CN106932572A - For the kit and its detection method of infertile antibody test - Google Patents
For the kit and its detection method of infertile antibody test Download PDFInfo
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- CN106932572A CN106932572A CN201710283171.3A CN201710283171A CN106932572A CN 106932572 A CN106932572 A CN 106932572A CN 201710283171 A CN201710283171 A CN 201710283171A CN 106932572 A CN106932572 A CN 106932572A
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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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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
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Abstract
The invention provides a kind of kit for infertile antibody test, the lid being connected including box body and with box body, is provided with infertile antigenic membrane bar, reactive tank, enzyme conjugates reagent bottle, Sample dilution reagent bottle, chromogenic substrate reagent bottle and concentrated cleaning solution reagent bottle in box body；Infertile antigenic membrane bar is provided with detection zone and quality control region, and detection zone is respectively equipped with the detection line of the detection line of coating sperm antigen, the detection line of coating Endometrial Antigen, the detection line of coating oolemma antigen, the detection line of coating ovary antigen, the detection line of coating human chorionic gonadotrophin antigen, the detection line of coating trophoblastic cell antigens, the detection line of coating cuorin antigen and coating beta2 Glycoprotein antigen.Kit is coated with eight kinds of detection lines of infertile antigen, the specific antibody of the infertile related antigen such as sperm being capable of achieving in half-quantitative detection human serum, has reached infertile eight joint-detections.
Description
Technical field
The present invention relates to field of biomedicine technology, and in particular to a kind of kit for infertile antibody test and
Its detection method.
Background technology
The inspections such as the product of infertile detection mainly has collaurum to detect, ELISA is detected are commonly used in the market
The product of survey form.Collaurum detection is to combine on nitrocellulose filter antigen or antibody, and will be marked with antibody or anti-
Former collaurum drying is on pad, if sample contains specific antibody or antigen can be prior to the antigen on collaurum or antibody knot
Close, and the direction of a blotting paper in the same direction chromatographs, and when chromatography is to detection line, then is combined with antigen or antibody in detection line, most
Yin and yang attribute is judged by color afterwards.Collaurum is mainly carries out single part list item detection, it is impossible to accomplish the multinomial visual inspection of single part
I.e. detection single channel is surveyed, while the sensitivity of collaurum detection relevant item is relatively low, above reason all limits such product and exists
Use in clinic.
And ELISA detections are then that antigen or antibody are incorporated into polystyrene plate hole, now antigen or antibody can still have
There is immunocompetence, antigen-antibody reaction can be carried out, coordinate substrate for enzymatic activity color reaction (sensitivity is high), you can display is specific anti-
Former or antibody whether there is, and the depth using colour generation carries out qualitative or quantitative analysis.Although the test format of ELISA can be with
Accomplish the detection of many person-portions, but such product is equally single channel list item detection, and need the professional operator to carry out
Clinical detection and related supporting instrument such as ELIASA, board-washing machine, water bath etc. are cooperated；Meanwhile, such Product checking
Substrate be made up of A/B liquid, troublesome poeration easily fails after mixing, and the non-specific colour developing of negative control is relatively low compared with strong, signal to noise ratio；
Finally limiting such product uses ELISA to detect that needs take nearly 3 hours, and reagent reacting is slow, in actual clinic
Cause inconvenience in use.
Based on this, it is necessary to design it is a kind of can the various infertile antibody of quick detection kit.
The content of the invention
To solve the above problems, the invention provides a kind of kit for infertile antibody test and its detection side
Method.Infertile antigenic membrane bar in the kit for infertile antibody test be coated with eight kinds it is infertile anti-
Original, is capable of achieving infertile eight joint-detections, and detection is quick and is easy to automation.
First aspect present invention provide a kind of kit for infertile antibody test, including box body and with it is described
The lid of box body connection, is provided with infertile antigenic membrane bar, reactive tank, enzyme conjugates reagent bottle, Sample Dilution in the box body
Liquid reagent bottle, chromogenic substrate reagent bottle and concentrated cleaning solution reagent bottle；The infertile antigenic membrane bar be provided with detection zone and
Quality control region, it is saturating that the detection zone is respectively equipped with the detection line of coating sperm antigen, the detection line of coating Endometrial Antigen, coating
The detection line of oolemma antigen, the detection line of coating ovary antigen, the detection line of coating human chorionic gonadotrophin antigen, coating
The detection line of the detection line, the detection line of coating cuorin antigen and coating beta2 Glycoprotein antigen of trophoblastic cell antigens.
Wherein, each detection line is parallel to each other and the interval between each adjacent detection line is wide.
Wherein, the quality control region is provided with the nature controlling line of coating anti-human IgG antibodies.
Wherein, nature controlling line, the bag of coating anti-human IgG antibodies are sequentially provided with along the infertile antigenic membrane bar length direction
Resisted by the detection line of sperm antigen, the detection line of coating Endometrial Antigen, the detection line of coating oolemma antigen, coating ovary
Former detection line, the detection line of coating human chorionic gonadotrophin antigen, the detection line of coating trophoblastic cell antigens, coating
The detection line of cuorin antigen and the detection line of coating beta2 Glycoprotein antigen.
Wherein, the nature controlling line of the coating anti-human IgG antibodies is parallel to each other with each detection line, and the coating anti-human igg resists
Between the detection line of the nature controlling line of body and the coating sperm antigen at intervals of 2.0mm, between each adjacent detection line
At intervals of 2.0mm.
Wherein, the length of the infertile antigenic membrane bar is 80mm, and width is 2.0mm-5.0mm.
Wherein, the infertile antigenic membrane bar is porous nitrocellulose film that pore size is 0.22 μm.
Wherein, alkali phosphatase enzyme mark anti-human IgG antibodies, alkali phosphatase enzyme mark are housed in the enzyme conjugates reagent bottle
The mixture that anti-human IgA antibody and alkali phosphatase enzyme mark anti-human IgM antibodies are formed；It is equipped with the Sample dilution reagent bottle
Tris buffer solutions containing the bovine serum albumin(BSA) that mass concentration is 3%；It is chloro- equipped with the bromo- 4- of 5- in the chromogenic substrate reagent bottle
The mixture of 3- indoles-phosphate and NBT；In the concentrated cleaning solution reagent bottle equipped with 20 times concentration
Tris buffer solutions.
Wherein, reaction temperature of the kit in detection process is 37 DEG C of constant temperature.
The kit for infertile antibody test that first aspect present invention is provided, is capable of achieving half-quantitative detection human blood
Sperm, endometrium, oolemma, ovary, human chorionic gonadotrophin, trophocyte, cuorin, beta2 Glycoprotein in clear
The specific IgG antibodies or IgA antibody or IgM antibody of the infertile related antigen such as 1, have reached infertile eight joints inspection
Survey, detection is comprehensive, detection is quick and is easy to the effect of automation.
Second aspect present invention provides the detection of the kit for infertile antibody test described in first aspect
Method, comprises the following steps：
(1) prepare before detecting：To be balanced to room temperature for the kit and test serum sample of infertile antibody test,
The test serum sample to room temperature has been balanced by default dilution proportion by described, has obtained test serum sample A；Will be infertile
Antigenic membrane bar is put into reactive tank；By concentrated cleaning solution according to default dilution proportion, cleaning solution A is obtained；
(2) it is incubated test serum sample A：The test serum sample A is added in reactive tank, the test serum sample
This A and coated sperm antigen, Endometrial Antigen, oolemma antigen, ovary antigen, people on the infertile antigenic membrane bar
Human chorionic gonadtropin antigen, trophoblastic cell antigens, cuorin antigen and beta2 Glycoprotein antigen are in 37 DEG C of constant-temperature incubations
10min is reacted, and obtains detection film bar A；
(3) washing detection film bar A：The test serum sample A in reactive tank is outwelled, cleaning solution A washing detection film bars A is added
1min, outwells, and rinses 3 times repeatedly, obtains detection film bar B；
(4) it is incubated detection film bar B：Enzyme conjugates is added in reactive tank, the enzyme conjugates and the detection film bar B
Reacted in 37 DEG C of constant-temperature incubation 10min, obtained detection film bar C；
(5) washing detection film bar C：The enzyme conjugates in reactive tank is outwelled, cleaning solution A washing detection film bars C is added
1min, outwells, and rinses 3 times repeatedly, obtains detection film bar D；
(6) color developing detection film bar D：Chromogenic substrate is added in reactive tank, the chromogenic substrate and the detection film bar D
Reacted in 37 DEG C of constant-temperature incubation 10min, obtained detection film bar E；
(7) chromogenic substrate in reactive tank is outwelled, distillation water washing detection film bar E is added, is outwelled, obtain detection film bar F.
The detection method of the kit for infertile antibody test that second aspect present invention is provided, method is simply easy
Operation.
To sum up, beneficial effect of the present invention includes the following aspects：
1st, provided by the present invention for the kit of infertile antibody test, antigenic membrane bar includes that eight kinds of coating is different
The detection line of infertile antigen, is capable of achieving sperm, endometrium, oolemma, ovary, people's suede in half-quantitative detection human serum
The specific IgG antibodies of infertile related antigen such as Chorionic Gonadotropin, trophocyte, cuorin, beta2 Glycoprotein 1 or
IgA antibody or IgM antibody, have reached infertile eight joint-detections, have detected that comprehensive, detection is quick and is easy to the effect of automation
Really；
2nd, provided by the present invention for infertile antibody test kit detection method, method is simple to operation.
Brief description of the drawings
The schematic diagram of the kit for infertile antibody test that Fig. 1 is provided for an embodiment of the present invention；
The schematic diagram of the infertile antigenic membrane bar that Fig. 2 is provided for an embodiment of the present invention.
Specific embodiment
As described below is the preferred embodiment of the present invention, it is noted that for those skilled in the art
For, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications are also considered as
Protection scope of the present invention.
As shown in figure 1, first aspect present invention provides a kind of kit for infertile antibody test, including box
Infertile antigenic membrane bar 11, reactive tank 12, enzyme is provided with body 1 and the lid 2 being connected with the box body, the box body 1 to combine
Thing reagent bottle 13, Sample dilution reagent bottle 14, chromogenic substrate reagent bottle 15 and concentrated cleaning solution reagent bottle 16；It is described it is infertile not
Educate antigenic membrane bar and be provided with detection zone and quality control region, the detection zone is provided with the detection line of coating sperm antigen, coating intrauterine
The detection line of membranous antigen, the detection line of coating oolemma antigen, the detection line of coating ovary antigen, coating human chorionic gonadotropin's gland
The detection line of hormone antigen, the detection line for being coated with trophoblastic cell antigens, the detection line of coating cuorin antigen and coating β 2
The detection line of glycoprotein antigen.
In embodiment of the present invention, in the kit, the infertile antigenic membrane bar can be stored in a holding bottle
In, the infertile antigenic membrane bar in the form of the holding bottle in be located at the box body of the kit.Alternatively, the box body
Inside it is additionally provided with the fixture for fixing the reagent bottle, reaction film bar and reactive tank.The fixture can be cardboard, plastics
Plate or sponge.Alternatively, the reactive tank is used to place infertile antigenic membrane bar when detecting, the structure of the reactive tank is normal
Rule technology, will not be repeated here.
In embodiment of the present invention, each detection line is parallel to each other and the interval between each adjacent detection line is wide.Can
Selection of land, the quality control region is provided with the nature controlling line of coating anti-human IgG antibodies.As shown in Fig. 2 alternatively, along described infertile anti-
Former film bar length direction is sequentially provided with the nature controlling line 0 of coating anti-human IgG antibodies, the detection line 1 of coating sperm antigen, coating uterus
The detection line 2 of interior membranous antigen, the detection line 3 of coating oolemma antigen, the detection line 4 of coating ovary antigen, coating human chorionic
The detection line 5 of promoting sexual gland hormone antigen, the detection line 6 of coating trophoblastic cell antigens, the and of detection line 7 of coating cuorin antigen
It is coated with the detection line 8 of beta2 Glycoprotein antigen.Alternatively, the nature controlling line of the coating anti-human IgG antibodies is mutually put down with each detection line
OK, it is described coating anti-human IgG antibodies nature controlling line and the detection line of the coating sperm antigen between at intervals of 2.0mm, institute
State between each adjacent detection line at intervals of 2.0mm.Alternatively, the nature controlling line be parallel to each other with the detection line and with it is described
Infertile antigenic membrane bar length direction is vertical.Specifically, the nature controlling line is equal to described infertile with the length of the detection line
The width of sterile antigenic membrane bar.Alternatively, the nature controlling line is equal with the width of each detection line.Still optionally further, institute
State nature controlling line and be 0.5mm-0.7mm with the width of each detection line.Specifically, the nature controlling line and each detection line
Width is 0.6mm.Alternatively, the infertile antigenic membrane article is provided with the first end and being oppositely arranged along its length
Two ends, the nature controlling line is 10mm-12mm with the distance of the first end.Still optionally further, the nature controlling line and described
The distance of one end is 11mm.
In embodiment of the present invention, the infertile antigenic membrane bar is porous cellulose nitrate that pore size is 0.22 μm
Plain film.Alternatively, the length of the infertile antigenic membrane bar is 80mm, and width is 2.0mm-5.0mm.Still optionally further,
The width of the infertile antigenic membrane bar is 3mm.
In embodiment of the present invention, the enzyme conjugates reagent bottle is equipped with alkali phosphatase enzyme mark anti-human IgG antibodies, alkalescence
The mixture that the anti-human IgA antibody of phosphatase enzyme mark and alkali phosphatase enzyme mark anti-human IgM antibodies are formed.Alternatively, the enzyme knot
Compound reagent bottle is also equipped with Sample dilution, and in the enzyme conjugates reagent bottle, the alkali phosphatase enzyme mark anti-human igg resists
Body：The anti-human IgA antibody of alkali phosphatase enzyme mark：The alkali phosphatase enzyme mark anti-human IgM antibodies：The body of Sample dilution
Product is than being 1:1:1:2000.Three kinds of enzyme conjugates are housed simultaneously in the enzyme conjugates reagent bottle that the present invention is provided.
In embodiment of the present invention, equipped with pure containing the ox blood that mass concentration is 3% in the Sample dilution reagent bottle
The Tris buffer solutions of albumen.
In embodiment of the present invention, 5-bromo-4-chloro-3-indolylphosphate salt and chlorine are housed in the chromogenic substrate reagent bottle
The mixture of NBT, both mass ratioes are 2:1.
In embodiment of the present invention, equipped with 20 times of Tris buffer solutions of concentration in the concentrated cleaning solution reagent bottle.
In embodiment of the present invention, reaction temperature of the kit in detection process is 37 DEG C of constant temperature.Institute of the present invention
Reaction temperature of the kit in detection process is stated for 37 DEG C of constant temperature, the association reaction of antigen-antibody, the inspection of kit can be accelerated
Survey process is rapider.
In embodiment of the present invention, the infertile antigenic membrane bar is coated with sperm, endometrium, oolemma, ovum respectively
Nest, human chorionic gonadotrophin, trophocyte, cuorin and beta2 Glycoprotein antigen this eight kinds of antigens；Sperm is to women
It is an alloantigen, female genital tract has the enzyme system of the sperm antigen that can degrade under normal circumstances, does not cause to essence
The immune response of sub- antigen.When this kind of enzyme system defect, the sperm antigen for injecting vagina can be absorbed by vaginal mucosa, induce complete
The immune response of body or part, causes female organism to produce AsAb.AsAb influences sperm in female genital tract
Interior transfer.AsAb trembles its original place, brakes with the feature of sperm surface antigen Binding change sperm motility, aggegation,
Even cannot be by Ovulation prediction, the sperm that can reach the site of fertilization is very few, makes the hardly possible generation of fertilization.Anti- essence
Sub- antibody can also influence capacitation and the acrosome reaction of sperm, disturb Sperm penetration ovarian cumulus, oolemma and combined with ovum, even if
Become pregnant, often dysontogenesis, only blastular and without plumule etc., or even miscarriage or infertile.AsAb is also found in it
His reason, such as damage of obstruction of vas deferen and testis and epididymis and inflammation.In view of AsAb heterogeneity and wherein very
The target antigen that many AsAbs are directed to fertility and it is uncorrelated.Therefore, the positive findings of AsAb must be combined and is faced
Bed performance considers；Endometrium is embryo nidation and the ground for growing, be also sperm it is up must be through path.Normal
In the case of, the endometrium that the women of child-bearing age periodically come off is external with menstrual blood outflow, does not induce body typically and produces and itself exempts from
Epidemic disease is reacted.But under pathological state, such as endometritis, endometriosis and uterus adenomyosis, menstrual period vagina operation or
Menstrual period sexual intercourse etc., can change into antigen or haptens, stimulate body itself to produce corresponding antibody.Additionally, induced abortion dilatation and curettage
When, blastular is likely to produce antibody as antigenic stimulus body.Once with the presence of AEA in women body, will lead
Cause it is infertile, stop pregnant or miscarry.Many women are no longer pregnant because having made the stream of people in primigravid, this secondary infertility
Disease most patients because generate AEA in vivo.AEA belongs to autoantibody, is normally educating
Be may also detect that in age women, but in infertility crowd, it is particularly more common in the women with endometriosis.
In addition, the generation of AEA be also possible to it is relevant with body immune system imbalance, reason fail to understand Sterility patient,
The recall rate of AEA is high；Ovary tissue or cell stimulate and itself produce anti-ovary as a kind of special antigen
Antibody.Premature ovarian failure person AOAb is positive, and some many body of gland autoimmune disease patients can have AOAb
Level is raised, and can also have specificity or non-specific autoantibody to raise in the Unexplained sterility patient body of part.Anti- ovary
The immune response that antibody is induced with corresponding antigen binding, may cause barrier by two approach of cell factor and hormone metabolism
Hinder, such as a long time after can cause paramenia, corpus luteum and other sex hormone hyposecretions, do not ovulate, premature ovarian failure, secondary amenorrhea
Menopause is failed to understand with reason, as infertile potential cause.There is the women follicular development of ovary antibody abnormal：Ovarian follicle is long less than receiving
Pregnant advantage, or grow to the advantage of becoming pregnant and can not but naturally drain, or even there is premature ovarian failure phenomenon, make many women of child-bearing age's ends
Life is difficult to develop the ovarian follicle of maturation, and causes primary infertility and secondary infertility.In vitro culture discovery, high-level anti-ovary
The presence of antibody can cause ovum oolemma crude, and after fertilization embryo division is slow；Human chorionic gonadotrophin is to remain early
The major hormone of phase gestation.For the women for having history of spontaneous abortion, history of artificial abortion and biochemical pregnancies history, during miscarriage,
Human chorionic gonadotrophin in chorionic villi produces antibody possibly as antigenic stimulus parent.This anti-human chorionic antibody
Human trophocyte cell's fusion and the synthesis of progesterone are prevented from, so as to cause the infertile or habitual abortion of women.It is anti-
HCG antibodies are used for threatened abortion, vesicular mole, the auxiliary diagnosis of ectopic pregnancy；Anti- vitellary membrane antibody with it is transparent
Band is combined, and can cover the specific sperm acceptor on oolemma, makes sperm None- identified ovum and in combination, can also be in complement
In the presence of directly produce CDCC, destroy oolemma, so as to destroy egg cell, cause infertile；Anti- trophocyte resists
Auxiliary diagnostic index of the body mainly as recurrent abortion patient；ACLA is one kind with blood platelet and endothelial cell membrane
Negatively charged cuorin belongs to anti-phospholipid antibody together as the autoantibody of target antigen with lupus anticoagulant, with Disadvantage pregnancy knot
Office such as Recurrent abortion, stillborn foetus and systemic lupus erythematosus, neurolues, multiple sclerosis, actue infectious polyradiculoneuritis nerve
Systemic disease is in close relations, is played an important role in thrombotic genesis mechanism.If ACLA is positive, occur
The incidence of habitual abortion is high, and repeat abortion, stillborn foetus, placenta infraction, the reduction of fetus at perinatal stage survival rate can occur.The anti-heart
Phospholipid antibody can prevent the synthesis of prostacyclin, make thromboxane-prostacyclin out of proportion, and the relative of thromboxane is increased, and is drawn
Play the spasm ischemic of whole body and placenta blood vessel, thrombosis.Anti-phospholipid antibody is caused by fighting for the phospholipid receptors of placenta blood vessel
Decidua vascular lesion and placenta embolism；The coenzyme indispensable when being anti-phospholipid antibody combination of beta2 Glycoprotein 1, can with it is negatively charged
Material (such as cuorin and lipoprotein) knot, play its physiological function.Beta2 Glycoprotein 1 is formed with the antibody of anti-beta2 Glycoprotein 1
Compound inducing endothelial cell is activated, and is the important danger of thrombosis so as to induce the expression of inflammatory molecule and adhesion molecule etc.
The dangerous antibody of factor beta2 Glycoprotein 1 helps to diagnose suspicious antiphospholipid syndrome (especially ACLA negative patient), with
Thrombosis and morbid state gestation, the correlation of habitual abortion are better than ACLA；Immune factor is many women of influence
Fertility and a major reason of gestation, due to infertile, the even women of habitual abortion that sexual factor is caused is immunized,
Influence caused by internal different antibodies is all the key factor that result in disease, and infertile eight joint-detections can be more preferable
Detection cause infertile specific immune factor, be conducive to precisely treatment.
Kit of the present invention is applied to human serum pattern detection, preferably fresh human serum, it is to avoid use significant hemolysis
Serum or piarhemia serum；The preferred 2-8 DEG C of preservation of human serum sample.Before being detected using the human serum sample, it is necessary to
All people's serum sample is balanced to room temperature (22-28 DEG C).
The preparation method of infertile antigenic membrane bar is to enter according to the conventional method for preparing film bar in kit of the present invention
It is prepared by row, wherein, it is to be configured to be coated with concentration accordingly by eight kinds of infertile antigen of the invention in preparation spotting solution, so
The spotting solution that will be prepared afterwards is fixed on diaphragm with certain ordering, forms independent detection line, is subsequently carried out again
Drying, pad pasting, cut film, assembling, slitting etc. operation to prepare infertile antigenic membrane bar.
The kit for infertile antibody test that first aspect present invention is provided, is capable of achieving half-quantitative detection human blood
Sperm, endometrium, oolemma, ovary, human chorionic gonadotrophin, trophocyte, cuorin, beta2 Glycoprotein in clear
The specific IgG antibodies or IgA antibody or IgM antibody of the infertile related antigen such as 1, have reached infertile eight joints inspection
Survey, detection is comprehensive, detection is quick and is easy to the effect of automation.
Second aspect present invention provides a kind of detection method of kit for infertile antibody test, the inspection
Survey method is comprised the following steps：
S10：Prepare before detection：To be balanced to room for the kit and test serum sample of infertile antibody test
Temperature, the test serum sample to room temperature has been balanced by default dilution proportion by described, obtains test serum sample A；Will detection
Film bar is put into reactive tank；By concentrated cleaning solution according to default dilution proportion, cleaning solution A is obtained；
In step slo, test serum sample refers to human serum sample to be measured, and kit and test serum sample are balanced
To room temperature；To balance to the test serum sample by volume 1 of room temperature:10 dilution proportions, preferably with pipettor by 900 μ l samples
This dilution is added in reactive tank, adds 100 μ l test serum samples, obtains test serum sample A；Detection film bar is put
In entering reactive tank；By concentrated cleaning solution distilled water or deionized water, by volume 1:20 are diluted to working concentration cleaning solution, excellent
Select 25ml concentrated cleaning solutions to add 475ml distilled water or deionized water, obtain cleaning solution A.
S20：It is incubated test serum sample A：Test serum sample A is added in reactive tank, test serum sample A and institute
State coated sperm antigen, Endometrial Antigen, oolemma antigen, ovary antigen, human chorionic on infertile antigenic membrane bar
Promoting sexual gland hormone antigen, trophoblastic cell antigens, cuorin antigen and beta2 Glycoprotein antigen enter in 37 DEG C of constant-temperature incubation 10min
Row reaction, obtains detection film bar A；
In step S20,1mL test serum samples A is added in reactive tank, test serum sample A and the reaction
Detection film bar in groove is reacted in 37 DEG C of constant-temperature incubation 10min, and coated sperm antigen, endometrium resist on detection film bar
Original, oolemma antigen, ovary antigen, human chorionic gonadotrophin antigen, trophoblastic cell antigens, cuorin antigen and β 2
Anti- sperm IgG antibody or anti-sperm IgA antibody or anti-sperm IgM antibody, blood to be measured in glycoprotein antigen, with test serum sample
Anti- endometrial immunoglobulin G antibodies or anti-endometrium IgA antibody or anti-endometrium IgM antibody, anti-oolemma IgG resist in final proof sheet
Body or anti-oolemma IgA antibody or anti-oolemma IgM antibody, anti-ovary IgG antibody or anti-ovary IgA antibody or anti-ovary IgM
Antibody, anti-hCG IgG antibody or anti-hCG IgA antibody or anti-human chorionic rush property
Glandular hormone IgM antibody, anti-human trophocyte's IgG antibody or anti-human trophocyte's IgA antibody or anti-human trophocyte IgM
Antibody, anti-human cuorin IgG antibody or anti-human cuorin IgA antibody or anti-human cuorin IgM antibody and anti-human beta2 Glycoprotein
There is specific antigen-antibody and combine instead in IgG antibody or anti-human beta 2 glycoprotein I gA antibody or anti-human beta 2 glycoprotein I gM antibody
Should, antigen-antibody conjugate is obtained, obtain detection film bar A, sperm antigen antibody conjugates A, endometrium on detection film bar A
Antigen-antibody conjugate A, oolemma antigen-antibody conjugate A, ovary antigen-antibody conjugate A, human chorionic gonadotrophin
Antigen-antibody conjugate A, trophoblastic cell antigens antibody conjugates A, cuorin antigen-antibody conjugate A and beta2 Glycoprotein resist
Original antibody conjugate A.
S30：Washing detection film bar A：The test serum sample A in reactive tank is outwelled, cleaning solution A washing detection film bars are added
A 1min, outwell, and rinse 3 times repeatedly, obtain detection film bar B；
In step s 30, outwell or blot reacted test serum sample A in reactive tank, add 2ml cleaning solutions A punchings
Detection film bar A 1min are washed, cleaning solution A in reactive tank is outwelled or blot, rinsed 3 times repeatedly, obtain detection film bar B, detect film bar
Sperm antigen antibody conjugates B, Endometrial Antigen antibody conjugates B, oolemma antigen-antibody conjugate B, ovary on B resist
Original antibody conjugate B, human chorionic gonadotrophin antigen-antibody conjugate B, trophoblastic cell antigens antibody conjugates B, the heart
Phospholipid antigen antibody conjugates B and beta2 Glycoprotein antigen-antibody conjugate B.
S40：It is incubated detection film bar B：Enzyme conjugates is added in reactive tank, the enzyme conjugates and it is described detection film bar
B is reacted in 37 DEG C of constant-temperature incubation 10min, obtains detection film bar C；
In step s 40,1mL enzyme conjugates is added in reactive tank, the alkali phosphatase enzyme mark in enzyme conjugates resists
In human IgG antibody or the anti-human IgA antibody of alkali phosphatase enzyme mark or alkali phosphatase enzyme mark anti-human IgM antibodies and the reactive tank
Detection film bar B reacted in 37 DEG C of constant-temperature incubation 10min, sperm antigen antibody conjugates B and enzyme knot on detection film bar B
Alkali phosphatase enzyme mark anti-human IgG antibodies or the anti-human IgA antibody of alkali phosphatase enzyme mark or alkali phosphatase enzyme mark in compound
There is specific antigen-antibody reaction in anti-human IgM antibodies, obtain sperm antigen antibody conjugates C, intrauterine on detection film bar C
Alkali phosphatase enzyme mark anti-human IgG antibodies or alkali phosphatase enzyme mark in membranous antigen antibody conjugates B and enzyme conjugates are anti-human
There is specific antigen-antibody reaction in IgA antibody or alkali phosphatase enzyme mark anti-human IgM antibodies, obtain the son on detection film bar C
Alkali phosphatase enzyme mark in Endometrium antigen-antibody conjugate C, oolemma antigen-antibody conjugate B and enzyme conjugates is anti-human
There is specific antigen and resist in IgG antibody or the anti-human IgA antibody of alkali phosphatase enzyme mark or alkali phosphatase enzyme mark anti-human IgM antibodies
Precursor reactant, in oolemma antigen-antibody conjugate C, the ovary antigen-antibody conjugate B and enzyme conjugates on acquisition detection film bar C
Alkali phosphatase enzyme mark anti-human IgG antibodies or the anti-human IgA antibody of alkali phosphatase enzyme mark or the anti-human IgM of alkali phosphatase enzyme mark
There is specific antigen-antibody reaction in antibody, obtain ovary antigen-antibody conjugate C, human chorionic gonadotropin on detection film bar C
Alkali phosphatase enzyme mark anti-human IgG antibodies or alkali phosphatase enzyme mark in glandular hormone antigen-antibody conjugate B and enzyme conjugates
There is specific antigen-antibody reaction in anti-human IgA antibody or alkali phosphatase enzyme mark anti-human IgM antibodies, obtain on detection film bar C
Human chorionic gonadotrophin antigen-antibody conjugate C, trophoblastic cell antigens antibody conjugates B and enzyme conjugates in alkali
Acid phosphatase marks anti-human IgG antibodies or the anti-human IgA antibody of alkali phosphatase enzyme mark or alkali phosphatase enzyme mark anti-human IgM antibodies
Generation specific antigen-antibody reaction, obtains trophoblastic cell antigens antibody conjugates C, the cuorin antigen on detection film bar C
Alkali phosphatase enzyme mark anti-human IgG antibodies or the anti-human IgA antibody of alkali phosphatase enzyme mark in antibody conjugates B and enzyme conjugates
Or alkali phosphatase enzyme mark anti-human IgM antibodies occur specific antigen-antibody reaction, the cuorin antigen on detection film bar C is obtained
Alkali phosphatase enzyme mark anti-human IgG antibodies in antibody conjugates C and beta2 Glycoprotein antigen-antibody conjugate B and enzyme conjugates
Or there is specific antigen-antibody reaction in the anti-human IgA antibody of alkali phosphatase enzyme mark or alkali phosphatase enzyme mark anti-human IgM antibodies,
Obtain the beta2 Glycoprotein antigen-antibody conjugate C on detection film bar C.
S50：Washing detection film bar C：The enzyme conjugates in reactive tank is outwelled, cleaning solution A washing detection film bars C is added
1min, outwells, and rinses 3 times repeatedly, obtains detection film bar D；
In step s 50, outwell or blot reacted enzyme conjugates in reactive tank, add 2ml cleaning solutions A to rinse detection
Film bar A 1min, outwell or blot cleaning solution A in reactive tank, rinse 3 times repeatedly, detection film bar D are obtained, on detection film bar D
Sperm antigen antibody conjugates D, Endometrial Antigen antibody conjugates D, oolemma antigen-antibody conjugate D, ovary antigen resist
Body conjugate D, human chorionic gonadotrophin antigen-antibody conjugate D, trophoblastic cell antigens antibody conjugates D, cuorin
Antigen-antibody conjugate D and beta2 Glycoprotein antigen-antibody conjugate D.
S60：Color developing detection film bar D：Chromogenic substrate is added in reactive tank, the chromogenic substrate and it is described detection film bar
D is reacted in 37 DEG C of constant-temperature incubation 10min, obtains detection film bar E；
In step S60,1mL chromogenic substrates are added in reactive tank, the chloro- 3- indoles of the bromo- 4- of the 5- in chromogenic substrate-
Detection film bar D in the mixture of phosphate and NBT and the reactive tank is in 37 DEG C of constant-temperature incubation 10min
Reacted, sperm antigen antibody conjugates D, Endometrial Antigen antibody conjugates D, oolemma antigen on detection film bar D
Antibody conjugates D, ovary antigen-antibody conjugate D, human chorionic gonadotrophin antigen-antibody conjugate D, trophocyte
Antigen-antibody conjugate D, cuorin antigen-antibody conjugate D and beta2 Glycoprotein antigen-antibody conjugate D respectively with colour developing bottom
There is chromogenic reaction in the 5-bromo-4-chloro-3-indolylphosphate salt in thing, detected with the mixture of NBT
Film bar E, sperm antigen antibody conjugates E, Endometrial Antigen antibody conjugates E, oolemma antigen-antibody on detection film bar E
Conjugate E, ovary antigen-antibody conjugate E, human chorionic gonadotrophin antigen-antibody conjugate E, trophoblastic cell antigens
Antibody conjugates E, cuorin antigen-antibody conjugate E and beta2 Glycoprotein antigen-antibody conjugate E.
S70：Chromogenic substrate in reactive tank is outwelled, distillation water washing detection film bar E is added, outwelled, obtain detection film bar F.
In step S70, chromogenic substrate in reactive tank is outwelled, adds 2mL distillation water washing detection film bar E1min, outwelled,
Obtain detection film bar F, hair dryer drying or natural drying detection film bar F.
Can also interpretation be carried out to detection film bar F, dry detection film bar F is put into immunoblotting assay in the present embodiment
In instrument film bar fixed mount, interpretation is carried out to detection film bar F by immunoblotting assay instrument, as a result can determine whether to be divided into feminine gender, face
Dividing value, 2 times of values of critical value, 4 times of values, 8 times of values, 5 grades, are output as the numerical value for quantifying.
The detection method of the kit for infertile antibody test that second aspect present invention is provided, method is simply easy
Operation, has reached infertile eight joint-detections, has detected that comprehensive, detection is quick and is easy to the effect of automation.
Embodiment described above only expresses several embodiments of the invention, and its description is more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Shield scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (10)
1. a kind of kit for infertile antibody test, it is characterised in that be connected including box body and with the box body
Lid, is provided with infertile antigenic membrane bar, reactive tank, enzyme conjugates reagent bottle, Sample dilution reagent bottle, aobvious in the box body
Color substrate reagent bottle and concentrated cleaning solution reagent bottle；The infertile antigenic membrane bar is provided with detection zone and quality control region, described
Detection zone is respectively equipped with the detection line of coating sperm antigen, the detection line of coating Endometrial Antigen, is coated with oolemma antigen
Detection line, the detection line of coating ovary antigen, the detection line of coating human chorionic gonadotrophin antigen, coating trophocyte
The detection line of the detection line, the detection line of coating cuorin antigen and coating beta2 Glycoprotein antigen of antigen.
2. the kit of infertile antibody test is used for as claimed in claim 1, it is characterised in that each detection line phase
Interval between mutual parallel and each adjacent detection line is wide.
3. the kit of infertile antibody test is used for as claimed in claim 1, it is characterised in that the quality control region is provided with
It is coated with the nature controlling line of anti-human IgG antibodies.
4. the kit of infertile antibody test is used for as claimed in claim 3, it is characterised in that along described infertile
Antigenic membrane bar length direction is sequentially provided with the nature controlling line of coating anti-human IgG antibodies, the detection line of coating sperm antigen, coating uterus
The detection line of interior membranous antigen, the detection line of coating oolemma antigen, the detection line of coating ovary antigen, coating human chorionic gonadotropin
The detection line of glandular hormone antigen, the detection line for being coated with trophoblastic cell antigens, the detection line of coating cuorin antigen and coating β 2
The detection line of glycoprotein antigen.
5. the kit of infertile antibody test is used for as claimed in claim 4, it is characterised in that the coating is anti-human
The nature controlling line of IgG antibody is parallel to each other with each detection line, and the nature controlling line of the coating anti-human IgG antibodies resists with the coating sperm
Between former detection line at intervals of 2.0mm, between each adjacent detection line at intervals of 2.0mm.
6. the kit of infertile antibody test is used for as claimed in claim 1, it is characterised in that described infertile anti-
The length of former film bar is 80mm, and width is 2.0mm-5.0mm.
7. the kit of infertile antibody test is used for as claimed in claim 1, it is characterised in that described infertile anti-
Former film bar is porous nitrocellulose film that pore size is 0.22 μm.
8. the kit of infertile antibody test is used for as claimed in claim 1, it is characterised in that the enzyme conjugates examination
Alkali phosphatase enzyme mark anti-human IgG antibodies, the anti-human IgA antibody of alkali phosphatase enzyme mark and alkali phosphatase enzyme mark are housed in agent bottle
The mixture that anti-human IgM antibodies are formed；It is equipped with containing the bovine serum albumin that mass concentration is 3% in the Sample dilution reagent bottle
White Tris buffer solutions；Equipped with 5-bromo-4-chloro-3-indolylphosphate salt and the nitrogen of chlorination nitro four in the chromogenic substrate reagent bottle
The blue mixture of azoles；Equipped with 20 times of Tris buffer solutions of concentration in the concentrated cleaning solution reagent bottle.
9. the kit of infertile antibody test is used for as claimed in claim 1, it is characterised in that the kit is in inspection
Reaction temperature during survey is 37 DEG C of constant temperature.
10. the detection method of the kit for infertile antibody test as described in claim any one of 1-9, its feature
It is to comprise the following steps：
(1) prepare before detecting：To be balanced to room temperature for the kit and test serum sample of infertile antibody test, by institute
State and balanced the test serum sample to room temperature by default dilution proportion, obtain test serum sample A；By infertile antigen
Film bar is put into reactive tank；By concentrated cleaning solution according to default dilution proportion, cleaning solution A is obtained；
(2) it is incubated test serum sample A：The test serum sample A is added in reactive tank, the test serum sample A
With coated sperm antigen, Endometrial Antigen, oolemma antigen, ovary antigen, people's suede on the infertile antigenic membrane bar
Chorionic Gonadotropin antigen, trophoblastic cell antigens, cuorin antigen and beta2 Glycoprotein antigen are in 37 DEG C of constant-temperature incubations
10min is reacted, and obtains detection film bar A；
(3) washing detection film bar A：The test serum sample A in reactive tank is outwelled, cleaning solution A washing detection film bars A is added
1min, outwells, and rinses 3 times repeatedly, obtains detection film bar B；
(4) it is incubated detection film bar B：Enzyme conjugates is added in reactive tank, the enzyme conjugates is with the detection film bar B in 37
DEG C constant-temperature incubation 10min is reacted, and obtains detection film bar C；
(5) washing detection film bar C：The enzyme conjugates in reactive tank is outwelled, cleaning solution A washing detection film bar C 1min is added,
Fall, rinse 3 times repeatedly, obtain detection film bar D；
(6) color developing detection film bar D：Chromogenic substrate is added in reactive tank, the chromogenic substrate is with the detection film bar D in 37
DEG C constant-temperature incubation 10min is reacted, and obtains detection film bar E；
(7) chromogenic substrate in reactive tank is outwelled, distillation water washing detection film bar E is added, is outwelled, obtain detection film bar F.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201710283171.3A CN106932572A (en) | 2017-04-26 | 2017-04-26 | For the kit and its detection method of infertile antibody test |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710283171.3A CN106932572A (en) | 2017-04-26 | 2017-04-26 | For the kit and its detection method of infertile antibody test |
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| Publication Number | Publication Date |
|---|---|
| CN106932572A true CN106932572A (en) | 2017-07-07 |
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ID=59438384
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| Application Number | Title | Priority Date | Filing Date |
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| CN201710283171.3A Pending CN106932572A (en) | 2017-04-26 | 2017-04-26 | For the kit and its detection method of infertile antibody test |
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| Country | Link |
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| CN (1) | CN106932572A (en) |
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| CN107290547A (en) * | 2017-08-07 | 2017-10-24 | 广州市微米生物科技有限公司 | A kind of infertile quick detection kit and preparation method thereof |
| CN108037290A (en) * | 2017-11-13 | 2018-05-15 | 深圳市博卡生物技术有限公司 | A kind of kit and its detection method of the detection of respiratory pathogen antigen |
| CN116298249A (en) * | 2023-03-24 | 2023-06-23 | 深圳市博卡生物技术有限公司 | Kit for detecting tuberculosis antibody and detection method thereof |
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Inventor after: Huang Zhijian Inventor after: Liu Xiao Inventor after: Lei Junping Inventor after: Zeng Fanbing Inventor after: Yan Lingjuan Inventor after: Xu Xinxin Inventor after: Deng Yonghai Inventor after: Li Chang Inventor before: Liu Xiao Inventor before: Huang Zhijian Inventor before: Lei Junping Inventor before: Zeng Fanbing Inventor before: Yan Lingjuan Inventor before: Xu Xinxin Inventor before: Deng Yonghai |
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Application publication date: 20170707 |