CN103728459A - Preparation method and application of kit for double-sandwich immunofluorescence quantitative detection of human anti-Mullerian hormone (AMH) on basis of quantum dots - Google Patents
Preparation method and application of kit for double-sandwich immunofluorescence quantitative detection of human anti-Mullerian hormone (AMH) on basis of quantum dots Download PDFInfo
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- CN103728459A CN103728459A CN201310738206.XA CN201310738206A CN103728459A CN 103728459 A CN103728459 A CN 103728459A CN 201310738206 A CN201310738206 A CN 201310738206A CN 103728459 A CN103728459 A CN 103728459A
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Abstract
The invention discloses a kit for double-sandwich immunofluorescence quantitative detection of human anti-Mullerian hormone (AMH) on the basis of quantum dots and application thereof. The original kit contains an AMH protein standard substance, an ELISA plate coated with an AMH specific polyclonal antibody and a CdTe quantum dot labeled AMH monoclonal antibody. The detection antibody used in the kit is a CdTe quantum dot label for monoclonal antibodies, and can better remove background signals; the detection method is simple and convenient, and has high practicality; the detection result directly determined by a fluorescent ELIASA indicates that the emitted fluorescence has the advantages of narrow spectrum peak, weak autofluorescence and high sensitivity; the kit has the advantages of high fluorescence intensity and long stabilization time, and overcomes the actual states of low sensitivity and poor specificity in the traditional ELISA detection kit; and the kit can enhance the resolution, sensitivity and specificity of ovary reservation function detection.
Description
Technical field
The invention belongs to immune diagnostic technique field, the immunofluorescence relating to based on quantum dot quantitatively detects, and is specifically related to a kind of double fastener heart immunofluorescence based on quantum dot and quantitatively detects kit and preparation and the application of the anti-gyneduct hormone of people (AMH).
Background technology
Domestic each diagnosis and treatment unit assesses ovarian function mainly according to the number of age, hole ovarian follicle (Antral follicle count), FSH (FSH) and estrogen (E2) level etc. clinically at present.But above-mentioned classic method inspection is easily subject to various factors impact and produces error, is easily subject to tester's subjective judgement and the bias that bears results simultaneously; In addition, the detection of These parameters is restricted by the time limit of menstrual cycle, and it detects numerical value and be subject to the fluctuation of menstrual cycle and fluctuate, thereby cannot accurately and timely judge.Can find a certain effective stability index, all can Efficient Evaluation ovarian function be one of subject matter of facing of current gynaecology reproductive endocrine at menstruation different times.In American-European countries, bring into use gradually clinically the index of anti-gyneduct hormone (Anti-Mullerian hormone:AMH) as ovarian function assessment in recent years.AMH is the glycoprotein dimer that is formed by connecting by disulfide bond, only in sexual gland, expresses, and it is mainly secreted by granular cell in growth period ovarian follicle, and AMH, in regulation and control ovarian follicular growth and growth, maintains in ovarian reserve function and plays a significant role.Research shows: AMH level and ovarian reserve function are obvious positive correlation, at selection of dominant follicles fermentation, has also played vital role, therefore detects the content of the AMH in women's blood, can be used as one of efficiency index of assessment ovarian reserve ability.
America and Europe, most of clinic diagnosis center is the conventional index using AMH level as assessment ovarian reserve at present.What adopt is mainly enzyme immunoassay (EIA).But enzyme immunoassay (EIA) sensitivity is low, influence factor is more, easily causes false negative and false positive results.Quantum dot (QDs) has continuous and wide exciting light spectrum.Any light source that its fluorescence can be less than its quantum confinement peak by wavelength excites, and fluorescence spectra position can regulate and control by changing quantum dot physical size.So only with a kind of excitation source of wavelength, just can excite the quantum dot of multiple different colours fluorescence to carry out multiplex fluorescence detection.Resolution and sensitivity have effectively been improved.Therefore, quantum dot immune analytic approach is far superior to enzyme immunoassay (EIA).
The domestic AMH the detection kit that there is no independent intellectual property right at present.Some Domestic diagnosis and treatment mechanism has introduced this kit line correlation clinical detection of going forward side by side, but its price is relatively costly, cannot obtain effective clinical and promote.The at present domestic registration kit supply that still also has no employing quantum dot immune analytical approach detection AMH, this detection research clinically does not yet extensively launch.And the many employings of at present external AMH detection kit is enzyme-linked immunoassay method, and still, enzyme immunoassay (EIA) sensitivity is low, and influence factor is many, easily causes false negative and false positive.And according to a large amount of test findings and clinical practice data, from practicality, stability, accuracy and potential applicability in clinical practice, quantum dot immune analytic approach is far superior to enzyme immunoassay (EIA).Therefore, be necessary to develop there is independent intellectual property right, in conjunction with quantum dot immune analysis method, there is high resolving power, sensitivity and specific AMH diagnostic kit, for reserve function accurate, rapid evaluation ovary simultaneously.
Summary of the invention
The object of the present invention is to provide a kind of quantum dot immune fluorescent quantitatively to detect the kit of anti-gyneduct hormone (AMH).
Another object of the present invention is to provide mentioned reagent box in the application detecting in ovarian reserve function.
The technical solution used in the present invention is:
Double fastener heart immunofluorescence based on quantum dot quantitatively detects a kit of the anti-gyneduct hormone of people AMH, and this kit comprises AMH protein standard substance, the ELISA Plate of coated AMH specific polyclonal antibody, the AMH monoclonal antibody that CdTe is quantum dot-labeled.
Further, the recombinant protein that above-mentioned AMH protein standard substance is procaryotic cell expression, its amino acid sequence is as shown in SEQ ID NO.1.
Further, above-mentioned AMH specific polyclonal antibody is that AMH recombinant protein using prokaryotic expression is as the anti-human AMH polyclonal antibody of the synthetic rabbit of immunogene.
Further, above-mentioned AMH monoclonal antibody is that AMH recombinant protein using prokaryotic expression is as the synthetic mouse-anti people AMH monoclonal antibody of immunogene.
Further, kit of the present invention also comprises: labelled antibody dilution, wash plate liquid, confining liquid and sample diluting liquid.
The invention has the beneficial effects as follows:
In kit, as detecting the monoclonal antibody of antibody, because the quantum dot-labeled method adopting has exciting light spectrum width, a little less than autofluorescence, the features such as the stable and long half time of fluorescence, have improved the resolution, sensitivity and the specificity that detect greatly.
Kit of the present invention, using the quantum dot-labeled mouse-anti people of CdTe AMH monoclonal antibody as detecting antibody, is better removed background signal, improves the resolution and the sensitivity that detect; And by fluorescence microplate reader, measure fluorescence intensity and obtain testing result, the fluorescence spectra of launching is narrow, a little less than autofluorescence, highly sensitive; Fluorescence intensity is high, and stabilization time is long; Overcome the sensitivity of existing inhibin B enzyme immunoassay kit low, the present situation of poor specificity, improves the resolution, sensitivity and the specificity that detect, more effectively early stage ovarian reserve function is detected and risk assessment.
It is elder generation and then effective method that the present invention detects AMH with quantum dot immunoassay, can effectively provide resolution, sensitivity and the specificity of clinical detection, and detection method is easy and simple to handle, practical.
Accompanying drawing explanation
Fig. 1 western blotting picture before and after AMH recombinant protein purification of behaving;
Fig. 2 is the typical curve of the anti-human AMH polyclonal antibody of the rabbit after purifying;
Fig. 3 is the typical curve of the mouse-anti people AMH monoclonal antibody after purifying;
Fig. 4 is the test zone half interval contour of AMH quantum dot immune analyzing and testing kit.
Embodiment
Below in conjunction with tool embodiment, the invention will be further described, but be not limited to this.
1) structure of AMH prokaryotic expression carrier
Select the immunogene district of AMH, its gene order as shown in SEQ ID NO.2,297bp altogether, corresponding amino acid sequence as shown in SEQ ID NO.1, totally 93 amino acid.
The Auele Specific Primer of design amplification AMH immunogene region sequence is as follows:
EcoR I Up primer:CCG
gAATTCaGCGTAGACCTCCGCGCCGC(underscore mark part is the recognition sequence of restriction endonuclease EcoR I) (SEQ ID NO.3),
Xho I Down primer:CCG
cTCGAGcCGGCAGCCACACTCGGTGG(underscore mark part is the recognition sequence of restriction endonuclease Xho I) (SEQ ID NO.4).
Utilize the total RNA of chloroform extraction method Proliferation of Human Ovarian Cell, by reverse transcription and PCR primer amplification, go out AMH immunogene region sequence, after being cut, AMH sequence enzyme after amplification is connected on pGEX-4T-1 prokaryotic expression carrier, be converted in DH5 α competent cell, with spreading rod, transformant is inoculated into the LB solid medium of ammonia benzyl resistance, 37 ℃ of overnight incubation, picking monoclonal bacterium colony also carries out a small amount of amplification cultivation, extract plasmid, adopt after EcoR I and Xho I double digestion plasmid, agarose electrophoresis checking, if energy enzyme cuts out the band of about 300bp, by the bacterium colony being initially identified as containing genes of interest, further sequence verification, order-checking is correct, successfully obtain the RT-PCR fusion protein expression vector of AMH, be designated as pGEX-4T-1-AMH.
2) the immunogenic Expression and purification of AMH
The expression of AMH destination protein
PGEX-4T-1-AMH is transformed to Escherichia coli RG2,37 ℃ of picking clones, 250rpm concussion is cultivated until OD value reaches 0.4～0.6,10 ℃, 150rpm overnight incubation under 0.5mM IPTG induction, then the centrifugal 10min collection of 5000g thalline is standby.The dry precipitation of bacterium can be stored in-80 ℃.
The detection of expression of AMH destination protein
In the dry precipitation of bacterium, add appropriate lysate, under ice bath, in ultrasonic degradation thalline ultrasonic procedure, guarantee that albumen is all the time in ice bath, prevent that ultrasonic to cross heat affecting protein stabilized.12000rpm, 4 ℃ are centrifugal, albumin in collection, Western Blot identifies albumen and detects expressing quantity.
The separation and purification of AMH destination protein
In the dry precipitation of bacterium, add appropriate lysate, under ice bath, in ultrasonic degradation thalline ultrasonic procedure, guarantee albumen
In ice bath, prevent that the heat affecting of ultrasonic mistake is protein stabilized eventually.12000rpm, 4 ℃ are centrifugal, albumin in collection, 0.44um filters.The protein lysate of centrifugal collection and prerinse GSH – agarose are fully mixed, and 4h is hatched in 4 ℃ of concussions altogether.Cell pyrolysis liquid cleans 3 times, and TBS cleans 3 times, adds 10mM glutathione eluent eluted protein.Albumen after wash-out can and be removed the glutathione in liquid by the albumen evaporating column protein concentrate of 10KD, and washs with PBS.Albumen after purifying is measured protein concentration and identifies its purity (see figure 1) by western blot by Coomassie brilliant blue method.
Two, the anti-human AMH polyclonal antibody preparation of rabbit and evaluation
1) the anti-human AMH polyclonal antibody preparation of rabbit
Animal immune
Prepare two adult rabbits, the recombined human AMH antigen 1 of purifying 00 μ g is dissolved in 1ml phosphate buffer solution stand-by.In 1ml freund 's incomplete adjuvant, add mycobacterium to make Freund's complete adjuvant, and add 1ml antigenic solution, it is fully emulsified that concuss makes it, and extracts this emulsion with 3ml syringe, connects 25G syringe needle, the bubble in Inside Syringe.From cage, take out rabbit and be placed on smoothly, at 4 different positions, carry out hypodermic injection, two are in back, and two are in thigh place.The skin of comforting the rabbit hair of injection place and exposing with ethanol disinfection.Pinch out skin, the angle inserting needle that syringe needle is spent with relative skin 15, depth of needle is 1cm～2cm, does not carefully thrust in muscle, at 4 different parts, injects respectively approximately 500 μ l antigenic solutions.After injection finishes, pin is extracted gently after several seconds are placed in injection place again, and in injection place, sterilized with ethanol.At 4 positions, repeat aforesaid operations.Every 4～6 weeks injections of antigens, and after injection 7 days～10 days according to collecting blood.Front to the blood of collection and the injection blood of collecting is compared, check and whether have antibody to produce.After generation antibody to be determined, can collect in a large number blood, but every rabbit collection blood can not be more than 40ml to prevent shock.
Collect serum
Rabbit is put on fixed mount gently, and dimethylbenzene is applied to the upper middle part of ear's blood vessel, with blade tilt 45 ° of otch that cut out 0.23cm～0.3cm at this place blood can be flowed out freely.With the pipe after sterilization, collect the blood oozing, if there is solidifying available warm water before finishing, dab incision, then continue to collect.Collect the gauze after available sterilization after appropriate blood and dab affected part, can after 10 seconds～20 seconds definite blood flows in flicking affected part stop.Blood is placed 30 minutes in 37 ℃ of constant temperature ovens, then spent the night 4 ℃ of placements.Medication shovel is dialled blood clot fall from tube wall, and by blood transfer, to plastic centrifuge tube, 4 ℃, centrifugal 10 minutes of 10,000g, collects supernatant and be antiserum, can preserve the several years at-20 ℃.
Antibody purification
Antiserum is put into frozen water or 4 ℃ of refrigerators and is slowly thawed to avoid the gathering of protein.The gathering occurring in protein course of defrosting can be dissolved by 37 ℃ of preheatings.Adding solid sodium azide to concentration is 0.05%, 4 ℃, centrifugal 5 minutes of 15,000g, and the antiserum that shifts out clarification is filtered again removes unnecessary fat.
Antibody is diluted with the ratio of 1:5 with TBS buffer solution, then filter with filtrator.With the speed of per minute 0.5ml by antiserum to post, be the combination that guarantees antiserum and filler, need continuous upper prop 2 times also retains loading efflux.With TBS buffer solution, clean pillar and add pH2.7 elution buffer solution to A λ 280nm<0.008, with the speed of 0.5ml/min, be eluted to all albumen and all flow down.With the 1.5ml EP pipe that adds 100ul neutralization buffer solution, be in charge of collection eluent, mix the rear pH that checks eluent with pH test paper, if pH can utilize neutralization buffer to be adjusted to about pH7.4 to prevent the sex change of antibody lower than 7.In post, add 10ml, pH1.9 elution buffer solution, collects eluent as stated above to A λ 280nm<0.008.
Utilize the content of protein in the each pipe of spectrophotometric determination, antibody concentration is 2.9mg/ml, by after the antibody packing of purifying 2～8 ℃ of preservations.
2) evaluation of anti-AMH polyclonal antibody
The Preliminary detection of the anti-human AMH polyclonal antibody of rabbit sensitivity
Adopt indirect Elisa method, recombinant protein A MH is coated with by the amount of 1ug, 100ng, 10ng, 1ng, 100pg, 10pg, 1pg respectively, add respectively by 1:500,1:1000,1:2000,1:5000, the rabbit immune serum of 1:10000 dilution is as primary antibodie, add by the goat anti-rabbit igg of the HRP mark of 1:80000 dilution anti-as two, TMB colour developing, 450nm surveys OD value.Take albumen dilutability as horizontal ordinate, take the OD value at 450nm place as ordinate, the figure that runs a curve, result shows that the serum of 1:10000 dilution exists 100pg～100ng between the detection zone of good linear relationship.
The Western blotting of the anti-human AMH polyclonal antibody of rabbit identifies
Recombinant protein A MH is carried out to SDS-PAGE glue point sample, electrophoresis by the amount of 1ng, 100pg, 10pg, 1pg respectively, while incubating antibody, add respectively polyclonal antibody that 1:500,1:1000,1:2000 doubly dilute as primary antibodie, add the goat anti-rabbit igg of the HRP mark of 1:10000 dilution to resist as two, by Western, identify antibody titer, the polyclonal antibody of result demonstration 1:2000 dilution can well be identified 1ng AMH recombinant protein.
The titration of anti-AMH polyclonal antibody after purifying
Adopt indirect Elisa method, coated with recombinant protein A MH antigen 1 μ g/100 μ l, 4 ℃ are spent the night; After washing, 5% skimmed milk power sealing is spent the night; The polyclonal antibody of purifying is carried out to serial dilution as primary antibodie, 37 ℃ of incubation 2h; After washing, add the goat anti-rabbit igg of the HRP-mark of 1 ︰ 50,000 dilutions to resist as two, 37 ℃ of incubation 1h; After washing, add substrate colour developing and stop in time, surveying OD
450nmvalue.
Testing result as shown in Figure 2, the tiring as 1:25600 of polyclonal antibody, wherein horizontal ordinate is dilution Log value, ordinate is OD value.
Three, the preparation of mouse-anti people AMH monoclonal antibody and evaluation
1) preparation of mouse-anti people AMH monoclonal antibody
I animal immune
By fully emulsified to the recombined human AMH antigen of purifying and isopyknic Freund's complete adjuvant, make Water-In-Oil antigen emulsion, 6～8 week age of SPF level pure lines BALB/c female mice, the subcutaneous multi-point injection in back, 100 μ g/ only, after fortnight, with equivalent antigen, add the incomplete Freund's adjuvant that volume is identical, carry out emulsification, immunity.Respectively at carrying out booster immunization the 2nd, 4,6 weekends after first immunisation, use the destination protein of same dose at every turn.With tiring of indirect Elisa method detection serum antibody.First 3 days mouse tail vein injections of Fusion of Cells or lumbar injection 100 μ g AMH albumen are with booster immunization.
The separation of ii mouse boosting cell
Get the BALB/c mouse of booster immunization, pluck eyeball blood sampling and put to death, 75% alcohol-pickled 5～10 minutes, be fixed on cake wax; With after 75% alcohol disinfecting skin, cut off skin of abdomen, expose peritonaeum also with 75% alcohol wipe sterilization; With glass syringe, draw serum-free DMEM nutrient solution 5ml and inject mouse peritoneal, with syringe suction (attention can not puncture the digestive organs of mouse) repeatedly in abdominal cavity; With this syringe, extract abdomen intracavity liquid out, inject in 50ml centrifuge tube; Change tweezers, mention peritonaeum, change scissors, expose abdominal cavity, the aseptic spleen of winning, carefully cut off fast periphery fat and manadesma, wash 1～2 time with serum-free DMEM nutrient solution, then spleen is put into the plate of containing 200 order copper sieves, break coating, with piston, grind, push splenocyte and cross net, draw serum-free DMEM nutrient solution 5ml piping and druming copper sieve, the splenocyte of collecting after net is put into the aseptic centrifuge tube of 50ml; By two centrifuge tube l000rpm, centrifugal 5min; Abandon supernatant, add 5ml serum-free DMEM nutrient solution re-suspended cell, cell count, stand-by; With macrophage in the abdominal cavity of the resuspended precipitation of complete culture solution, add 96 well culture plates, 100 μ l/ holes, are then placed in 37 ℃, 5%CO
2in incubator, cultivate, standby.
Recovery and the cultivation of iii SP2/0 cell
From liquid nitrogen container, take out and contain SP2/0 cell cryopreservation tube, drop into immediately in 37 ℃ of water-baths, after thawing, in the centrifugal 5～10min of 1000rpm, abandon supernatant; Preparation is cultivated recovery cell containing the DMEM nutrient solution of 10% calf serum.Cell is inoculated in to both sides, normal BALB/c mouse back subcutaneous, treat that knurl grows to 3～5cm left and right, carry out the aseptic knurl of plucking, with after serum-free DMEM nutrient solution washing 3 times, be cut into diameter 2mm left and right fritter with little scissors, add in the 200 order copper sieves that added in advance 2～3ml serum-free DMEM nutrient solution, grind, squeeze out single tumour cell with piston, put containing cellar culture in the DMEM nutrient solution of 10%FBS, make cell maintain exponential phase.Merge and to SP2/0 cell, change a not good liquor in first 1 day, regulating cell density is 1～5 × 10
5/ ml, merges and got approximately 1～5 × 10 the same day
7individual SP2/0 cell harvesting is to the aseptic centrifuge tube of 50ml, and the centrifugal supernatant of abandoning, adds 5ml serum-free DMEM nutrient solution, mix, and cell count, standby.
Iv Fusion of Cells and selection are cultivated
SP2/0 cell is mixed in the aseptic centrifuge tube of 50ml to l000rpm, centrifugal 5min in 1:5 ratio with splenocyte; Abandon supernatant, flick the pipe end, make precipitation loosening, along centrifugal tube wall, slowly drip the 45%PEG solution lml of 37 ℃ of pre-temperature, slowly rotate centrifuge tube to mix cell simultaneously, in 1min, PEG4000 is added; Put in 37 ℃ of water-baths after 1min, then in 5min, slowly add the serum-free DMEM nutrient solution 8ml of 37 ℃ of pre-temperature, stir and make cell become the suspension of homogeneous gently simultaneously; Put in 37 ℃ of water-baths after 5min, l000rpm, centrifugal 5min, abandons supernatant; Add the HAT nutrient solution of 37 ℃ of pre-temperature, gently outstanding cell, by 1 × 10
5individual splenocyte/hole is added drop-wise in the 96 porocyte culture plates containing feeder cells, is placed in 37 ℃, 5%CO
2in incubator, cultivate.Within after merging 5～10 days, can by HAT nutrient solution half amount, change liquid according to clonal growth situation; Interchangeable HT nutrient solution after 2 weeks, interchangeable complete culture solution after 3 weeks; Cultivating 3～5 days is that visible little clone occurs, hybrid cell is larger, rounded and transparent, and other cell light transmissions are poor and dead gradually; Cultivate 8～2 days, clonal growth is to 1/3～1/2 of hole floorage, and now desirable culture supernatant, carries out antibody test; Once detect secretion predetermined antibody clone cell, in time by positive colony transferred species to 24 well culture plates, more further proceed to culture flask expand cultivate, frozen part clone cell carries out cloning cultivation simultaneously.
The screening of v hybridoma
After Fusion of Cells, once the clone who grows suitable size, should select in time the hybrid cell clone of the predetermined antibody of sensitive, quick, reliable immunological method screening secretion.This experiment adopts the indirect Elisa method of recombined human ES antigen to detect, the supernatant that wherein primary antibodie is hybridoma, and two resist for HRP-mountain sheep anti-mouse igg (1:80,000).Concrete operation step detects with mouse serum titer.
Vi subcloning is cultivated
Cloning is cultivated for the hybridoma cell strain that obtains secretion monospecific antibody most important, generally need to carry out 3～4 subclonings and cultivate, to guarantee to secrete the stability of sex clone growth.Adopt limiting dilution assay, detailed step is as follows: prepare feeder layer, get normal mouse abdominal cavity cell, make cell suspension, and inoculation 96 well culture plates, every hole 50 μ l, approximately containing 2 × 10
4cell, 37 ℃, 5%CO
2overnight incubation in incubator; Clone in inferior daily micropipettor piping and druming Hybridoma Cell Culture plate mesopore, is suspended in complete culture solution; Sampling, with blood counting chamber counting, adjusts cell concentration and is respectively 20/ml, 5/ml; Respectively by the hybridoma suspension inoculation of two kinds of density in containing in the culture plate of feeder cells, every hole 50 μ l, make every hole respectively containing 2,1,0.5 cells; Be placed in 37 ℃, 5%CO
2in incubator, cultivate, after one week, add 100 μ l nutrient culture media.Cultivate 12～15 days, the culture supernatant that has clone is carried out to antibody test; To the cloning cultivation again of positive monoclonal cell, until clone's secreting specificity antibody of 100%; Positive colony is further expanded to cultivation, frozen simultaneously.
A large amount of preparations and the purifying of vii mouse-anti people AMH monoclonal antibody
In cell cultivation process, hybridoma can produce and secrete monoclonal antibody approximately 10～100 μ g/ml.In order to obtain a large amount of high-titer antibodies, conventionally hybridoma is implanted in BALB/c mouse body, prepare and collect the ascites containing monoclonal antibody specific, method is as follows: select 8～10 weeks BALB/c female mices, inoculation hybridoma before 1～2 week, first, to mouse peritoneal injection 0.5ml freund 's incomplete adjuvant, pretreated mouse is 2～3 monthly uses; Collect well-grown hybridoma, centrifuge washing 1 time, is resuspended in serum-free medium, and adjusting cell density is 1～2 × 10
6/ ml, every mouse peritoneal injection 0.5ml cell suspension; The health status of close observation mouse and ascites sign, inoculating cell 7～12 days, visible mouse web portion obviously expands, and while touching with hand, skin has tension, the skin of abdomen of can sterilizing, connect syringe needle No. 8 with 5ml syringe, thrust abdominal cavity, unload syringe, raise mouse head, ascites is splashed in centrifuge tube; 2～3 days, interval, after ascites regeneration is gathered, gets with method again, and a mouse generally can extract 2～3 times.The centrifugal 15min of 3000rpm, discards upper strata grease, cell component and other sediments, draws faint yellow ascites; Adopt the thick purifying of saturated ammonium sulphate method, Protein G Sepharose4FF affinity column method purifying is containing the ascites of hybridoma, and the antibody concentration after purifying is 1.2mg/ml.
2) evaluation of anti-AMH monoclonal antibody
The sensitivity of the anti-AMH monoclonal antibody of the indirect Elisa Preliminary detection of i
Recombinant protein A MH is coated with by the amount of 1ug, 100ng, 10ng, 1ng, 100pg, 10pg, 1pg respectively, add respectively by the ascites containing hybridoma of 1:500,1:1000,1:2000,1:4000 dilution as primary antibodie, add the goat anti-mouse IgG of the HRP mark of 1:80000 dilution to resist as two, TMB colour developing, 450nm surveys OD value.Take albumen dilutability as horizontal ordinate, with OD
450nmvalue is for ordinate, the figure that runs a curve, and result shows that the ascites of 1:4000 dilution exists 100pg～100ng between the detection zone of good linear relationship.
The Western blotting of ii mouse-anti people AMH monoclonal antibody identifies
Recombinant protein A MH is carried out to SDS-PAGE glue point sample, electrophoresis by the amount of 1ng, 100pg, 10pg, 1pg respectively, add respectively the monoclonal antibody ascites of doubly diluting by 1:500,1:1000 as primary antibodie, add the goat anti-mouse IgG of the HRP mark of 1:10000 dilution to resist as two, Western detects antibody titer.The monoclonal antibody ascites of testing result demonstration 1:1000 dilution can well be identified 100pg AMH recombinant protein.
The titration of anti-AMH monoclonal antibody after iii purifying
Adopt indirect Elisa method, coated with recombined human ES antigen 1 μ g/100 μ l, 4 ℃ are spent the night; After washing, 5% skimmed milk power sealing is spent the night; After purifying, monoclonal antibody is carried out to serial dilution as primary antibodie, 37 ℃ of incubation 2h; After washing, by 1:80, the goat anti-mouse IgG of the HRP-mark of 000 dilution is anti-as two, 37 ℃ of incubation 1h; After washing, add substrate colour developing and stop in time, surveying OD
450nmvalue.Testing result as shown in Figure 3, the tiring as 1:512 of monoclonal antibody.
The immunoglobulin class of iv mouse-anti people AMH monoclonal antibody and hypotype are identified
With reference to Sigma company antibody subtype detection kit instructions, adopt Antigen-Mediated Elisa method to measure Ig classification and the hypotype of monoclonal antibody.
Four, quantum dot-labeled
1) CdTe quantum dot is synthetic
CdCl
22.5H
2o(2.5 × 10
-4mol) be dissolved in the ultrapure water of 25ml, add glutathione GSH(3 × 10
-4mol), two hydration trisodium citrates (0.1g), Na
2teO
3(0.5 × 10
-4mol) and NaBH
4(2.4 × 10
-4mol), under the condition of magnetic agitation, with NaOH, regulate pH to 10.5, it is all under room temperature environment that institute responds, Cd
2+, TeO
3 2-with the mol ratio of GSH be 5:1:6, put into the microwave device (power is made as 600W) that band refluxes, when solution colour becomes light green, start to reflux, along with the difference of return time forms the quantum dot take glutathione as stabilizing agent of a series of different-grain diameters.Select suitable quantum dot reacted solution to be cooled to after room temperature and to be precipitated with absolute ethyl alcohol, centrifugal 5min under the condition of 4000rpm.Remove excessive Cd in supernatant
2+, TeO
3 2-deng impurity, repeat 3 times, after ethanol volatilizees completely, precipitation is resuspended in the PBS of pH7.4.
2) coupling of CdTe quantum dot and mouse-anti people AMH monoclonal antibody and purifying (quantum dot-labeled two anti-)
Get quantum dot (QDs) the CdTe600 μ l of above-mentioned concentration, add the EDC(1mg/ml of 18ul) and the methyl alcohol of 800 μ l mix after lucifuge concussion 30min, add again the beta-mercaptoethanol of 8ul to stop, getting two of 0.75mg is anti-ly diluted in the QDs that proper volume adds activation and mixes with it with PBS, lucifuge concussion 2h, after reaction finishes, add again mercaptoethanol to stablize quantum dot, dialyse.After dialysis under 16300g condition centrifugal 3min, remove supernatant, precipitation (coupling that is purifying has the AMH recombinant protein antibody of quantum dot) is resuspended in PBS 4 ℃ of preservations.
Through indirect elisa method, detect and find that mark two resists that under the diluting condition of 1:10000, to detect effect best.
The concrete steps of all described indirect Elisa methods are:
A wrapper sheet: pH9.6 carbonate buffer solution dilution antigen is to optimum concentration 1 μ g/ hole, and 100 μ l/ holes, puts into wet box, and 4 ℃ are spent the night;
B sealing: discard coating buffer next day, PBST detersive enzyme mark reacting hole 4 times, each 5min, dries, and 5% bovine serum albumin(BSA) 300 μ l/ hole sealings, put into wet box, and 4 ℃ are spent the night;
C discards confining liquid, the same washing, and blank is set up in PBS doubling dilution rabbit anteserum sample 100 μ l/ holes simultaneously, the positive and negative control, 37 ℃ of wet box incubation 1.5h;
D discards blood serum sample, the same washing, and PBS is diluted to 1:80,000 HRP-goat anti-rabbit igg 100 μ l/ holes, 37 ℃ of incubation 1h;
E discards ELIAS secondary antibody, the same washing, and TMB colour developing, the each 50 μ l/ holes of substrate A and B, 37 ℃ of lucifuge colour developing 10min, every hole adds 50 μ L2mol/L H
2sO
4stop buffer, full-automatic microplate reader is measured OD
450nmvalue.
Five, the double fastener heart immunofluorescence based on quantum dot quantitatively detects people AMH kit and detection method thereof
1) kit test method
Utilize the anti-human AMH polyclonal antibody of rabbit of purifying as capture antibody, coupling has the mouse-anti people AMH monoclonal antibody of quantum dot as detecting antibody, sets up monoclonal antibody and the how anti-double fastener heart quantum dot detection method of AMH.(whether correctly please check)
Coated and the sealing of i ELISA Plate
PH9.6 carbonate buffer solution dilution polyclonal antibody is to optimum concentration 1 μ g/ hole, and 100 μ l/ holes, puts into wet box, and 4 ℃ are spent the night; Discard coating buffer next day, PBST detersive enzyme mark reacting hole 4 times, each 5min, dries, and 5% bovine serum albumin(BSA) 300 μ l/ hole sealings, put into wet box or dry metal foil bag, and 4 ℃ are spent the night.
The processing of ii standard items or tested serum
AMH protein standard substance (positive criteria product) or tested sample diluting liquid for serum (containing the PBS of the Tween-20 of 1%BSA and 0.05%) do the dilution of proper proportion, the sample diluting liquid of increase serum is not as negative standard items, 100 μ l/ holes, 37 ℃ of reactions were washed plate 3 times with washing plate liquid after 1 hour, and ELISA Plate is patted dry.
Iii monoclonal antibody detects
After AMH monoclonal antibody 1:5000 dilution, use, now with the current.100 μ l/ holes, 37 ℃ of reactions were washed plate 3 times with washing plate liquid after 1 hour, added 100 μ l PBS after patting dry.The ELISA Plate of handling well is measured to fluorescence intensity level via fluorescence microplate reader.Criterion curve, obtains detectability.
2) kit performance index detect
Between the detection zone of i kit
The ELISA Plate of getting the coated anti-human AMH polyclonal antibody of good rabbit and sealing, does AMH protein standard substance the dilution of proper proportion, and each sample does 3 repeating holes.100 μ l/ holes, 37 ℃ of reactions were washed plate 3 times with washing plate liquid after 1 hour, and ELISA Plate is patted dry.With using after PBS dilution mouse-anti people AMH monoclonal antibody 1:10000 dilution, now with the current, 100 μ l/ holes, 37 ℃ of reactions were washed plate 3 times with washing plate liquid after 1 hour, after patting dry, add 100 μ l PBS, the ELISA Plate of handling well is measured to fluorescence intensity level via fluorescence microplate reader.Detection data are as shown in table 1 below, take the standard items albumen of the AMH of variable concentrations as horizontal ordinate, take corresponding glimmering light intensity value as ordinate, curve plotting as shown in Figure 4, it is 10pM～1000pM that this kit has between the detection zone of good linear relationship, at this interval typical curve equation, is y=2.8858x+271.42(R
2=0.9985).
The sensitivity of ii kit detects
With zero-dose standard items (being that blank is same, containing albumen), be used as sample and measure 20 times, calculate its fluorescent value average and standard deviation.With the average of measured value, add the fluorescent value substitution typical curve equation y=2.8858x+271.42(R of the standard deviation gained of 2 times
2=0.9985).The concentration calculating is its detection limit.
Table 2 kit of the present invention is measured the fluorescent value of the standard items that AMH protein concentration is zero
| Mark this |
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
| Fluorescent value | 258.472 | 269.592 | 255.659 | 267.396 | 259.599 | 243.693 | 268.794 | 246.793 | 261.293 | 258.393 | 261.194 |
| Mark this |
12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | Mean value | Standard deviation |
| Fluorescent value | 268.091 | 254.329 | 251.239 | 257.547 | 257.593 | 265.194 | 262.193 | 261.091 | 255.921 | 259.204 | 6.938 |
Testing result sees the above table, and detection limit result substitution typical curve equation being calculated by method is above 0.575pg/ml, shows that kit of the present invention and detection method have the sensitivity of height to the detection of people AMH.
The specific detection of iii kit
1. AMH detection kit of the present invention only detects the AMH albumen in human serum
With detection agent box of the present invention, detect respectively the AMH content (ng/ml) in people, the mice serum of 24 parts, detect data as following table 3.
Table 3 detection agent box of the present invention detects respectively the AMH content (ng/ml) in people, the mice serum of 24 parts
| Human |
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| AMH（ng/ml） | 3.597 | 3.435 | 3.615 | 3.52 | 3.435 | 3.483 | 3.607 | 3.555 | 3.479 | 3.756 | 3.495 | 3.625 |
| Human serum sample number | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| AMH（ng/ml） | 3.493 | 3.334 | 3.373 | 3.358 | 3.381 | 3.709 | 3.554 | 3.633 | 3.752 | 3.759 | 3.43 | 3.553 |
| Mouse serum |
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| AMH（pg/ml） | 0.012 | 0.020 | 0.135 | 0.450 | -0.131 | -0.031 | 0.010 | 0.402 | 0.080 | 0.351 | -0.122 | -0.301 |
| Mouse serum sample Article Number | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| AMH（pg/ml） | 0.214 | 0.157 | 0.313 | 0.210 | -0.022 | -0.020 | 0.103 | 0.201 | 0.304 | 0.209 | -0.130 | 0.082 |
The content of the AMH albumen of result demonstration human serum is greatly between 3.334ng/ml～3.759ng/ml, and the content detection of mouse, lower than detectability 0.575pg/ml, shows that this kit is only to human serum sensitivity, insensitive to other animal blood serums.
2. other haemocyanins do not affect the detection of AMH detection kit to AMH
With serum (AMH concentration is lower than detecting lower limit) the dilution AMH standard items antigen of postmenopausal women, with kit of the present invention, detect the content of AMH, and compare with the AMH standard items testing result after normal dilution, detect data as shown in table 4 below.
The detection of the AMH standard items of table 4 AMH detection kit of the present invention to different modes dilution
Result shows, (the AMH standard items testing result after testing result and the normal dilution of the AMH standard items of (AMH concentration is lower than detecting lower limit) dilution is basically identical, illustrates that other albumen in serum do not affect the detection of AMH detection kit of the present invention to AMH to the serum with postmenopausal women for AMH detection kit of the present invention.
The stability of iv detection kit of the present invention
Three batches of reagent of self-control kit are positioned over respectively to 4 ℃ of half a year and 1 year, place after 7 days for 37 ℃, linear relationship and zero standard product fluorescence intensity level relatively and between the front normative reference product each point fluorescence intensity level of placement, detect the each batch of stability that reagent closes, before result demonstration and placement, do not have bright difference.
The clinical application of v detection kit of the present invention
Detecting 220 examples is the content of AMH in 31 years old women's serum the mean aves, wherein 100 example mean aves be the healthy women of health check-up in 28 years old as Normal group (n=100), 120 examples are carried out supplementary reproduction because of ovarian reserve function reduction and are helped pregnant patient (n=120).Testing result is: in Normal group serum, the scope of AMH content is 0.24～11.78ng/ml, and average is 3.58 ± 1.32ng/ml; In ovarian reserve function reduction group serum, the scope of AMH content is 0.12～1.41ng/ml, and average is 0.24 ± 0.27ng/ml.Difference has statistical significance (p<0.01).
Above-mentioned this kit of clinical detection presentation of results has higher sensitivity and good accuracy, non-false positive or false-negative result.
SEQUENCE LISTING
<110> Li Zhi honor
Mono-kind of the <120> double fastener heart immunofluorescence based on quantum dot quantitatively detects the kit of the anti-gyneduct hormone of people AMH
Preparation method and application thereof
<130>
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 93
<212> PRT
<213> people
<400> 1
Ser Val Asp Leu Arg Ala Glu Arg Ser Val Leu Ile Pro Glu Thr Tyr
1 5 10 15
Gln Ala Asn Asn Cys Gln Gly Val Cys Gly Trp Pro Gln Ser Asp Arg
20 25 30
Asn Pro Arg Tyr Gly Asn His Val Val Leu Leu Leu Lys Met Gln Ala
35 40 45
Arg Gly Ala Ala Leu Ala Arg Pro Pro Cys Cys Val Pro Thr Ala Tyr
50 55 60
Ala Gly Lys Leu Leu Ile Ser Leu Ser Glu Glu Arg Ile Ser Ala His
65 70 75 80
His Val Pro Asn Met Val Ala Thr Glu Cys Gly Cys Arg
85 90
<210> 2
<211> 279
<212> DNA
<213> people
<400> 2
agcgtagacc tccgcgccga gcgctccgta ctcatccccg agacctacca ggccaacaat 60
tgccagggcg tgtgcggctg gcctcagtcc gaccgcaacc cgcgctacgg caaccacgtg 120
gtgctgctgc tgaagatgca ggcccgtggg gccgccctgg cgcgcccacc ctgctgcgtg 180
cccaccgcct acgcgggcaa gctgctcatc agcctgtcgg aggagcgcat cagcgcgcac 240
cacgtgccca acatggtggc caccgagtgt ggctgccgg 279
<210> 3
<211> 29
<212> DNA
The artificial primer of <213>
<400> 3
ccggaattca gcgtagacct ccgcgccgc 29
<210> 4
<211> 29
<212> DNA
The artificial primer of <213>
<400> 4
ccgctcgagc cggcagccac actcggtgg 29
Claims (5)
1. the double fastener heart immunofluorescence based on quantum dot quantitatively detects the kit of the anti-gyneduct hormone of people AMH, it is characterized in that: this kit comprises AMH protein standard substance, the ELISA Plate of coated AMH specific polyclonal antibody, the AMH monoclonal antibody that CdTe is quantum dot-labeled.
2. a kind of double fastener heart immunofluorescence based on quantum dot according to claim 1 quantitatively detects the kit of the anti-gyneduct hormone of people AMH, it is characterized in that: the recombinant protein that described AMH protein standard substance is procaryotic cell expression, its amino acid sequence is as shown in SEQ ID NO.1.
3. a kind of double fastener heart immunofluorescence based on quantum dot according to claim 1 quantitatively detects the kit of the anti-gyneduct hormone of people AMH, it is characterized in that: described AMH specific polyclonal antibody is that AMH recombinant protein using prokaryotic expression is as the anti-human AMH polyclonal antibody of the synthetic rabbit of immunogene.
4. a kind of double fastener heart immunofluorescence based on quantum dot according to claim 1 quantitatively detects the kit of the anti-gyneduct hormone of people AMH, it is characterized in that: described AMH monoclonal antibody is that AMH recombinant protein using prokaryotic expression is as the synthetic mouse-anti people AMH monoclonal antibody of immunogene.
5. a kind of double fastener heart immunofluorescence based on quantum dot according to claim 1 quantitatively detects the kit of the anti-gyneduct hormone of people AMH, it is characterized in that: this kit also comprises: labelled antibody dilution, wash plate liquid, confining liquid and sample diluting liquid.
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| CN201310738206.XA CN103728459A (en) | 2013-12-25 | 2013-12-25 | Preparation method and application of kit for double-sandwich immunofluorescence quantitative detection of human anti-Mullerian hormone (AMH) on basis of quantum dots |
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| CN104730247A (en) * | 2015-03-12 | 2015-06-24 | 广州市丰华生物工程有限公司 | Kit suitable for rapidly detecting AMH and INHB by using double-tagging time resolution fluorescence immunoassay method and use method of kit |
| CN104865391A (en) * | 2015-05-06 | 2015-08-26 | 薛金锋 | Anti-Mulerian hormone (AMH) enzyme-linked immunosorbent assay reagent |
| CN105527448A (en) * | 2015-12-31 | 2016-04-27 | 苏州市博纳泰科生物技术有限公司 | A fluorescence immunochromatographic detecting method for anti-mullerian hormone and a kit |
| CN105891490A (en) * | 2016-04-05 | 2016-08-24 | 付国亮 | Test strip for quantitatively detecting anti-mullerian hormone, preparation method thereof and determination method for concentration of anti-mullerian hormone |
| CN106053791A (en) * | 2016-06-30 | 2016-10-26 | 深圳市亚辉龙生物科技股份有限公司 | Anti-mullerian hormone chemiluminescence immunoassay kit and preparation method and application thereof |
| CN106674348A (en) * | 2017-01-06 | 2017-05-17 | 刘玲 | Anti-mullerian hormone (AMH) antibody and preparation method thereof |
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| CN108254577A (en) * | 2018-04-10 | 2018-07-06 | 安徽金标点生物科技有限公司 | A kind of anti-mullerian duct hormone AMH diagnostic kits and preparation method thereof |
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| CN104730247A (en) * | 2015-03-12 | 2015-06-24 | 广州市丰华生物工程有限公司 | Kit suitable for rapidly detecting AMH and INHB by using double-tagging time resolution fluorescence immunoassay method and use method of kit |
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| CN106674348A (en) * | 2017-01-06 | 2017-05-17 | 刘玲 | Anti-mullerian hormone (AMH) antibody and preparation method thereof |
| CN106970057A (en) * | 2017-05-02 | 2017-07-21 | 浙江星博生物科技股份有限公司 | Flow cytometer detection reagent of the anti-gyneduct hormone of people and its preparation method and application |
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