CN108152515A - A kind of test paper detected for heat bitch progesterone and preparation method thereof - Google Patents
A kind of test paper detected for heat bitch progesterone and preparation method thereof Download PDFInfo
- Publication number
- CN108152515A CN108152515A CN201711431534.XA CN201711431534A CN108152515A CN 108152515 A CN108152515 A CN 108152515A CN 201711431534 A CN201711431534 A CN 201711431534A CN 108152515 A CN108152515 A CN 108152515A
- Authority
- CN
- China
- Prior art keywords
- progesterone
- concentration
- detection
- colloid gold
- line
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- 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
- G01N33/531—Production of immunochemical test materials
-
- 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
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
Abstract
The present invention provides a kind of for test paper of heat bitch progesterone detection and preparation method thereof, the technical solution is based on colloidal gold immunochromatographimethod technology, using competition law, by the progesterone antigen composition of improvement, the anti-mouse IgG of sheep (rabbit) is fixed on ribbon on nitrocellulose filter (NC), antiprogestin monoclonal antibody colloid gold label object is fixed on bonding pad, when sample to be checked adds to test strips well, it is moved forward by capillarity, it reacts to each other after colloid gold label object on dissolving bonding pad, it is moved to the region of fixed antigen or antibody again, object to be checked occurs specific binding therewith again with colloid gold label object and is trapped, detection is gathered in take, colour developing result can be observed by the naked eye.Present invention detection is simple and quick, without complex operations skill and special installation, and easy to carry, in addition, the present invention can realize progesterone semi-quantitative analysis, it is as a result sensitive, accurate, it is more objective effectively that bitch heat condition is evaluated with it.
Description
Technical field
The present invention relates to colloidal gold immunochromatographimethod technical fields, and in particular to a kind of examination for the detection of heat bitch progesterone
Paper and preparation method thereof.
Background technology
In canine feeding work, judge whether heat is that the important prerequisite of breeding work is unfolded to bitch.Bitch is carried out
Detection of oestrus can effectively judge the heat stage of bitch, to determine suitable breeding opportunity, improve conception rate and farrowing rate,
It is horizontal with important production practices meaning to improving dog reproductive performance and breeding.The conventional method of detection of oestrus has external observation
Method, examination feelings method, vaginal epithelial cell detection method, electrical measurement resistance method etc..External observation method needs testing staff to have abundant theory
And practical experience, it is not high for atypical estrus or the unconspicuous bitch judging nicety rate of symptom of estrus.Feelings method is tried easily by public dog
Desire of breeding and ambient environmental factors influence, and utilization rate is not high in real work.Vaginal epithelial cell detection method, electrical measurement resistance method
Corresponding instrument and equipment and reagent are needed, easily bitch is damaged, therefore is difficult to promote in real work.
Progesterone is the biologically active main progestational hormone by ovarian secretion, and bitch is in different heat periods, in vivo
Progesterone value is significantly altered, and certain rule is presented, therefore the estrus behavior available for evaluating bitch on a molecular scale.
At present, the modern instrument method for applying to detect progesterone has high performance liquid chromatography (HPLC), liquid chromatography mass combined instrument (LC-
MS/MS), the methods of radio immunoassay (RIA), Chemiluminescence immunoassay (CLIA), wherein due to HPLC, LC-MS/MS sample
The reasons such as present treatment hardly possible, testing cost height, are not suitable for field quick detection.Progesterone is detected using RIA, CLIA, although detection
High sensitivity, but expensive necessary instrument is needed to detect.
Invention content
The present invention is directed to be directed to the technological deficiency of the prior art, a kind of test paper for the detection of heat bitch progesterone is provided,
To solve the technical issues of heat bitch progesterone detection method in the prior art is complex, detection is inconvenient.
Another technical problem to be solved by the present invention is that how to ensure detection effect during easy detection method is established
The sensitivity and accuracy of fruit.
The invention solves another technical problem be to lack a kind of preparation method of above-mentioned test paper in the prior art.
For realization more than technical purpose, the present invention uses following technical scheme：
A kind of test paper for the detection of heat bitch progesterone, including sample pad, bonding pad, detection line T, nature controlling line C, nitric acid
Cellulose membrane, blotting paper and PVC backings, wherein nitrocellulose filter are attached on PVC backings, and the one of the nitrocellulose filter
End, which is sticked, sample pad and bonding pad, and the other end, which sticks, blotting paper, and detection line T and nature controlling line C are located at the nitrocellulose
On the surface of film, the distribution of the sample pad, bonding pad, detection line T, nature controlling line C, blotting paper on nitrocellulose filter
Position is followed successively by sample pad, bonding pad, detection line T, nature controlling line C, blotting paper, and the spacing of the detection line T and nature controlling line C is
0.7cm；
The heparin of a concentration of 15mg/mL, the matrimony vine acid sodium of a concentration of 22mg/mL, dense is fixed in the sample pad simultaneously
Spend the ammonium sulfate for 400mg/mL；
Antiprogestin monoclonal antibody-colloid gold label object of a concentration of 6 μ g/mL is fixed on the bonding pad；
The detection line T contain the progesterone holoantigen of a concentration of 4mg/mL, a concentration of 12mg/mL Sodium Hyaluronate and
The 1,2- pentanediols of a concentration of 7mg/mL；
The nature controlling line C is the sheep anti-mouse igg or rabbit anti-mouse igg of a concentration of 0.4mg/mL.
Preferably, the antiprogestin monoclonal antibody-colloid gold label object is prepared by the following method：
1) gold chloride is reduced into the colloid gold particle solution that grain size is 25~35nm with reducing agent；
2) the colloid gold particle solution and antiprogestin monoclonal antibody are pressed 1:0.001(mL:G) ratio mixing, obtains
To colloidal gold composite, through closing and being concentrated to give progesterone monoclonal antibody-colloid gold label object.More preferably, the colloidal gold
The grain size of particle is 30nm.
Preferably, the reducing agent is trisodium citrate.
Preferably, step 1) specifically includes following operation：The gold chloride for taking 100mL a concentration of 0.01% (w/v) is water-soluble
Liquid is heated to boiling, and adds in a concentration of 1% (w/v) trisodium citrate aqueous solutions of 1.45mL thereto and is stirred continuously, treats solution
After color becomes aubergine, stop heating after continuing heating two minutes, be cooled to room temperature.
Preferably, the colloid gold particle solution obtained by step 1) has maximum absorption peak under 525nm wavelength conditions.
Preferably, step 2) specifically includes following operation：The pH value of the colloid gold particle solution is adjusted to 6, with glue
Body gold particle solution and antiprogestin monoclonal antibody 1:0.001(mL:G) ratio, under agitation by antiprogestin monoclonal
Antibody is added in by liquid into the colloid gold particle solution, stirs 60min, then adds in colloid gold particle solution usage thereto
The PEG20000 aqueous solutions of 0.1 times of volume, a concentration of 1% (w/v) continue to add in colloidal gold thereto after stirring 30min
The BSA aqueous solutions of grain 0.1 times of volume of solution usage, a concentration of 10% (w/v), close 30min, are then turned with 8000rpm
Speed centrifuges 30min under the conditions of 4 DEG C, takes solid phase, is resuspended to a concentration of 6 μ g/mL with re-suspension liquid and resisted to get to progesterone monoclonal
Body-colloid gold label object；
The re-suspension liquid is containing 1%BSA, 2% sucrose, 0.05%PEG20000,0.1%NaN3, it is a concentration of
The phosphate buffer solution of 0.02mol/L；The pH of the phosphate buffer solution is 7.4.
The present invention additionally provides the preparation method of above-mentioned test paper simultaneously, includes the following steps：Will contain 15mg/mL heparin,
22mg/mL matrimony vine acid sodium, 400mg/mL ammonium sulfate mixed solution be fixed in sample pad, by the antiprogestin of a concentration of 6 μ g/mL
Monoclonal antibody-colloid gold label object is fixed on bonding pad, using three-dimensional specking platform will contain 4mg/mL progesterone holoantigen,
12mg/mL Sodium Hyaluronates, 7mg/mL1, the mixed solution of 2- pentanediols are fixed on nitrocellulose filter as detection line T,
The sheep anti-mouse igg of a concentration of 0.4mg/mL or rabbit anti-mouse igg are fixed on nitrocellulose filter using three-dimensional specking platform and made
For nature controlling line C, detection line T is kept then to be attached on nitrocellulose filter with nature controlling line C spacing 0.7cm, 37 DEG C of dry 8h
On PVC backings, sample pad and bonding pad are attached into one end of nitrocellulose filter, blotting paper is attached into the cellulose nitrate
The other end of plain film, point of the sample pad, bonding pad, detection line T, nature controlling line C, blotting paper on nitrocellulose filter
Cloth position is followed successively by sample pad, bonding pad, detection line T, nature controlling line C, blotting paper, will integrally be cut into several bars.
The present invention additionally provides above-mentioned test paper for detecting the application of heat bitch progesterone simultaneously, includes the following steps：It takes
Tested animal serum is added in the sample pad of the test paper, and the colour developing situation of detection line T, nature controlling line C are observed after 15min；Work as inspection
When survey line T colored intensities are more than nature controlling line C, progesterone content is less than or equal to 5ng/mL in serum；When detection line T colored intensities are less than
Progesterone content is 10~20ng/mL in serum during nature controlling line C；Progesterone content is more than 20ng/ in serum when detection line T does not develop the color
mL。
In above technical scheme, the detection line T colored intensities are more than nature controlling line C, refer to visually observe lower color inspection
The color depth of survey line T is more than the color depth of nature controlling line C.
Preferably, above-mentioned test paper preserves before use in hermetically drying condition.
The present invention provides a kind of for test paper of heat bitch progesterone detection and preparation method thereof, which is based on
Using competition law, the progesterone antigen composition of improvement, the anti-mouse IgG of sheep (rabbit) are consolidated with ribbon for colloidal gold immunochromatographimethod technology
It is scheduled on nitrocellulose filter (NC), antiprogestin monoclonal antibody-colloid gold label object is fixed on bonding pad, when sample to be checked
Test strips well is added to, is moved forward by capillarity, is reacted to each other after dissolving the colloid gold label object on bonding pad,
It is moved to the region of fixed antigen or antibody again, object to be checked occurs specific binding therewith again with colloid gold label object and cut
It stays, is gathered in detection and takes, colour developing result can be observed by the naked eye.
It was found that, since the blood serum sample of gained under the conditions of routine sampling is there are still certain solidification phenomenon, thus
The capillary rise of sample is acted on there are certain influence, and since the coagulation grade of different samples is unequal, thus is difficult to
Ensure the repeatability of color developing effect.For this problem, the present invention is secured in sample pad with good external anticoagulation
The heparin of effect and matrimony vine acid sodium, have been effectively ensured the repeatability of test paper testing result.It is in addition, present invention discover that big in serum
Protein ingredient is measured on progesterone antigen and antibody association reaction there may be influence, therefore the present invention fixes excess of sulfur in sample pad
Sour ammonium inactivates most of albuminous degeneration in sample, so as to not influence subsequent luminescence-producing reaction effect.In addition, present invention meaning
Outer discovery carries out compounding improvement on the basis of progesterone holoantigen, and may exist on color developing effect influences, and thinks in line with this experiment
Road, the present invention investigated detection line T Multiple components combination, finally determine containing progesterone holoantigen, Sodium Hyaluronate and
The assembled scheme of 1,2- pentanediols；By this improvement, colour developing degree is exaggerated under the premise of testing result is not influenced, is convenient for
Observe the aberration of detection line T and nature controlling line C.
The present invention mainly has following technical advantage：(1) it does not need to bind bitch in detection process, avoids bitch
In the case that stress be to adverse effect that its physiology is brought；(2) it is not required to, to sampling inside bitch vagina, keep away in detection process
Exempt to heat bitch vagina internal contamination or infection；(3) it is not required to by large-scale instrument and equipment, it is spent with low；(4) blood is alleviated
Final proof product condense phenomenon, while reduce influence of the other compositions to testing result in sample；(5) detection process is without progesterone mark
Product are easy to operate, quick, it is only necessary to 15-20min as instruction；(6) sensitivity is higher, and up to 5ng/mL, testing result is more just
In observation；(7) room temperature preservation, the detection of single dog, can scene be detected.(8) bitch is judged by the detection to progesterone content
The method of heat is more objective, accurate.
Description of the drawings
Fig. 1 is the detection principle diagram of test paper of the present invention.
Fig. 2 is the structure diagram of test paper of the present invention.
Fig. 3 is pattern detection result figure in the specific embodiment of the invention；Wherein, mark-on serum sample concentration from left to right
Respectively 0,5,10,15,20,25,30ng/mL.
Specific embodiment
The specific embodiment of the present invention will be described in detail below.In order to avoid excessive unnecessary details,
It will not be described in detail in following embodiment to belonging to well known structure or function.
Approximating language used in following embodiment can be used for quantitative expression, show in the feelings for not changing basic function
Quantity is allowed to have certain variation under condition.Therefore, it is not limited to this accurately with the modified numerical value of the language such as " about ", " left and right " institute
Numerical value is in itself.In some embodiments, the range for allowing its modified numerical value in positive and negative 10 (10%) " about " is represented
Interior variation, for example, what " about 100 " represented can be any numerical value between 90 to 110.In addition, " the about first numerical value arrives
In the statement of second value ", the first and second numerical value two values are at about corrected.In some cases, approximating language
It may be related with the precision of measuring instrument.
In addition to being defined, technical and scientific term used in following embodiment has and fields technology people of the present invention
The identical meanings that member is commonly understood by.
Test reagent consumptive material used in following embodiment is conventional biochemical reagent unless otherwise specified；The experiment
Method is conventional method unless otherwise specified；Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result
It is averaged；% in following embodiment is mass percentage unless otherwise instructed.
Embodiment 1
1st, it is a kind of for test paper of heat bitch progesterone detection and preparation method thereof
The preparation of 1.1 colloidal golds
Colloidal gold be gold chloride under the action of reducing agent, can be grouped to a certain size gold particle, and since electrostatic is made
With and stablize electronegative hydrophobic sol solution.The basic principle of immune colloidal gold technique is that gold colloid surface is negatively charged, with egg
The positive charge group electrostatical binding of white matter molecule, the compound of formation do not interfere with the biological nature of protein.Present invention selection
Trisodium citrate prepares colloidal gold as reducing agent, and detailed process is as follows：100mL0.01% aqueous solution of chloraurate is taken in cleaning
It is heated to boiling in conical flask, rapidly joins 1.45mL1% trisodium citrates and is stirred continuously, treat that solution colour becomes aubergine
Afterwards, stop heating after continuing heating two minutes, be cooled to room temperature, 4 DEG C save backup.Whether this colloidal gold meets production requirement,
Except the color that detects by an unaided eye, uniformity and permeability, it is also necessary to be analyzed by ultra-violet and visible spectrophotometer, colloidal gold exists
Have maximum absorption peak at 525nm, meanwhile, electron microscope colour developing prepare colloid gold particle uniformity is good, granular size about 30nm.
The preparation of 1.2 colloidal gold labeled monoclonal antibodies
Colloidal gold pH value is adjusted to 6, under the action of constant speed stirrer uniform stirring, ultra-pure water dilution experiment room is homemade
Antiprogestin monoclonal antibody is added dropwise to 1/10 volume of colloidal gold in colloidal gold solution, and colloidal gold 1/10 is pressed after 60min
Volume adds in 1%PEG20000, and pressing 1/10 volume of colloidal gold after 30min adds in 10%BSA, after closing 30min, passes through centrifugation
(8kr/min, 4 DEG C, 30min) obtain the gold labeling antibody concentrated.Re-suspension liquid is added (containing 1%BSA, 2% sucrose, 0.05%
PEG20000,0.1%NaN3PH be 7.4 0.02mol/L phosphate buffer solution) be resuspended gold labeling antibody.
The preparation of 1.3 progesterone colloidal gold immune chromatography rapid detecting test paper strips
Progesterone colloidal gold immuno-chromatography test paper strip is detected, including sample pad, bonding pad, nitrocellulose filter (NC), water suction
Paper and PVC backings；Nitrocellulose filter is attached on PVC backings, and bonding pad and sample pad are attached on nitrocellulose filter and lean on
One end of nearly detection line, blotting paper are attached on nitrocellulose filter close to one end of nature controlling line；
Sample pad, bonding pad, detection line T, nature controlling line C, blotting paper are sequentially distributed on nitrocellulose filter；
In sample pad it is fixed be the heparin of a concentration of 15mg/mL, a concentration of 22mg/mL matrimony vine acid sodium, a concentration of
The ammonium sulfate of 400mg/mL；
Fixed on bonding pad is antiprogestin monoclonal antibody-colloid gold label object, a concentration of 6 μ g/mL；
Fixed on detection line T is the mixed solution containing progesterone holoantigen, wherein a concentration of 4.0mg/ of progesterone holoantigen
ML, hyaluronic acid na concn 12mg/mL, 1,2- pentanediol concentration 7mg/mL；
Fixed on nature controlling line C is the anti-mouse IgG of sheep (rabbit), a concentration of 0.4mg/mL；
The spacing of detection line T and nature controlling line C is 0.7cm；
Progesterone holoantigen composition, the anti-mouse IgG of sheep (rabbit) are passed through into instrument (BioDot XYZ, Irvine, CA, USA) point
It is not fixed on NC films and is used as detection line T and nature controlling line C, 37 DEG C of dry 8h；NC films are attached on PVC backings, sample pad, tied
It closes pad to be attached on close to one end of detection line T on NC films, blotting paper is attached on NC films close to one end of nature controlling line C；It will glue
PVC material be cut into the reagent strip of one fixed width；It is finally made test strips.
Progesterone colloidal gold immuno-chromatography test paper strip structure chart is shown in Fig. 2.
During detection, serum sample is added into test paper well, naked eye colour developing result after 15min.
The present invention is based on competition laws, i.e., progesterone antigen, the anti-mouse IgG of sheep (rabbit) are fixed on nitrocellulose filter with ribbon
(NC) on, antiprogestin monoclonal antibody-colloid gold label object is fixed on bonding pad, when sample to be checked adds to test strips sample-adding
Hole is moved forward by capillarity, is reacted to each other, then be moved to fixed after dissolving the colloid gold label object on bonding pad
The region of antigen or antibody, object to be checked occur specific binding therewith again with colloid gold label object and are trapped, and are gathered in detection
It takes, colour developing result can be observed by the naked eye.C lines have colour developing always in experimentation, when progesterone content is less than in serum
During 5ng/mL, enough colloid gold label objects and the T lines being fixed on NC films react to form strong red stripes, and C is compared in the colour developing of T lines
Line colour developing is deep；When progesterone content is higher than 10ng/mL in serum, appropriate or no anti-object of colloid gold label is with being fixed on NC films
T lines form weak red stripes or without band.It is specifically shown in Fig. 1.
2nd, experimental condition optimization and compliance test result
2.1 orthogonal test L9(3)4Determine the test strips optimal conditions used in this patent
Progesterone colloid gold immune Rapid detection test strip experimental factor water-glass in 1 serum of table
Note：+++ colour developing is stronger；++ colour developing is strong；+ colour developing is weak；Disappear line
Pass through orthogonal test L9(3)4Optimal conditions, which is, to be obtained to the optimization of experiment condition：6 μ g/mL of gold labeling antibody concentration；Knot
Close the upper 5 μ L of gold labeling antibody discharge rate of pad；The fixed progesterone holoantigen concentration 4mg/mL of detection line.
2.2 in optimal conditions test strips of the present invention actual sample is detected
2.2.1 serum mark-on
Progesterone mark product are separately added into negative serum sample, the concentration of mark-on serum is respectively：0、5、10、15、20、
25、30ng/mL.The mark-on serum sample of each concentration is taken to add to test strips well, naked eye colour developing result after 15min.Tool
Body result is shown in Fig. 3.
2.2.2 actual sample measures
Positive serum sample is taken to add to test strips well, naked eye colour developing result after 15min：The colour developing of detection line T
Intensity is weaker than nature controlling line C.
2.3 colloidal gold strip sensitivity tests
Take test strips made of the above, 15 samples of serum mark-on, chemiluminescence detection authentic specimen concentration, each
Sample is repeated 3 times, and is judged the detection sensitivity of test strips, as a result be see the table below：
2 colloidal gold strip sensitivity test of the present invention of table
Note：+++ colour developing is stronger；++ colour developing is strong；+ colour developing is weak；Disappear line
As can be seen from the above results, progesterone colloid gold immune of the present invention quickly detects the detection of progesterone test strips in serum
It is limited to 5ng/mL.
The specific test of 2.4 colloidal gold strips
Prepared test strips more than taking are separately added into tumer hydroxyl in negative serum (chemical luminescent detecting is feminine gender)
Progesterone, serine progesterone acetate, megestrol acetate, hydrocortisone, make its final concentration of 5,10,50,100,500ng/mL blood
Clear processing solution.Above-mentioned treatment fluid is added into test strips well, observation test strips develop the color situation so as to judge test paper after 15min
The specificity of item detection, the serum processing sample of each concentration do 3 repetitions.The results are shown in table below：
Progesterone colloid gold immune Rapid detection test strip specific test result in 3 serum of table
As can be seen from the above results, medroxyprogesterone acetate, serine progesterone acetate, megestrol acetate, hydrocortisone
These analogues do not generate cross reaction in serum of the present invention in the detection of progesterone colloid gold immune Rapid detection test strip.
The accuracy test of 2.5 colloidal gold strips
Progesterone colloidal gold immuno-chromatography test paper strip of the present invention is sxemiquantitative card.Prepared test strips, work as serum more than taking
Middle progesterone content is less than 5ng/mL, and sample detection result is considered as feminine gender, is considered as bitch and is in diestrum, proestrum or enters hair
Early stage feelings phase；When progesterone content reaches 5ng/mL, it is suitable to be considered as progesterone content in sample, at this time for the breeding of heat bitch it is best when
Machine can arrange heat bitch to breed；Hereafter dog progesterone content will continue to rise to 10ng/mL-20ng/mL, and maintain
4-6d.When progesterone content is more than 20ng/mL in serum, it is considered as that progesterone content in sample is higher, and dog has been enter into the heat later stage.
2.6 colloidal gold strip storage life is tested
Prepared test strips more than taking, kit preservation condition are 2-8 DEG C, and through the measure of 6 months, progesterone addition was practical
Serum measured value is within normal range (NR).Consider during transport and use, have improper preservation condition and occur, will try
Agent box is placed 6 days under 37 DEG C of preservation conditions, carries out accelerated stability test, the results showed that the colloidal gold strip indices
Comply fully with requirement.
Embodiment 2
A kind of test paper for the detection of heat bitch progesterone, including sample pad, bonding pad, detection line T, nature controlling line C, nitric acid
Cellulose membrane, blotting paper and PVC backings, wherein nitrocellulose filter are attached on PVC backings, and the one of the nitrocellulose filter
End, which is sticked, sample pad and bonding pad, and the other end, which sticks, blotting paper, and detection line T and nature controlling line C are located at the nitrocellulose
On the surface of film, the distribution of the sample pad, bonding pad, detection line T, nature controlling line C, blotting paper on nitrocellulose filter
Position is followed successively by sample pad, bonding pad, detection line T, nature controlling line C, blotting paper, and the spacing of the detection line T and nature controlling line C is
0.7cm；
The heparin of a concentration of 15mg/mL, the matrimony vine acid sodium of a concentration of 22mg/mL, dense is fixed in the sample pad simultaneously
Spend the ammonium sulfate for 400mg/mL；
Antiprogestin monoclonal antibody-colloid gold label object of a concentration of 6 μ g/mL is fixed on the bonding pad；
The detection line T contain the progesterone holoantigen of a concentration of 4mg/mL, a concentration of 12mg/mL Sodium Hyaluronate and
The 1,2- pentanediols of a concentration of 7mg/mL；
The nature controlling line C is the sheep anti-mouse igg or rabbit anti-mouse igg of a concentration of 0.4mg/mL.
On the basis of above technical scheme, meet the following conditions：
Antiprogestin monoclonal antibody-colloid gold label the object is prepared by the following method：
1) gold chloride is reduced into the colloid gold particle solution that grain size is 25~35nm with trisodium citrate；
2) the colloid gold particle solution and antiprogestin monoclonal antibody are pressed 1:0.001(mL:G) ratio mixing, obtains
To colloidal gold composite, through closing and being concentrated to give progesterone monoclonal antibody-colloid gold label object.
Meanwhile the present embodiment additionally provides above-mentioned test paper for detecting the application of bitch progesterone, includes the following steps：It takes and treats
Animal blood serum is surveyed, is added in the sample pad of the test paper, the colour developing situation of detection line T, nature controlling line C are observed after 15min；Work as detection
When line T colored intensities are more than nature controlling line C, progesterone content is less than or equal to 5ng/mL in serum；When detection line T colored intensities are less than matter
Progesterone content is 10~20ng/mL in serum when controlling line C；Progesterone content is more than 20ng/ in serum when detection line T does not develop the color
mL。
Embodiment 3
A kind of test paper for the detection of heat bitch progesterone, including sample pad, bonding pad, detection line T, nature controlling line C, nitric acid
Cellulose membrane, blotting paper and PVC backings, wherein nitrocellulose filter are attached on PVC backings, and the one of the nitrocellulose filter
End, which is sticked, sample pad and bonding pad, and the other end, which sticks, blotting paper, and detection line T and nature controlling line C are located at the nitrocellulose
On the surface of film, the distribution of the sample pad, bonding pad, detection line T, nature controlling line C, blotting paper on nitrocellulose filter
Position is followed successively by sample pad, bonding pad, detection line T, nature controlling line C, blotting paper, and the spacing of the detection line T and nature controlling line C is
0.7cm；
The heparin of a concentration of 15mg/mL, the matrimony vine acid sodium of a concentration of 22mg/mL, dense is fixed in the sample pad simultaneously
Spend the ammonium sulfate for 400mg/mL；
Antiprogestin monoclonal antibody-colloid gold label object of a concentration of 6 μ g/mL is fixed on the bonding pad；
The detection line T contain the progesterone holoantigen of a concentration of 4mg/mL, a concentration of 12mg/mL Sodium Hyaluronate and
The 1,2- pentanediols of a concentration of 7mg/mL；
The nature controlling line C is the sheep anti-mouse igg or rabbit anti-mouse igg of a concentration of 0.4mg/mL.
The embodiment of the present invention is described in detail above, but the content is only presently preferred embodiments of the present invention,
It is not intended to limit the invention.All all any modification, equivalent and improvement done in the application range of the present invention etc., should all
It is included within protection scope of the present invention.
Claims (8)
1. a kind of test paper for the detection of heat bitch progesterone, it is characterised in that including sample pad, bonding pad, detection line T, Quality Control
Line C, nitrocellulose filter, blotting paper and PVC backings, wherein nitrocellulose filter are attached on PVC backings, the cellulose nitrate
One end of plain film, which is sticked, sample pad and bonding pad, and the other end, which sticks, blotting paper, and detection line T and nature controlling line C are located at the nitre
On the surface of acid cellulose film, the sample pad, bonding pad, detection line T, nature controlling line C, blotting paper are in nitrocellulose filter
On distributing position be followed successively by sample pad, bonding pad, detection line T, nature controlling line C, blotting paper, the detection line T and nature controlling line C's
Spacing is 0.7cm；
The heparin of a concentration of 15mg/mL, the matrimony vine acid sodium of a concentration of 22mg/mL, a concentration of is fixed in the sample pad simultaneously
The ammonium sulfate of 400mg/mL；
Antiprogestin monoclonal antibody-colloid gold label object of a concentration of 6 μ g/mL is fixed on the bonding pad；
The detection line T contains the Sodium Hyaluronate and concentration of the progesterone holoantigen of a concentration of 4mg/mL, a concentration of 12mg/mL
1,2- pentanediols for 7mg/mL；
The nature controlling line C is the sheep anti-mouse igg or rabbit anti-mouse igg of a concentration of 0.4mg/mL.
A kind of 2. test paper for the detection of heat bitch progesterone according to claim 1, it is characterised in that the antiprogestin
Monoclonal antibody-colloid gold label object is prepared by the following method：
1) gold chloride is reduced into the colloid gold particle solution that grain size is 25~35nm with reducing agent；
2) the colloid gold particle solution and antiprogestin monoclonal antibody are pressed 1:0.001(mL:G) ratio mixing, obtains glue
Body Au composite, through closing and being concentrated to give progesterone monoclonal antibody-colloid gold label object.
A kind of 3. test paper for the detection of heat bitch progesterone according to claim 2, it is characterised in that the reducing agent
For trisodium citrate.
4. a kind of test paper for the detection of heat bitch progesterone according to claim 3, it is characterised in that step 1) is specific
Including following operation：The aqueous solution of chloraurate of 100mL a concentration of 0.01% (w/v) is taken to be heated to boiling, is added in thereto
A concentration of 1% (w/v) trisodium citrate aqueous solutions of 1.45mL are simultaneously stirred continuously, and after solution colour becomes aubergine, continue to add
Heat stops heating after two minutes, be cooled to room temperature.
5. a kind of test paper for the detection of heat bitch progesterone according to claim 4, it is characterised in that obtained by step 1)
Colloid gold particle solution under 525nm wavelength conditions have maximum absorption peak.
6. a kind of test paper for the detection of heat bitch progesterone according to claim 3, it is characterised in that step 2) is specific
Including following operation：The pH value of the colloid gold particle solution is adjusted to 6, is resisted with colloid gold particle solution and antiprogestin monoclonal
Body 1:0.001(mL:Antiprogestin monoclonal antibody is added dropwise to the colloid gold particle by ratio g) under agitation
In solution, 60min is stirred, is then added in thereto colloid gold particle 0.1 times of volume of solution usage, a concentration of 1% (w/v)
PEG20000 aqueous solutions, continue stir 30min after add in thereto 0.1 times of volume of colloid gold particle solution usage, it is a concentration of
The BSA aqueous solutions of 10% (w/v) close 30min, then centrifuge 30min under the conditions of 4 DEG C with the rotating speed of 8000rpm, take solid
Phase is resuspended to a concentration of 6 μ g/mL with re-suspension liquid to get to progesterone monoclonal antibody-colloid gold label object；
The re-suspension liquid is containing 1%BSA, 2% sucrose, 0.05%PEG20000,0.1%NaN3, a concentration of 0.02mol/L
Phosphate buffer solution；The pH of the phosphate buffer solution is 7.4.
7. the preparation method of any one of claim 1~6 test paper, it is characterised in that include the following steps：15mg/ will be contained
ML heparin, 22mg/mL matrimony vine acid sodium, 400mg/mL ammonium sulfate mixed solution be fixed in sample pad, by a concentration of 6 μ g/mL
Antiprogestin monoclonal antibody-colloid gold label object be fixed on bonding pad, using three-dimensional specking platform will contain 4mg/mL it is pregnant
Ketone holoantigen, 12mg/mL Sodium Hyaluronates, 7mg/mL1,2- pentanediols mixed solution be fixed on conduct on nitrocellulose filter
The sheep anti-mouse igg of a concentration of 0.4mg/mL or rabbit anti-mouse igg are fixed on cellulose nitrate by detection line T using three-dimensional specking platform
As nature controlling line C on plain film, detection line T and nature controlling line C spacing 0.7cm, 37 DEG C of dry 8h, then by nitrocellulose filter are kept
It is attached on PVC backings, sample pad and bonding pad is attached into one end of nitrocellulose filter, blotting paper is attached into the nitre
The other end of acid cellulose film, the sample pad, bonding pad, detection line T, nature controlling line C, blotting paper are in nitrocellulose filter
On distributing position be followed successively by sample pad, bonding pad, detection line T, nature controlling line C, blotting paper, will integrally be cut into several bars.
8. any one of claim 1~6 test paper is used to detect the application of bitch progesterone, it is characterised in that including following step
Suddenly：Tested animal serum is taken, is added in the sample pad of the test paper, the colour developing feelings of detection line T, nature controlling line C are observed after 15min
Condition；When detection line T colored intensities are more than nature controlling line C, progesterone content is less than or equal to 5ng/mL in serum；When detection line T develops the color
Progesterone content is 10~20ng/mL in serum when intensity is less than nature controlling line C；The progesterone content in serum when detection line T does not develop the color
More than 20ng/mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711431534.XA CN108152515A (en) | 2017-12-26 | 2017-12-26 | A kind of test paper detected for heat bitch progesterone and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711431534.XA CN108152515A (en) | 2017-12-26 | 2017-12-26 | A kind of test paper detected for heat bitch progesterone and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108152515A true CN108152515A (en) | 2018-06-12 |
Family
ID=62462336
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711431534.XA Pending CN108152515A (en) | 2017-12-26 | 2017-12-26 | A kind of test paper detected for heat bitch progesterone and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108152515A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108802395A (en) * | 2018-06-20 | 2018-11-13 | 广州质量监督检测研究院 | Detect the detection card and its preparation method and application of Triclosan |
| CN112578127A (en) * | 2020-11-16 | 2021-03-30 | 爱若维生物科技(苏州)有限公司 | Canine progesterone immunochromatography test strip, preparation method and detection method |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003028582A2 (en) * | 2001-09-28 | 2003-04-10 | Aspenbio, Inc. | Bovine pregnancy test |
| US20060121626A1 (en) * | 2004-12-03 | 2006-06-08 | Genzyme Corporation | Diagnostic assay device |
| US20090011436A1 (en) * | 2003-02-24 | 2009-01-08 | Binax, Inc. | Dry Chemistry, Lateral Flow-Reconstituted Chromatographic Enzyme-Driven Assays |
| CN102297966A (en) * | 2011-05-27 | 2011-12-28 | 天津农学院 | Progesterone semi-quantitative determining colloidal gold test paper and preparation method thereof |
| JP2013181870A (en) * | 2012-03-02 | 2013-09-12 | Tanaka Kikinzoku Kogyo Kk | Test strip for immunochromatography |
| CN104237538A (en) * | 2014-07-07 | 2014-12-24 | 黑龙江八一农垦大学 | Dairy cow milk progesterone colloidal gold test paper strip and preparation method thereof |
| CN105424937A (en) * | 2015-10-05 | 2016-03-23 | 华中农业大学 | Progesterone colloidal gold immunochromatography test strip for monkey early pregnancy detection |
| CN105467115A (en) * | 2015-11-17 | 2016-04-06 | 南昌大学 | Immunochromatographic colloidal gold test strip for detecting aflatoxin M1 |
| CN107144693A (en) * | 2017-05-02 | 2017-09-08 | 暨南大学 | Detect CD4 in blood+Lateral chromatography kit of T cell quantity and preparation method thereof |
-
2017
- 2017-12-26 CN CN201711431534.XA patent/CN108152515A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003028582A2 (en) * | 2001-09-28 | 2003-04-10 | Aspenbio, Inc. | Bovine pregnancy test |
| US20090011436A1 (en) * | 2003-02-24 | 2009-01-08 | Binax, Inc. | Dry Chemistry, Lateral Flow-Reconstituted Chromatographic Enzyme-Driven Assays |
| US20060121626A1 (en) * | 2004-12-03 | 2006-06-08 | Genzyme Corporation | Diagnostic assay device |
| CN102297966A (en) * | 2011-05-27 | 2011-12-28 | 天津农学院 | Progesterone semi-quantitative determining colloidal gold test paper and preparation method thereof |
| JP2013181870A (en) * | 2012-03-02 | 2013-09-12 | Tanaka Kikinzoku Kogyo Kk | Test strip for immunochromatography |
| CN104237538A (en) * | 2014-07-07 | 2014-12-24 | 黑龙江八一农垦大学 | Dairy cow milk progesterone colloidal gold test paper strip and preparation method thereof |
| CN105424937A (en) * | 2015-10-05 | 2016-03-23 | 华中农业大学 | Progesterone colloidal gold immunochromatography test strip for monkey early pregnancy detection |
| CN105467115A (en) * | 2015-11-17 | 2016-04-06 | 南昌大学 | Immunochromatographic colloidal gold test strip for detecting aflatoxin M1 |
| CN107144693A (en) * | 2017-05-02 | 2017-09-08 | 暨南大学 | Detect CD4 in blood+Lateral chromatography kit of T cell quantity and preparation method thereof |
Non-Patent Citations (7)
| Title |
|---|
| J.V.SAMSONOVA ET AL.,: "Pretreatment-free lateral flow enzyme immunoassay for progesterone detection in whole cows" milk", 《TALANTA》 * |
| VINAYTRIPATHI ET AL.,: "A competitive immunochromatographic strip assay for 17-α-hydroxy progesterone using colloidal gold nanoparticles", 《CLINICA CHIMICA ACTA》 * |
| 万积成 等: "奶牛早孕诊断试纸条的研制及临床试验", 《中国奶牛》 * |
| 张改平主编: "《免疫层析试纸快速检测技术》", 31 August 2015, 郑州：河南科学技术出版社 * |
| 李光鹏: "《动物解剖学、组织学与生殖生物学简明教程》", 30 September 2016, 呼和浩特：内蒙古大学出版社 * |
| 杨建生 等主编: "《麻醉基本技能与药物》", 31 October 2014, 太原：山西科学技术出版社 * |
| 杨红莉: "进行血清β－HCG及孕酮检测在诊断早期异位妊娠方面的临床价值分析", 《当代医药论丛》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108802395A (en) * | 2018-06-20 | 2018-11-13 | 广州质量监督检测研究院 | Detect the detection card and its preparation method and application of Triclosan |
| CN112578127A (en) * | 2020-11-16 | 2021-03-30 | 爱若维生物科技(苏州)有限公司 | Canine progesterone immunochromatography test strip, preparation method and detection method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108362688A (en) | A kind of 25(OH)VD magnetic microparticle chemiluminescence detection kit | |
| DE60124705T2 (en) | AUTOMATIC MEASURING CARTRIDGE AND MEASURING PROCESS THEREOF | |
| CN104237538B (en) | A kind of milk progesterone of cow colloidal gold colloidal gold detection test paper strip and preparation method thereof | |
| DE60308259T2 (en) | Immunological methods, devices and reagents | |
| CN104614511B (en) | Immune chromatography analysis method, immune chromatography analysis device, and immune chromatography analysis reagent box | |
| CN108152515A (en) | A kind of test paper detected for heat bitch progesterone and preparation method thereof | |
| CN113125696B (en) | Estradiol homogeneous chemiluminescence detection kit and application thereof | |
| NO149864B (en) | IMMUNOLOGICAL REAGENT. | |
| EP0671006B1 (en) | Immunologic rapid test for the optical determination of progesterone in fluids | |
| CN101329280A (en) | Fast testing method of hydrogen peroxide residue in milk-like liquid and testing reagent kit thereof | |
| CN111051887B (en) | Analysis device and analysis method | |
| CN109254145A (en) | For improving the dilution of matrix effect between fresh serum and third party's Quality Control | |
| JPH01210863A (en) | Counting of spermatid and ratio of living spermatid or fast determination method and apparatus for either thereof | |
| KR20020078032A (en) | Kit for diagnosing non-pregnancy, and method for diagnosing non-pregnancy using the same | |
| CN103163291A (en) | Kit and operation method thereof | |
| CN114660293A (en) | SERS immunoassay kit for detecting mycotoxin exposure marker in blood plasma or urine | |
| JPH0658935A (en) | Immunoassay and reagent therefor | |
| CN111537751A (en) | Myocardial troponin I, creatine kinase isoenzyme and myoglobin joint detection kit and detection method | |
| CN110794139A (en) | Aldosterone latex enhanced detection kit and preparation method thereof | |
| CN110456044A (en) | A kind of kit for prostatitis detection | |
| CN117169518B (en) | Method and kit for detecting CD3 antibody residues in T lymphocyte preparation | |
| CN108562735A (en) | A kind of enzyme-catalyzed chemical luminescence substrate liquid of alkaline phosphatase and preparation method thereof | |
| JP3828614B2 (en) | Protein quantification by turbidimetry and turbidity analysis unaffected by excess antigen | |
| CN113668069B (en) | Preparation method of protein chip board | |
| EP4206671A1 (en) | Ferritin measuring reagent |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180612 |
|
| RJ01 | Rejection of invention patent application after publication |