-24 | 上海惠宝生物科技有限公司 | Gold immnnochromatography sperm density detection kit |
| CN203206892U (en) * | 2013-04-27 | 2013-09-25 | 全胜 | Mouse pregnancy testing feeding cage |
| CN104865381A (en) * | 2015-04-22 | 2015-08-26 | 河南省康星药业股份有限公司 | Pig early pregnancy factor colloidal gold test paper card and preparation method thereof |
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