CN103344761A - Double sandwich immunoassay test kit labeled by HE4 quantum dots and application thereof - Google Patents

Abstract

The invention discloses a double sandwich immunoassay test kit labeled by HE4 quantum dots. The kit comprises HE4 protein standards, an ELISA plate coated with HE4 specific polyclonal antibodies, and CdTe quantum dots labeled monoclonal antibodies in specific binding with HE4 proteins. The invention further discloses an application of the kit in diagnosis of ovarian tumor. According to the invention, the test kit can directly determine test results through fluorescent ELISA, emitted fluorescence spectral peaks are narrow, autofluorescence is weak, and a sensitivity is high. The kit is high in fluorescence intensity and long in stable time, and can relatively effectively facilitate early detection and risk assessment of the ovarian tumor.

Description

Double fastener heart immunoassay detection kit and application thereof that a kind of HE4 is quantum dot-labeled

Technical field

The present invention relates to the immune diagnostic technique field, particularly a kind of people's epididymal proteins 4(HE4) quantum dot double fastener heart immunoassay detection kit and application thereof.

Background technology

At present, oophoroma is generally used two kinds of detection methods, but its limitation is arranged: 1) Transvaginal Ultrasound inspection (TUV) can not detect the good, pernicious of lump exactly, also needs to have the clinical technology personnel of the experience of being rich in that the result is carried out correct deciphering simultaneously; 2) this biomarker of CA125 now is identified as " goldstandard " of ovarian cancer diagnosis, but its limitation is arranged also, about 50% oophoroma I phase patient does not have the phenomenon that the CA125 level raises, and some optimum disease of ovary also can cause the CA125 level to raise.CA125 can cause occurring false negative or false positive than low sensitivity and specificity.People's epididymal proteins 4(HE4) belong to whey acidic 4-curing center (WFDC) protein family, this albumen probably is 20kD～25Kd.There is report to think that HE4 all has expressed at the last intracutaneous of a plurality of normal structures (comprise respiratory tract and reproductive tract tissue, and ovarian cancer tissue).Secreting type HE4 has detected high level expression in the serum of ovarian cancer patients, HE4 rising phenomenon all can appear in 88% ovarian cancer patients.Subsequently one HE4 has the highest sensitivity when detecting oophoroma to oophoroma in the evaluation study of relevant large number of biological mark, especially at the commitment of disease.The early stage HE4 diagnostic sensitivity of disease is 82.7% and CA125 only has 45.9%, and the susceptibility of HE4 surmounts CA125 greatly.Therefore, HE4 is as a kind of new tumor markers, HE4 measured kit and the CA125 detection kit is united use, can help to carry out early detection and the risk assessment of oophoroma.

Present people's epididymal proteins 4(HE4 that is used for) immunization method is mainly enzyme immunoassay (EIA).But enzyme immunoassay (EIA) sensitivity is low, and influence factor is many, easily causes false negative and false positive.Quantum dot (QDs) has continuous and wide excitation spectrum " its fluorescence can be excited by any light source of wavelength less than its quantum confinement peak " and the fluorescence spectra position can be regulated and control by the physical size that changes QDs, so only just can excite the QDs of multiple different colours fluorescence with a kind of excitation source of wavelength " carrying out multiplex fluorescence detects; and organic fluorescent dye " every kind of color all needs the excitation source of different wave length "; in addition " because this optical characteristics of QDs " we can in its continuous excitation spectrum, choose more suitable excitation wavelength " thereby the autofluorescence that makes biological specimen drops to minimum point, improves resolution and sensitivity.Therefore, the quantum dot immune analytic approach is far superior to enzyme immunoassay (EIA).Detect people's epididymal proteins 4(HE4 with the quantum dot immunoassay) be elder generation and then effective method, sensitivity and the specificity of clinical detection can effectively be provided.

Summary of the invention

One of purpose of the present invention has provided a kind of people's epididymal proteins 4(HE4) quantum dot-labeled double fastener heart immunoassay detection kit, it is low that this kit has overcome the sensitivity of existing HE4 enzyme immunoassay kit, the technological deficiency of poor specificity.

Two of purpose of the present invention has provided the application of above-mentioned detection kit in oophoroma detects, and this application can improve resolution, sensitivity and the specificity that oophoroma detects, and more effectively carries out early detection and the risk assessment of oophoroma.

The double fastener heart immunoassay detection kit that a kind of HE4 disclosed by the invention is quantum dot-labeled comprises the HE4 protein standard substance, wraps by the ELISA Plate of HE4 specific polyclonal antibody the quantum dot-labeled monoclonal antibody of CdTe that can be combined with the HE4 protein-specific.

Described HE4 protein standard substance is the recombinant protein of procaryotic cell expression, the HE4 albumen of expressing in prokaryotic is the soluble protein that possesses native conformation, namely can be used as the used HE4 protein standard substance of kit simultaneously as monoclonal or the polyclonal antibody of immunogen preparing high specific.Its peptide sequence is: EKTGVCPELQADQNCTQECVSDSECADNLKCCSAGCATFCSLPNDKEGSCPQVNIN FPQLGLCRDQCQVDSQCPGQMKCCRNGCGKVSCVTP.

Described polyclonal antibody is the anti-people's polyclonal antibody of rabbit, is used for coated elisa plate as capture antibody, and described monoclonal antibody is mouse-anti people HE4 monoclonal antibody, as detecting antibody.

The anti-people's polyclonal antibody of described rabbit is synthetic as immunogene with the HE4 recombinant protein of procaryotic cell expression.

Described mouse-anti people HE4 monoclonal antibody is synthetic as immunogene with the HE4 recombinant protein of procaryotic cell expression, screening obtains the hybridoma cell strain of the anti-HE4 monoclonal antibody of 2 strains energy stably excreting when synthesizing mouse-anti people HE4 monoclonal antibody, difference is called after 3B4 voluntarily, 1D10, and protected kind frozen.

Described kit also comprises confining liquid (5% bovine serum albumin(BSA)), sample diluting liquid (PBS that contains the Tween-20 of 1%BSA and 0.05%), the monoclonal antibody dilution (PBS,, PH7.2), wash plate liquid (the PBS+0.5% Tween-20, PH7.2).

The invention also discloses the application of above-mentioned detection box in diagnosis on ovarian tumors, can utilize fluorescence microplate reader fluorescence intensity value to obtain final detection result.

Carry out mark as the monoclonal anti body and function CdTe quantum dot that detects antibody in the kit disclosed by the invention, because the quantum dot-labeled method that adopts has the exciting light spectrum width, a little less than the autofluorescence, characteristics such as the stable and long half time of fluorescence, big raising the oophoroma resolution, sensitivity and the specificity that detect, early detection and the risk assessment of oophoroma are carried out in more effective help.

Description of drawings

Fig. 1 western blotting figure before and after the HE4 recombinant protein purification that behaves;

Fig. 2 is the typical curve of the anti-people HE4 of the rabbit behind purifying polyclonal antibody;

Fig. 3 is the typical curve of the mouse-anti people HE4 monoclonal antibody behind the purifying;

Fig. 4 is the test zone half interval contour of HE4 quantum dot immune analyzing and testing kit.

Embodiment

Embodiment 1:HE4 prokaryotic expression carrier makes up and the expression and purification of albumen is identified

1, the immunogenic selection of HE4

(1) the former district of HE4 genetic immunization

GAGAAGACTGGCGTGTGCCCCGAGCTCCAGGCTGACCAGAACTGCACGCAAGAGTGCGTCTCGGACAGCGAATGCGCCGACAACCTCAAGTGCTGCAGCGCGGGCTGTGCCACCTTCTGCTCTCTGCCCAATGATAAGGAGGGTTCCTGCCCCCAGGTGAACATTAACTTTCCCCAGCTCGGCCTCTGTCGGGACCAGTGCCAGGTGGACAGCCAGTGTCCTGGCCAGATGAAATGCTGCCGCAATGGCTGTGGGAAGGTGTCCTGTGTCACTCCCAATTTC

(2) immunogene district translation

EKTGVCPELQADQNCTQECVSDSECADNLKCCSAGCATFCSLPNDKEGSCPQVNINFPQLGLCRDQCQVDSQCPGQMK?CCRNGCGKVSCVTP

2, the HE4 prokaryotic expression carrier makes up

(1) restriction enzyme site and primer sequence

EcoRI?Up?primer：CCG?gaattc?gagaagactggcgtgtgcccc

XhoI?Down?primer：CCG?ctcgag?gaaattgggagtgacacagga

(2) the HE4 prokaryotic expression carrier makes up

Select the mucous ovarian cancer cell as the RNA extraction source.Utilize chloroform extraction method extracting cell total rna from cell, go out HE4 immunogene region sequence by reverse transcription and PCR primer amplification.HE4 sequence enzyme after the amplification is connected on the pGEX-4T1 prokaryotic expression carrier after cutting, transforms in the DH5 α competent cell, with spreading rod transformant is inoculated into the LB solid culture ware of ammonia benzyl resistance, 37 ℃ of overnight incubation.The single bacterium colony that picking transforms on the LB flat board of back carries out a small amount of amplification cultivation, extracts plasmid.Adopt agarose electrophoresis behind EcoR I and the Xho I double digestion, observe and have or not purpose 300bp place band to occur, the bacterium colony sample presentation after the Preliminary Identification is checked order, the correct bacterium colony that the checks order preservation that keeps sample.

3, the immunogenic expression of HE4 and purifying

(1) expression of HE4 destination protein

The fusion protein expression carrier pGEX-4T-1-HE4 transformed into escherichia coli RG2 that order-checking is correct, 37 ℃ of picking clones, the 250rpm concussion is cultivated and is reached 0.4-0.6 up to the OD value, induces 4h for IPTG1mM28 ℃.5000g10min is centrifugal, and the collection thalline is standby.Bacterium does precipitation can be stored in-80 ℃.

(2) detection of expression of HE4 destination protein

Bacterium does and to add an amount of lysate in the precipitation, guarantees in the ultrasonic degradation thalline ultrasonic procedure under the ice bath that albumen is in the ice bath all the time, prevents that ultrasonic to cross heat affecting protein stabilized.12000rpm, 4 ℃ centrifugal, albumin in the collection.Western identifies expressing quantity and albumen.

(3) separation and purification of HE4 destination protein

Bacterium does and to add an amount of lysate in the precipitation, guarantees in the ultrasonic degradation thalline ultrasonic procedure under the ice bath that albumen is in the ice bath eventually, prevents that ultrasonic to cross heat affecting protein stabilized.12000rpm, 4 ℃ centrifugal, albumin in the collection.With protein lysate and the abundant mixing of prerinse GSH – agarose of centrifugal collection, 2-4h is hatched in 4 ℃ of concussions altogether.Cell pyrolysis liquid cleans 3 times, and TBS cleans 3 times.Add 100mM glutathione eluent eluted protein.Albumen behind the wash-out can be by 3KD albumen evaporating column protein concentrate and remove glutathione in the liquid.Albumen behind the purifying is measured protein concentration and is identified its purity by western with the Coomassie brilliant blue method, sees accompanying drawing 1.

Embodiment 2: the preparation of the anti-people HE4 of rabbit polyclonal antibody and evaluation

1, animal immune

Prepare two adult rabbits, dissolve in the 1ml phosphate buffer solution 100 μ g antigen/rabbits stand-by.Add mycobacterium and make Freund's complete adjuvant in the 1ml freund 's incomplete adjuvant, and add the 1ml antigenic solution, it is fully emulsified that concuss makes it, and extracts this emulsion with the 3ml syringe, connects the 25G syringe needle, the bubble in the Inside Syringe.Take out rabbit and be placed on smoothly from cage, carry out hypodermic injection at 4 different positions, two are in back, and two are in the thigh place.Comfort the rabbit hair of injection place and the skin that exposes with ethanol disinfection.Pinch out skin, with the angle inserting needle of syringe needle with relative skin 15 degree, depth of needle is 1cm-2cm, does not carefully thrust in the muscle, respectively injects about 500 μ l antigenic solutions respectively at 4 different parts.Injection is extracted pin gently after several seconds are placed in injection place again, and is sterilized in injection place with ethanol after finishing.Repeat aforesaid operations at 4 positions.Every 4-6 week injections of antigens, and blood is collected according to step 2 in after injection 7 days-10 days.Whether the blood of collecting, checking has antibody to produce if being compared with the preceding blood of collecting of injection.Can collect blood in a large number behind the generation antibody to be determined, but every rabbit collection blood can not be more than 40ml to prevent shock.

2, collect serum

Rabbit is put on the fixed mount gently, and dimethylbenzene is applied to the middle part of going up of ear's blood vessel, with 45 ° of otch that cut out 0.23cm-0.3cm at this place of blade inclination blood can be flowed out freely.Collect the blood that oozes with the pipe after the sterilization, dab incision if before finishing, available warm water occurs solidifying, continue again to collect.Collect that the gauze after the available sterilization dabs the affected part behind an amount of blood, can finish after 10 seconds-20 seconds definite blood flows in flicking affected part stop.Blood was placed 30 minutes in 37 ° of C constant temperature ovens, placed at 4 ° of C again and spend the night.Medication shovel is dialled blood clot fall from tube wall, with blood transfer to plastic centrifuge tube, 4 ° of C, centrifugal 10 minutes of 10,000g collects supernatant and is antiserum, can preserve the several years at-20 ° of C.

3, antibody purification

Antiserum is put into the gathering that frozen water or 4 ° of C refrigerators slowly thaw to avoid protein.The gathering that occurs in the protein course of defrosting can be dissolved by 37 ° of C preheatings.Adding the solid sodium azide is 0.05%, 4 ° of C to concentration, centrifugal 5 minutes of 15,000xg, and the antiserum that shifts out clarification removes by filter unnecessary fat through filter again.

Antibody is diluted with the ratio of 1:5 with TBS buffer solution, filter with filtrator again.With the speed of per minute 0.5ml with on the antiserum to post, be the combination that guarantees antiserum and filler, need continuous upper prop 2 times and keep to go up sample outflow liquid.Clean pillar with TBS buffer solution and to A λ 280nm＜0.008, add pH2.7 elution buffer solution, be eluted to all albumen with the speed of 0.5ml/min and all flow down.Be in charge of the collection eluent with the 1.5ml EP pipe that adds 100ul neutralization buffer solution, check the pH of eluent behind the mixing with the pH test paper, can utilize neutralization buffer to transfer to about pH7.4 to prevent the sex change of antibody if pH is lower than 7.Add 10ml in post, pH1.9 elution buffer solution is collected eluent as stated above to A λ 280nm＜0.008.

Utilize Protein content in each pipe of spectrophotometric determination.Antibody concentration is 2.4mg/ml.With ° C preserves 2 ° of C～8 after the antibody purified packing.

4, the evaluation of anti-HE4 polyclonal antibody

(1) the anti-HE4 polyclonal antibody of indirect ELISA Preliminary detection serum sensitivity

Recombinant protein HE4 is wrapped quilt by the amount of 1ug, 100ng, 10ng, 1ng, 100pg, 10pg, 1pg respectively, primary antibodie adds presses 1:500,1:1000,1:2000,1:5000, the rabbit immune serum that 1:10000 doubly dilutes, two anti-be the anti-rabbit igg of HRP labelled goat of 1:8 ten thousand dilutions, the TMB colour developing, 450nm surveys the OD value.Being horizontal ordinate with the albumen dilutability, is ordinate with the 450nmOD value, and figure runs a curve.

As a result, serum 1:10000 dilutes 10pg～10ng between the detection zone that has good linear relationship.

(2) the western blotting of the anti-people HE4 of rabbit polyclonal antibody serum identifies

Recombinant protein HE4 is carried out SDS-PAGE glue point sample by the amount of 1ng, 100pg, 10pg, 1pg respectively, primary antibodie adds the polyclonal antibody serum that doubly dilutes by 1:500,1:1000,1:2000, two anti-be the anti-rabbit igg of HRP labelled goat of 1:1 ten thousand dilutions, western identifies antibody titer.

As a result, the polyclonal antibody serum that doubly dilutes of 1:2000 can well be identified 1ng HE4 recombinant protein.

(3) titration of anti-HE4 polyclonal antibody behind the purifying

Adopt indirect elisa method, with recombinant protein HE4 antigen 1 μ g/100 μ l bag quilt, 4 ℃ are spent the night; The 5% skimmed milk power sealing of washing back is spent the night; The polyclonal antibody serial dilution of purifying is as primary antibodie, 37 ℃ of incubation 2h; The anti-rabbit igg 1:80 of washing back HRP-labelled goat, 000 dilution is anti-as two, 37 ℃ of incubation 1h; Add the substrate colour developing after the washing and in time stop survey OD450 value.

The results are shown in accompanying drawing 2, tiring of polyclonal antibody is 1:51200.

The concrete steps of indirect ELISA:

1) wrapper sheet: pH9.6 carbonate buffer solution dilution antigen is measured 100 μ l/ holes to optimum concentration 1 μ g/ hole, puts into wet box, and 4 ℃ are spent the night;

2) sealing: discard coating buffer next day, PBST detersive enzyme mark reacting hole 4 times, each 5min dries, and wet box is put in the sealing of 5% bovine serum albumin(BSA), 300 μ l/ holes, and 4 ℃ are spent the night;

3) discard confining liquid, the same washing, 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;

4) discard blood serum sample, the same washing, PBS is diluted to 1:80,000 HRP-goat anti-rabbit igg 100 μ l/ holes, 37 ℃ of incubation 1h;

5) discard ELIAS secondary antibody, the same washing, the TMB colour developing, each 50 μ l/ hole of substrate A and B, 37 ℃ of lucifuge colour developing 10min, every hole adds 50 μ l2mol/L H2SO4 stop buffers, and full-automatic microplate reader is measured the OD450nm value.

Embodiment 3: mouse-anti people HE4 Monoclonal Antibody and evaluation

1, mouse-anti people HE4 MONOCLONAL ANTIBODIES SPECIFIC FOR

(1) animal immune

Immunization protocol: recombined human HE4 antigen and isopyknic Freund's complete adjuvant of purifying is fully emulsified, make Water-In-Oil antigen emulsion, 6～8 ages in week of SPF level pure lines BALB/c female mice, the subcutaneous multi-point injection in back.100 μ g/ only add the identical incomplete Freund of volume with equivalent antigen behind the fortnight, carry out emulsification, immunity.Respectively at the 2nd after the first immunisation, 4,6 weekends carrying out booster immunization, use the destination protein of same dose at every turn.Detect tiring of serum antibody with indirect elisa method.Preceding 3 days mouse tail vein injections of Fusion of Cells or lumbar injection 100 μ g HE4 albumen are with booster immunization.

(2) recovery of SP2/0 cell and cultivation

From liquid nitrogen container, take out and contain SP2/0 cell cryopreservation pipe, drop into immediately in 37 ℃ of water-baths, melt the back in the centrifugal 5～10min of 1000rpm, abandon supernatant; Preparation contains the DMEM nutrient solution of 10% calf serum and cultivates the recovery cell.It is subcutaneous that cell is inoculated in both sides, normal BALB/c mouse back.Treat that knurl grows to about 3～5cm, namely carry out the aseptic knurl of plucking, after serum-free DMEM nutrient solution washing 3 times, be cut into the about 2mm of diameter left and right sides fritter with little scissors, add in the 200 order copper sieve that has added 2～3ml serum-free DMEM nutrient solution in advance, grind, squeeze out single tumour cell with piston, put conventional cultivation the in the DMEM nutrient solution that contains 10%FBS, make cell keep exponential phase.Merge preceding 1 day for the SP2/0 cell changes a not good liquor, the adjusting cell density is 1～5 * 105/ml, merges to get in about 1～5 * 107 aseptic centrifuge tubes of SP2/0 cell harvesting to 1 50ml same day, the centrifugal supernatant of abandoning adds 5ml serum-free DMEM nutrient solution, mixing, cell count, standby.

(3) separate the fusion 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 in cake wax; Behind 75% alcohol disinfecting skin, cut off skin of abdomen, expose peritonaeum and also sterilize with 75% alcohol wipe; Draw serum-free DMEM nutrient solution 5ml with glass syringe and inject mouse peritoneal, with syringe suction (attention can not puncture the digestive organs of mouse) repeatedly in the abdominal cavity; Extract liquid in the abdominal cavity out with this syringe, inject in the 50ml centrifuge tube; Change tweezers, mention peritonaeum, change scissors, expose the abdominal cavity, the aseptic spleen of winning, carefully cut off peripheral fat and manadesma fast, wash 1～2 time with serum-free DMEM nutrient solution, again spleen is put into the plate of containing 200 order copper sieve, break coating, cross net with piston grinding, extruding splenocyte, draw serum-free DMEM nutrient solution 5ml piping and druming copper sieve, the splenocyte of collecting after netting is put into the aseptic centrifuge tube of 50ml; With 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 place 37 ℃, the interior cultivation of 5%CO2 incubator then, and are standby.

(4) selectivity of Fusion of Cells and hybridoma is cultivated

The SP2/0 cell is mixed in the aseptic centrifuge tube of 50ml l000rpm, centrifugal 5min with splenocyte in the 1:5 ratio; Abandon supernatant, flick the pipe end, make precipitation loosening, along the slow 45%PEG solution lml that drips 37 ℃ of pre-temperature of centrifugal tube wall, the slow centrifuge tube that rotates adds PEG4000 in the 1min with the mixing cell simultaneously; Put in 37 ℃ of water-baths behind the 1min, in 5min, slowly add the serum-free DMEM nutrient solution 8ml of 37 ℃ of pre-temperature again, stir gently simultaneously and make cell become the suspension of homogeneous; Put in 37 ℃ of water-baths behind the 5min, l000rpm, centrifugal 5min abandons supernatant; The HAT nutrient solutions that add 37 ℃ of pre-temperature, gently outstanding cell is added drop-wise in the 96 porocyte culture plates that contain the feeder layer cell by 1 * 105 splenocyte/hole, places 37 ℃, 5%CO2 incubator to cultivate.Merging the back can namely use HAT nutrient solution half amount to change liquid according to the clonal growth situation in 5～10 days; The interchangeable HT nutrient solution in 2 week backs, the interchangeable complete culture solution in 3 week backs; Cultivating 3～5 days is that visible little clone occurs, and hybrid cell is bigger, and is rounded and transparent, and other cell light transmission differences are also dead gradually; Cultivated 8～12 days, clonal growth is to 1/3～1/2 of the hole floorage, and this moment, desirable culture supernatant was carried out antibody test; In case detect the clone cell of the predetermined antibody of secretion, should be in time with the positive colony transferred species to 24 well culture plates, further change culture flask again over to and enlarge and cultivate, frozen part clone cell carries out cloning simultaneously and cultivates.

(5) hybridoma screening of secretion monoclonal antibody specific

After the Fusion of Cells, in case when growing the clone of suitable size, should in time select sensitivity, fast, the hybrid cell clone of the predetermined antibody of the secretion of immunological method screening reliably.This experiment adopts the indirect elisa method of recombined human ES antigen to detect, and wherein primary antibodie is the Hybridoma Cell Culture supernatant, and two anti-ly are HRP-mountain sheep anti-mouse igg (1:80,000).The concrete operations step detects with the mouse serum titer.

(6) subcloning is cultivated

Cloning is cultivated for the hybridoma cell strain that obtains the secretion monospecific antibody most important.Generally need carry out 3～4 subclonings and cultivate, to guarantee the stability of secretion sex clone growth.Adopt limiting dilution assay, detailed step is as follows: the preparation feeder layer: get the normal mouse abdominal cavity cell, make cell suspension, inoculate 96 well culture plates, every hole 50 μ l contain 2 * 104 cells approximately, overnight incubation in 37 ℃, 5%CO2 incubator; Clone in the inferior daily micropipettor piping and druming Hybridoma Cell Culture plate mesopore is suspended in the complete culture solution; Sampling with the blood counting chamber counting, is adjusted cell concentration to 20,5/ml; Respectively with the hybridoma suspension inoculation of two kinds of density in the culture plate that contains the feeder layer cell, every hole 50 μ l make every hole contain 2,1,0.5 cells respectively; Place 37 ℃, 5%CO2 incubator to cultivate, add 100 μ l nutrient culture media after the week.Cultivated 12～15 days, the culture supernatant that the clone is arranged is carried out antibody test; To the cloning cultivation again of positive monoclonal cell, till clone's secreting specificity antibody of 100%; Simultaneously positive colony is further enlarged cultivation, frozen.The final hybridoma cell strain that obtains the anti-HE4 monoclonal antibody of 2 strains energy stably excreting, difference called after 3B4,1D10.

(7) a large amount of preparations and the purifying of mouse-anti people HE4 monoclonal antibody

Hybridoma can produce and secrete monoclonal antibody in cell cultivation process, about 10～100 μ g/ml.In order to obtain a large amount of high-titer antibodies, usually hybridoma to be implanted in the BALB/c mouse body, preparation is also collected the ascites that contains monoclonal antibody specific, and method is as follows: select 8～10 all BALB/c female mices for use.1～2 week before the inoculation hybridoma is earlier 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 the serum-free medium, and the adjustment cell density is 1～2 * 106/ml, every mouse peritoneal injection 0.5ml cell suspension; The health status of close observation mouse and ascites sign, inoculating cell 7～12 days, as seen mouse web portion obviously expands, and when touching with hand, skin has nervous sense, the skin of abdomen of can sterilizing, connect syringe needle No. 8 with the 5ml syringe, thrust the abdominal cavity, unload syringe, raise mouse head, ascites is splashed in the centrifuge tube; 2～3 days at interval, treat that ascites regeneration is gathered after, get again with method, a mouse generally can extract 2-3 time.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 hybridoma ascites.Antibody concentration behind the purifying is 1.2mg/ml

2, the evaluation of anti-HE4 monoclonal antibody

(1) the anti-HE4 monoclonal antibody of indirect ELISA Preliminary detection sensitivity

Recombinant protein HE4 is wrapped quilt by the amount of 1ug, 100ng, 10ng, 1ng, 100pg, 10pg, 1pg respectively, primary antibodie adds the 3B4 that doubly dilutes by 1:500,1:1000,1:2000,1:4000,1D10 ascites, two anti-be the anti-mouse IgG of HRP labelled goat of 1:8 ten thousand dilutions, the TMB colour developing, 450nm surveys the OD value.Being horizontal ordinate with the albumen dilutability, is ordinate with the 450nmOD value, and figure runs a curve.

As a result, ascites 1:4000 dilutes 10pg～10ng between the detection zone that has good linear relationship

(2) the western blotting of mouse-anti people HE4 monoclonal antibody identifies

Recombinant protein HE4 is carried out SDS-PAGE glue point sample by the amount of 1ng, 100pg, 10pg, 1pg respectively, primary antibodie adds the monoclonal antibody ascites of doubly diluting by 1:500,1:1000, two anti-be the anti-mouse IgG of HRP labelled goat of 1:1 ten thousand dilutions, western identifies antibody titer.

As a result, the 1:1000 monoclonal antibody ascites of doubly diluting can well be identified 1ng HE4 recombinant protein.

(2) titration of anti-HE4 monoclonal antibody behind the purifying

Adopt indirect elisa method, with recombined human ES antigen 1 μ g/100 μ l bag quilt, 4 ℃ are spent the night; The 5% skimmed milk power sealing of washing back is spent the night; The monoclonal antibody serial dilution is as primary antibodie behind the purifying, 37 ℃ of incubation 2h; The anti-mouse IgG1:80 of washing back HRP-labelled goat, 000 dilution is anti-as two, 37 ℃ of incubation 1h; Add the substrate colour developing after the washing and in time stop survey OD450 value.

The results are shown in accompanying drawing 3, tiring of monoclonal antibody is 1:10240.

(3) immunoglobulin class of mouse-anti people HE4 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.

(4) the CdTe quantum dot is synthetic

CdCl22.5H2O(2.5 * 10-4mol) be dissolved in the ultrapure water of 25ml, adding glutathione GSH(3 * 10-4mol), two hydration trisodium citrates (0.1g), Na2TeO3(0.5 * 10-4mol) and NaBH4(2.4 * 10-4mol), under the condition of magnetic agitation, regulate PH to 10.5 with NaOH, it all is under room temperature environment that institute responds, Cd2+, the mol ratio of TeO32-and GSH is 5:1:6, put into the microwave device (power is made as 600W) of taking back stream, begin to reflux when solution colour becomes light green, what form a series of different-grain diameters along with the difference of return time is the quantum dot of stabilizing agent with the glutathione.Precipitate centrifugal 5min under the condition of 4000rpm with absolute ethyl alcohol after selecting suitable quantum dot that reacted solution is cooled to room temperature.Remove excessive impurity such as Cd2+, TeO32-in the supernatant, repeat 3 times, treat that ethanol volatilizees fully after, precipitation is resuspended among the PBS of PH7.4.

(5) coupling of CdTe quantum dot and mouse-anti people HE4 monoclonal antibody and purifying (serve as a mark two anti-)

Get the CdTeQDs600ul of above-mentioned concentration, the EDC(1mg/ml that adds 18ul) and the methyl alcohol of 800ul mix the back lucifuge and shake 30min, the beta-mercaptoethanol that adds 8ul again stops, getting two anti-being diluted among the QDs that proper volume adds activation with PBS of 0.75mg mixes with it, lucifuge concussion 2h, add mercaptoethanol again after reaction finishes and stablize quantum dot, dialyse.Dialysis back centrifugal 3min under the 16300g condition removes supernatant, and precipitation (coupling that is purifying has the P1 recombinant protein antibody of quantum dot) is resuspended among the PBS 4 ℃ of preservations.

Find that in fluorescence microplate reader fluorescence intensity value mark two resists that to detect effect under the diluting condition at 1:10000 best through immunofluorescence assay.

Embodiment 4: the preparation of anti-people HE4Elisa detection kit

1, detection method

In the present embodiment, utilize the anti-people HE4 of the rabbit polyclonal antibody of the purifying that obtains among the embodiment 2 as capture antibody, the quantum dot-labeled mouse-anti people HE4 monoclonal antibody of CdTe is set up the how anti-and monoclonal antibody double fastener heart quantum dot fluorescence immunodetection of oophoroma tumor label HE4 as detecting antibody among the embodiment 3.

2, the bag of ELISA Plate quilt and sealing

PH9.6 carbonate buffer solution dilution polyclonal antibody is measured 100 μ l/ holes to optimum concentration 1 μ g/ hole, 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 wet box or dry metal foil bag are put in the sealing of 5% bovine serum albumin(BSA), 300 μ l/ holes, and 4 ℃ are spent the night.

3, the processing of standard items or tested serum

HE4 protein standard substance (positive criteria product) or tested serum are done the dilution of proper proportion with sample diluting liquid (PBS that contains the Tween-20 of 1%BSA and 0.05%), and the sample diluting liquid of increase serum is not as negative standard items.The 100ul/ hole, 37 ℃ of reactions were washed plate 3 times with washing plate liquid after 1 hour, and ELISA Plate is patted dry.

4, the processing of the monoclonal antibody behind the mark

The quantum dot-labeled mouse-anti people HE4 monoclonal antibody of CdTe is dissolved among the PBS that contains 50% glycerine, uses with PBS dilution monoclonal antibody 1:10000 dilution back, and is now with the current.The 100ul/ hole, 37 ℃ of reactions were washed plate 3 times with washing plate liquid after 1 hour.Pat dry the back and add 100ul PBS.

5, analyzing and testing

The ELISA Plate of handling well is measured fluorescence intensity level via fluorescence microplate reader (excitation wavelength 511nm, emission wavelength 548nm).Set up typical curve, obtain detectability (LOD).

6, kit performance index examination

(1) between the detection zone of kit

Get bag by good rabbit anti-people HE4 polyclonal antibody and the ELISA Plate of sealing, the HE4 protein standard substance is done the dilution of proper proportion, each sample is done 3 repeating holes.The 100ul/ hole, 37 ℃ of reactions were washed plate 3 times with washing plate liquid after 1 hour, and ELISA Plate is patted dry.The quantum dot-labeled mouse-anti people HE4 monoclonal antibody of CdTe is dissolved among the PBS that contains 50% glycerine, uses with PBS dilution monoclonal antibody 1:10000 dilution back, and is now with the current.The 100ul/ hole, 37 ℃ of reactions were washed plate 3 times with washing plate liquid after 1 hour.Pat dry the back and add 100ul PBS.The ELISA Plate of handling well is measured fluorescence intensity level via fluorescence microplate reader (excitation wavelength 511nm, emission wavelength 548nm).Standard items albumen with the HE4 of variable concentrations is horizontal ordinate, is ordinate with corresponding glimmering light intensity value, curve plotting.

As a result, as shown in Figure 4, this kit has and is 10pM-1000pM. between the detection zone of good linear relationship, if detected serum surpasses 1000pM, suggestion detects after with the sample diluting liquid dilution again.

(2) kit sensitivity (lowest detection amount)

Be used as sample with zero normative reference product (A point) and measure 20 times, calculate its fluorescent value average and standard deviation.Average with A point measured value adds that the concentration that the fluorescent value substitution typical curve equation of 2 times standard deviation gained calculates is its lowest detection amount.

As a result, the lowest detection amount is＜5pM.

(3) stability of kit

Three batches of reagent of self-control kit are positioned over 4 ℃ of half a year and 1 year respectively, place after 7 days for 37 ℃, linear relationship and the zero standard product fluorescence intensity level between normative reference product each point fluorescence intensity level relatively and before placing, stable testing.

Embodiment 5: the evaluation of anti-people HE4Elisa detection kit

Detected for 200 example mean aves and be the content of HE4 in 41 years old women's serum, wherein 50 examples are that the healthy women of health check-up in 35 years old is as normal control group (n=50) mean aves, 150 examples are because of ovarian neoplasm hospitalization patient, the result classifies according to its Pathological diagnoses, wherein 86 examples are benign tumour, and 64 examples are malignant tumour.Testing result is: the scope of HE4 content is 18～40.0pmol/L in the normal control group serum, and average is 31 ± 4.39pmol/L; The scope of HE4 content is 25～73.0pmol/L in the benign tumour group serum, and average is 42 ± 9.2pmol/L; The scope of HE4 content is 54～1263.0pmol/L in the malignant tumors group serum, and average is 476 ± 356.6pmol/L; Malignant tumors group HE4 level is apparently higher than other two groups, and difference has statistical significance (p＜0.01).

In sum; although the specific embodiment of the present invention is described in detail the present invention; but persons skilled in the art should be understood that; above-described embodiment only is the description to the preferred embodiments of the present invention; but not limiting the scope of the invention; persons skilled in the art are in the disclosed technical scope of the present invention, and the variation that can expect easily is all within protection scope of the present invention.

Claims (10)

1. double fastener heart immunoassay detection kit that HE4 is quantum dot-labeled, it is characterized in that: described kit comprises the HE4 protein standard substance, bag is by the ELISA Plate of HE4 specific polyclonal antibody, the quantum dot-labeled monoclonal antibody of CdTe that can be combined with the HE4 protein-specific.

2. the quantum dot-labeled double fastener heart immunoassay detection kit of HE4 as claimed in claim 1, it is characterized in that: described HE4 protein standard substance is the recombinant protein of procaryotic cell expression.

3. the quantum dot-labeled double fastener heart immunoassay detection kit of HE4 as claimed in claim 2, it is characterized in that: its peptide sequence of described recombinant protein is: EKTGVCPELQADQNCTQECVSDSECADNLKCCSAGCATFCSLPNDKEGSCPQVNIN FPQLGLCRDQCQVDSQCPGQMKCCRNGCGKVSCVTP.

4. the quantum dot-labeled double fastener heart immunoassay detection kit of HE4 as claimed in claim 1, it is characterized in that: described polyclonal antibody is the anti-people's polyclonal antibody of rabbit, be used for coated elisa plate as capture antibody, described monoclonal antibody is mouse-anti people HE4 monoclonal antibody, as detecting antibody.

5. the quantum dot-labeled double fastener heart immunoassay detection kit of HE4 as claimed in claim 4 is characterized in that: the anti-people's polyclonal antibody of described rabbit is synthetic as immunogene with the HE4 recombinant protein of procaryotic cell expression.

6. the quantum dot-labeled double fastener heart immunoassay detection kit of HE4 as claimed in claim 4 is characterized in that: described mouse-anti people HE4 monoclonal antibody is synthetic as immunogene with the HE4 recombinant protein of procaryotic cell expression.

7. the quantum dot-labeled double fastener heart immunoassay detection kit of HE4 as claimed in claim 6 is characterized in that: the hybridoma cell strain of the anti-HE4 monoclonal antibody of screening acquisition 2 strains energy stably excreting in synthetic mouse-anti people HE4 monoclonal antibody.

8. the quantum dot-labeled double fastener heart immunoassay detection kit of HE4 as claimed in claim 1, it is characterized in that: described kit also comprises confining liquid, sample diluting liquid, the monoclonal antibody dilution is washed plate liquid.

9. utilize the application of the described detection box of claim 1 in diagnosis on ovarian tumors.

10. the application of detection box as claimed in claim 9 in diagnosis on ovarian tumors is characterized in that: utilize fluorescence microplate reader fluorescence intensity value to obtain final detection result.

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