CN109444435A - A kind of nanometer selenium kit of quick detection HE4 and CA125 - Google Patents
A kind of nanometer selenium kit of quick detection HE4 and CA125 Download PDFInfo
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- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57449—Specifically defined cancers of ovaries
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
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Abstract
The invention belongs to biology immunochromatography detection method fields, and in particular to a kind of nanometer selenium kit and preparation method thereof of quickly detection HE4 and CA125.The kit is made of sample pad, bonding pad, reacting pad, water absorption pad and PVC bottom plate；There is the detection line T1 for being coated with anti-CA 125 antibody A 2 on the reacting pad, be coated with the detection line T2 of anti-HE4 antibody B2 and be coated with the nature controlling line C of sheep anti-mouse igg；Rapid screening diagnosis by the bed of early ovarian cancer can be realized in chromogenic reaction by observing detection line.Nanometer selenium immune chromatography reagent kit prepared by the present invention has high specificity, high sensitivity, accuracy is good, easy quick, required detection sample size is few, be suitable for oophoroma people at highest risk screening and family's self-test and bed side is quick to detect.
Description
Technical field
The invention belongs to biology immunochromatography detection method fields, and in particular to a kind of quickly to detect HE4's and CA125
Nanometer selenium kit and preparation method thereof.
Background technique
Oophoroma is one of most common gynecologic malignant tumor, and the death rate is in first of gynecologic malignant tumor, is had simultaneously
Disease incidence is high, progression of the disease is very fast, prognosis is bad, the features such as easily recurring.5 years survival rates of early ovarian cancer patient are reachable
70%, but the 5 of Patients with Advanced Ovarian Carcinoma years survival rates only have 30%.Since the morbidity of oophoroma is hidden, early symptom is unknown
It is aobvious, it has been advanced stage when clinically Most patients are made a definite diagnosis, has missed best occasion for the treatment.And the reason of causing these to happen with
It is closely related to lack simple and effective early detection method.Clinically the diagnosis of oophoroma is mainly examined by laboratory at present
It looks into, the methods of gynecologial examination and imageological examination, these methods are complicated for operation, take a long time, and need large-scale instrument and equipment
Auxiliary.In contrast, the detection of serology tumor-marker have it is sensitive it is quick, traumatic it is small, detection is simple, cost is relatively low, is easy to
The advantages that acquisition is one of the method for the widest screening tumour of current clinical application, it can also be used to which the curative effect of tumor patient is supervised
The screening of survey, prognosis evaluation and people at highest risk.
Carbohydrate antigen CA125 is a kind of sugar-protein compound, is primarily present in ovary tissue.In normal human blood
CA125 content is lower, and CA125 height is expressed in epithelial ovarian carcinoma patients, and can be detected in blood samples of patients
To (Zwakman N, Journal of gynecologic oncology).Main blood as current oophoroma early screening
Clear tumor markers, CA125 is widely used in the prediction of the diagnosis of oophoroma, the assessment of therapeutic effect and recurrence, and is regarded
For " goldstandard " of oophoroma clinical diagnosis.But Serum tumor marker CA125 sensibility is lower, it is different in pelvic inflammatory disease, peritonitis, endometrium
Also there is certain expression in position disease and other tumours, be easy to cause false positive.Therefore, Serum tumor marker CA125 is examined in oophoroma clinic
Application in disconnected has certain limitation.
People's epididymal proteins 4 (HE4) are a kind of small points rich in cysteine found by scholars such as Kirchhoff in 1991
Sub- secreting glycoprotein has no its expression in ovary normal tissue, and highly expresses in ovarian carcinoma
(Steffensen K D,Oncology letters).It is now recognized that HE4 is a kind of novel blood for early ovarian cancer detection
Clear marker, at the same HE4 be also after CA125 first obtain Food and Drug Adminstration of the US approval can be used for monitoring ovum
The tumor markers of nest cancer recurrence patient.Studies have shown that the specificity of HE4 diagnosis is significantly higher than Serum tumor marker CA125, level variation
There is certain correlation with ovarian cancer progression's stage, on the one hand can be used as the reference of medical diagnosis on disease, on the other hand can make
For therapeutic effect monitoring index, on monitoring outcome, it may have significance.But the spirit of HE4 clinical diagnosis oophoroma
Sensitivity is lower than Serum tumor marker CA125.Serum tumor marker CA125 and HE4 detection are all the important tumor markers for detecting oophoroma, individually detect one
When a index, but respectively there is deficiency.Therefore, application of the two joint-detection in oophoroma clinical diagnosis is gradually extensive, CA125 connection
Close the exclusive use that the sensibility and specificity of HE4 both is superior to, significantly reduced while improving recall rate rate of missed diagnosis with
Misdiagnosis rate can be used for early diagnosis, identification, Treatment monitoring and prognosis evaluation (the Ghasemi N, Medical of oophoroma
Oncology)。
It is by enzyme-linked immunization and chemistry hair mostly in terms of oophoroma tumor marker early detection both at home and abroad at present
Light immunoassay realizes, using the principle of double-antibody sandwich combine several markers carry out quantitative detecting methods can to ovum
Nest cancerous swelling tumor markers quantitative detection, but need by instrument, complicated for operation, detection time is longer.It there is no and be directed in the market
The lateral chromatography kit of oophoroma tumor marker joint-detection.
Good, high sensitivity that the object of the present invention is to provide a species specificity, accuracy are strong, easy to operate, cost is relatively low simultaneously
Combine CA125 double antibody sandwich method nanometer selenium suitable for the HE4 of rapid screening diagnosis and family's self-test by the bed of early ovarian cancer
Immune chromatography reagent kit.The preparation of this kit is immunoreacted principle based on antigen-antibody, utilizes double antibody sandwich method.It is suitble to label
The nanometer selenium of immunoglobulin has the characteristics that other antigens or antibody can be marked.
The home-use lateral chromatography kit for being currently used for detection blood sample has the following deficiencies:
1. nanogold is with high costs: the marker that home-use lateral chromatography kit is mostly used at present is nanogold, preparation
Technique and detection effect are all preferable, but the cost is relatively high for nanogold, and preparation process is complex.
2. blood sample detects, false positive rate is high, and sensitivity is poor: lateral chromatography kit is for serum or blood at present
Detection relative difficulty, be easy to produce false positive, the detection of especially double detection lines, sensitivity, stability are more difficult to control, false sun
Property is also higher.
The formula that the present invention passes through optimization sample pad treatment fluid, it has unexpectedly been found that, at sample pad disclosed by the invention
It manages liquid (phosphate buffer, sucrose, 300mM sodium chloride, bovine serum albumin(BSA) and tween), blood sample inspection can be significantly reduced
The false positive of survey, hence it is evident that improve Detection accuracy.
Kit provided by the invention has simple, economic, accurate, quick, special, spirit using lateral chromatography immunization
It is quick and the features such as be not required to by pertinent instruments, and marker is using nanometer selenium preparation method, compared to common colloid
The advantages that preparation of the markers such as gold is easier, performance is more stable, detection is sensitiveer and more economical material benefit.Nanometer selenium method
The development of joint HE4 and CA125 oophoroma quick detection kit can satisfy oophoroma early detection, and this method has fastly
Speed, it is easy, economical, accurate, false positive rate is lower the features such as, be suitable for people at highest risk and screen, by family's self-test and bed quickly
Detection is of great significance to oophoroma early screening and raising survival.
Bibliography:
Zwakman N,van de Laar R,Van Gorp T,et al.Perioperative)changes in
serum CA125 levels:a prognostic factor for disease-specific survival in
patients with ovarian cancer[J].Journal of gynecologic oncology,2016,28(1).
Steffensen K D,M,Brandslund I,et al.Identification of
high-risk patients by human epididymis protein 4 levels during follow-up of
ovarian cancer[J].Oncology letters,2016,11(6):3967-3974.
Ghasemi N,Ghobadzadeh S,Zahraei M,et al.HE4 combined with CA125:
favorable screening tool for ovarian cancer[J].Medical Oncology,2014,31(1):1-
6.
Summary of the invention
The purpose of the present invention is to provide the nanometer selenium kits and preparation method thereof of quickly detection HE4 and CA125 a kind of.
The kit is made of sample pad 1, bonding pad 3, reacting pad 2, water absorption pad 4 and PVC bottom plate 5, the sample
Pad 1, bonding pad 3, reacting pad 2 and water absorption pad 4 are successively overlapped on PVC bottom plate；Be provided on the reacting pad 3 be coated with it is anti-
The detection line T1 of CA125 antibody ab2, the detection line T2 for being coated with anti-HE4 antibody ab2, the nature controlling line C for being coated with sheep anti-mouse igg.
The sample pad 1 is handled through sample pad treatment fluid, and treatment fluid is by phosphate buffer, neutral salt, sucrose, ox blood
Pure albumen and tween composition.
The neutral salt is selected from one of sodium chloride, potassium chloride, calcium chloride, barium chloride, calcium sulfate, potassium nitrate.
Preferably, neutral salt contained by the sample pad treatment fluid is sodium chloride or potassium chloride, concentration 300mM.
Preferably, the neutral salt in the sample pad treatment fluid is sodium chloride, concentration 300mM.
The compound of the nanometer selenium and anti-HE4 antibody and anti-CA 125 antibody coupling suspends solidification in knot through working solution
It closes on pad, the working solution is made of phosphate buffer, neutral salt, bovine serum albumin(BSA), carbohydrate and tween, the neutral salt
Selected from one of sodium chloride, potassium chloride, calcium chloride, barium chloride, calcium sulfate, potassium nitrate；The carbohydrate be selected from monosaccharide and disaccharide or
One of polysaccharide.
Preferably, the carbohydrate in the working solution is sucrose and trehalose.
Preferably, the neutral salinity in the working solution is 150-500mM.
Preferably, the concentration of the phosphate buffer of the treatment fluid and working solution is 0.01mol/L, pH 7.2.
The preparation method of the nanometer selenium kit of quick detection HE4 and CA125 of the present invention, comprising the following steps:
(1) preparation of nanometer selenium
It takes 16mL ultrapure water to be placed in 50mL small beaker, the PEG of the 2mol/L of 1mL is added, 20min is stirred at room temperature, disperse
Uniformly, PEG solution is obtained；The SDS of the 0.1mol/L of 1mL is dissolved in PEG solution, 20min is stirred at room temperature, is uniformly dispersed, mould is obtained
Plate solution；The selenous acid of the 0.04mol/L of 1mL is dissolved in template solution, 20min is stirred at room temperature, is uniformly dispersed, selenous acid is obtained
Solution；The vitamin C of the 0.32mol/L of 1mL is dissolved in selenous acid template solution, 20min is stirred at room temperature, is uniformly dispersed, i.e.,
Obtain nanometer selenium；
(2) antibody marks
The nanometer selenium for taking 1mL step (1) to be prepared is added the solution of potassium carbonate that 10 μ L concentration are 1mol/L, adjusts body
The pH value of system is 7.5；The Anti-HE4-Ab1 of the 2mg/mL of 2 μ L is added, mixed at room temperature 30min fills Anti-HE4-Ab1
That divides is integrated on nanometer Se particle, completes the label of Anti-HE4-Ab1；The nanometer selenium for taking 1mL step (1) to be prepared, adds
Enter the solution of potassium carbonate that 12 μ L concentration are 1mol/L, the pH value of regulation system is 8.0；Add the Anti- of the 2mg/mL of 3 μ L
CA125-Ab1, mixed at room temperature 30min are integrated to Anti-CA125-Ab1 adequately on nanometer Se particle, complete Anti-
The label of CA125-Ab1；
(3) it closes
It is added in the Anti-HE4-Ab1 for Anti-CA125-Ab1 and the nanometer selenium label that nanometer selenium marks in step (2)
BSA solution controls the quality final concentration of 1% of BSA, and room temperature mixes 30min, to close the exposed position on nanometer selenium nano particles
Point；
(4) it is resuspended
Nanometer selenium labelled antibody after closing is centrifuged 10min at 10000rpm, discards supernatant, the work of 120 μ L of precipitating
Make liquid resuspension；
The working solution is by phosphate buffer, sodium chloride, bovine serum albumin(BSA), sucrose, trehalose and Tween-20 group
At wherein phosphate buffering liquid concentration is 0.01mol/L, pH 7.2；Sodium chloride concentration is 150mM, protein amount
Final concentration of 2%, sucrose quality final concentration of 3%, final concentration of 3%, the Tween-20 volume of trehalose quality is final concentration of
0.1%；In phosphate buffer, Na2HPO4·12H2O mass content is 0.3%, NaH2PO4·2H2O mass content is
0.06%；
(5) preparation of sample pad
GF-08 glass fibre membrane is cut into 220mm × 100mm, 5min in sample pad treatment fluid is immersed in, then 40
DEG C air dry oven in dry 12h, take out up to sample pad, be put into spare in valve bag；
Sample pad treatment fluid is made of phosphate buffer, sodium chloride, bovine serum albumin(BSA), sucrose and Tween-20,
In, the concentration of phosphate buffer is 0.01mol/L, pH 7.2, sodium chloride concentration 300mM, the volume of bovine serum albumin(BSA)
The volume of final concentration of 10%, Tween-20 final concentration of 0.05%, the mass concentration of sucrose are 7%, in phosphate buffer
Na2HPO4·12H2The mass content of O is 0.3%, NaH2PO4·2H2The mass content of O is 0.06%；
(6) preparation of bonding pad
The Anti-HE4-Ab1 and Anti-CA125-Ab1 of step (2) nanometer selenium label are uniformly layered on GF-06 respectively
On glass fibre membrane, the glass fibre membrane is 2.8mm × 75mm, and the volumetric usage of selenium labeling antibody is 120 μ L, then at 40 DEG C
Air dry oven in dry 3h, be solidificated in the nanometer Se particle of labelled antibody on glass fibre membrane up to bonding pad, take out
It is put into valve bag, desiccant is added, is sealed；
(7) preparation of reacting pad
Nitrocellulose filter is cut into the specification of 25mm × 100mm, is attached in PVC board, 0.01mol/L is then used,
It is 2mg/mL that Anti-CA125-Ab2 is diluted to concentration by the PBS of pH7.2, as T1 detection line coating antigen；Anti-HE4-
It is 1mg/mL that Ab2, which is diluted to concentration, as T2 detection line coating antigen；Sheep anti-mouse igg is diluted to 2mg/mL, as nature controlling line C
Coating antigen；Film instrument is drawn with metal spraying, film is drawn with 1 μ L/cm, be placed in the dry 3h of 40 DEG C of air dry ovens up to reacting pad, taking-up is put into certainly
Envelope, it is spare；
(8) kit assembles
Bonding pad is attached to the front end of reacting pad, presses reacting pad 1mm, then sample pad is attached to the front end of bonding pad, is pushed down
The front end of 180mm × 100mm blotting paper is finally attached to the rear end of reacting pad by bonding pad 2mm, presses reacting pad 2mm, other end patch
In the end of PVC board, that is, the assembling of test strips is completed, assembled test strips are put into cutting machine, the test paper of 4mm wide is cut into
Item is put into getting stuck, and can obtain HE4 joint CA125 quick detection kit.
It is a further object to provide a kind of sample pad treatment fluid, the sample pad treatment fluid is by phosphate-buffered
Liquid, sucrose, 300mM sodium chloride, bovine serum albumin(BSA) and tween composition.
The detection method of kit of the present invention are as follows: by people's whole blood or serum sample and sample treatment solution (PBS+
Tween-20 it) is added drop-wise to each 1 drop of sample pad location, sample is by the nanometer selenium and antibody complex redissolution on bonding pad, by hair
Spy is chromatographed with towards water absorption pad direction, after chromatography is to detection line and nature controlling line, can according in sample whether containing purpose object and
It develops the color respectively.
The beneficial effects of the present invention are: 1. the preparation cost of nanometer selenium is low, it is economical and practical；2. HE4 combines CA125 double antibody
Screening rapid sensitive of the sandwich method to oophoroma, accuracy rate height；3. can be improved the sensitivity of testing result, enhance testing result
Specificity, the false positive occurred in detection process can be eliminated, testing result is more acurrate；4. the sample size for needing to detect is small,
Suitable for quickly detection by oophoroma people at highest risk screening and family's self-test and bed.
Detailed description of the invention
The structural schematic diagram of kit Fig. 1 of the invention；
The testing result process decision chart of kit Fig. 2 of the invention；
The sensitivity test testing result figure of the kit of Fig. 3 embodiment 1；
The specific test testing result figure of the kit of Fig. 4 embodiment 1；
The stability test testing result figure of the kit of Fig. 5 embodiment 1.
Fig. 6 kit of the present invention and conventional method reagent preparation box false positive contrast and experiment
Specific embodiment
Embodiment one quickly detects the preparation of the nanometer selenium kit of HE4 and CA125
Anti-HE4-Ab1, Anti-HE4-Ab2, Anti-CA125-Ab1 and Anti- that kit of the present invention uses
CA125-Ab2 is purchased from MEDIX company, and article No. is followed successively by 4501,4503,4601,4602.
Anti-HE4-Ab1, Anti-HE4-Ab2 are respectively the monoclonal antibody for being directed to HE4 difference epitope；
Anti-CA125-Ab1, Anti-CA125-Ab2 are respectively the monoclonal antibody for being directed to CA125 difference epitope.
(1) preparation of nanometer selenium
It takes 16mL ultrapure water to be placed in 50mL small beaker, the PEG of the 2mol/L of 1mL is added, 20min is stirred at room temperature, disperse
Uniformly, PEG solution is obtained；The SDS of the 0.1mol/L of 1mL is dissolved in PEG solution, 20min is stirred at room temperature, is uniformly dispersed, mould is obtained
Plate solution；The selenous acid of the 0.04mol/L of 1mL is dissolved in template solution, 20min is stirred at room temperature, is uniformly dispersed, selenous acid is obtained
Solution；The vitamin C of the 0.32mol/L of 1mL is dissolved in selenous acid template solution, 20min is stirred at room temperature, is uniformly dispersed, i.e.,
Obtain nanometer selenium；
(2) antibody marks
The nanometer selenium for taking 1mL step (1) to be prepared is added the solution of potassium carbonate that 10 μ L concentration are 1mol/L, adjusts body
The pH value of system is 7.5；The Anti-HE4-Ab1 of the 2mg/mL of 2 μ L is added, mixed at room temperature 30min fills Anti-HE4-Ab1
That divides is integrated on nanometer Se particle, completes the label of Anti-HE4-Ab1；The nanometer selenium for taking 1mL step (1) to be prepared, adds
Enter the solution of potassium carbonate that 12 μ L concentration are 1mol/L, the pH value of regulation system is 8.0；Add the Anti- of the 2mg/mL of 3 μ L
CA125-Ab1, mixed at room temperature 30min are integrated to Anti-CA125-Ab1 adequately on nanometer Se particle, complete Anti-
The label of CA125-Ab1；
(3) it closes
It is added in the Anti-HE4-Ab1 for Anti-CA125-Ab1 and the nanometer selenium label that nanometer selenium marks in step (2)
Bovine serum albumin solution, controls the quality final concentration of 1% of bovine serum albumin(BSA), and room temperature mixes 30min；
(4) it is resuspended
Nanometer selenium labelled antibody after closing is centrifuged 10min at 10000rpm, discards supernatant, the work of 120 μ L of precipitating
Make liquid resuspension；
The working solution is by phosphate buffer, sodium chloride, bovine serum albumin(BSA), sucrose, trehalose and Tween-20 group
At wherein phosphate buffering liquid concentration is 0.01mol/L, pH 7.2；Sodium chloride concentration is 150mM, protein amount
Final concentration of 2%, sucrose quality final concentration of 3%, final concentration of 3%, the Tween-20 volume of trehalose quality is final concentration of
0.1%；In phosphate buffer, Na2HPO4·12H2O mass content is 0.3%, NaH2PO4·2H2O mass content is
0.06%；
(5) preparation of sample pad
GF-08 glass fibre membrane is cut into 220mm × 100mm, 5min in sample pad treatment fluid is immersed in, then 40
DEG C air dry oven in dry 12h, take out up to sample pad, be put into spare in valve bag；
Sample pad treatment fluid is by phosphate buffer, sucrose, sodium chloride, bovine serum albumin(BSA), sucrose and Tween-20 group
At, wherein the concentration of phosphate buffer is 0.01mol/L, pH 7.2, sodium chloride concentration 300mM, bovine serum albumin(BSA)
Final concentration of 10%, the Tween-20 of volume volume final concentration of 0.05%, the mass concentration of sucrose is 7%, and phosphate is slow
Na in fliud flushing2HPO4·12H2The mass content of O is 0.3%, NaH2PO4·2H2The mass content of O is 0.06%；
(6) preparation of bonding pad
The Anti-HE4-Ab1 and Anti-CA125-Ab1 of step (2) nanometer selenium label are uniformly layered on GF-06 respectively
On glass fibre membrane, the glass fibre membrane is 2.8mm × 75mm, and the volumetric usage of selenium labeling antibody is 120 μ L, then at 40 DEG C
Air dry oven in dry 3h, be solidificated in the nanometer Se particle of labelled antibody on glass fibre membrane up to bonding pad, take out
It is put into valve bag, desiccant is added, is sealed；
(7) preparation of reacting pad
Nitrocellulose filter is cut into the specification of 25mm × 100mm, is attached in PVC board, 0.01mol/L is then used,
It is 2mg/mL that Anti-CA125-Ab2 is diluted to concentration by the PBS of pH7.2, as T1 detection line coating antigen；Anti-HE4-
It is 1mg/mL that Ab2, which is diluted to concentration, as T2 detection line coating antigen；Sheep anti-mouse igg is diluted to 2mg/mL, as nature controlling line C
Coating antigen；Film instrument is drawn with metal spraying, film is drawn with 1 μ L/cm, be placed in the dry 3h of 40 DEG C of air dry ovens up to reacting pad, taking-up is put into certainly
Envelope, it is spare；
(8) kit assembles
Bonding pad is attached to the front end of reacting pad, presses reacting pad 1mm, then sample pad is attached to the front end of bonding pad, is pushed down
The front end of 180mm × 100mm blotting paper is finally attached to the rear end of reacting pad by bonding pad 2mm, presses reacting pad 2mm, other end patch
In the end of PVC board, that is, the assembling of test strips is completed, assembled test strips are put into cutting machine, the test paper of 4mm wide is cut into
Item is put into getting stuck, and can obtain HE4 joint CA125 quick detection kit.
The testing result that embodiment two quickly detects the nanometer selenium kit of HE4 and CA125 determines
1. detection method:
People's whole blood or serum sample and sample treatment solution (PBS+Tween-20) are added drop-wise to the preparation of the embodiment of the present invention 1
Each 1 drop of the sample pad location of kit, sample is by the nanometer selenium and antibody complex redissolution on bonding pad, by capillarity court
Water absorption pad direction chromatography can develop the color respectively after chromatography arrives detection line and nature controlling line according to whether purpose object is contained in sample.
2. result judgement:
When nature controlling line C, detection line T1 and detection line T2 develop the color, then it is detected in sample more than containing sensitivity
HE4 and carbohydrate antigen CA125, as shown in 1 in Fig. 2；When nature controlling line C and detection line T1 develop the color, and detection line T2 does not develop the color, say
Containing CA125 more than sensitivity in bright test sample, but without or with sensitivity HE4 below, as shown in 2 in Fig. 2；When
Nature controlling line C and detection line T2 colour developing, and when detection line T1 does not develop the color, illustrate in test sample containing HE4 more than sensitivity, but
Without or with sensitivity CA125 below, as shown in 3 in Fig. 2；When nature controlling line C develops the color, detection line T1 and detection line T2 are not shown
When color, illustrate without or with sensitivity CA125 below and HE4 in test sample, as indicated with 4 in fig. 2；When nature controlling line C not
Colour developing, whether no matter detection line T1 and detection line T2 develop the color, detection effect is invalid, as shown in 5-8 in Fig. 2.
Three sensitivity technique of embodiment
The present embodiment detects the sensitivity of kit in embodiment 1.By the negative blood of HE4 and CA125 standard items
It is 100ng/mL+250U/mL, 10ng/mL+100U/mL, 2.5ng/mL+35U/mL, 0.25ng/mL+ that sorting, which is not diluted to concentration,
The standard solution of 3.5U/mL, feminine gender are the negative serum that HE4 and CA125 standard items are not added.The standard of 80 μ L various concentrations is molten
Liquid is separately added into kit, and negative findings are that T detection line does not develop the color, and C nature controlling line colour developing, positive findings are T detection line and C matter
Control line develops the color, and the minimum standard liquid concentration for detecting positive findings is the sensitivity of the kit, specific testing result such as Fig. 3
It is shown.
In Fig. 3,1 is negative serum testing result, is shown as negative, the concentration of 2-5 is respectively the detection of standard solution
As a result.As seen from the figure, with the sensitivity point of the HE4 joint CA125 nanometer selenium early detection kit of double antibody sandwich method preparation
Other HE4 is 2.5ng/mL (i.e. 100pmol/L), and CA125 35U/mL reaches China's Healthy female ovarian cancer diagnosis general reference
Value.
Example IV specific detection
The present embodiment detects the specificity of kit in embodiment 1.Prepare 250U/mL's respectively with negative serum
The HE4 standard solution of CA125 standard solution and 10ng/mL are used for the specific detection of kit.By the above-mentioned sample of 80 μ L
Solution is added dropwise in kit, and specific testing result is as shown in Figure 4.
In Fig. 4,1 is the testing result of negative serum, is shown as negative；2 be the testing result of CA125 standard solution,
It is shown as positive；3 be the testing result of HE4 standard solution, is shown as positive；4 be the inspection of CA125 and HE4 mixed standard solution
It surveys as a result, being shown as positive.Intersect instead the experimental results showed that Anti-CA125-Ab and Anti-HE4-Ab is not present between the two
It answers, the specificity of kit obtained by embodiment is good.
Five Detection of Stability of embodiment
The present embodiment detects the stability of kit in embodiment 1.It will be protected with a batch of kit at 40 DEG C
It deposits after two weeks, it is 100ng/mL+250U/mL, 10ng/mL that HE4 and CA125 standard items are diluted to concentration with negative serum respectively
+ 100U/mL, 2.5ng/mL+35U/mL, the standard solution of 0.25ng/mL+3.5U/mL, feminine gender are that HE4 and CA125 standard is not added
The negative serum of product, is detected, as a result as shown in Figure 5.
In Fig. 5,1 is the testing result of negative serum, is shown as negative, the concentration of 2-5 is respectively 100ng/mL+250U/
ML, 10ng/mL+100U/mL, 2.5ng/mL+35U/mL, the testing result of 0.25ng/mL+3.5U/mL standard solution.By scheming
5 it is found that the colour developing of the kit is normal, and sensitivity still is able to reach the standard of 2.5ng/mL+35U/mL, it was demonstrated that its stability
Well.
The kit of the present invention of embodiment six and conventional method reagent preparation box false positive contrast and experiment
Vacation of the present embodiment to being generated during kit of the present invention and conventional method reagent preparation box detection blood sample
Positive findings have carried out the kit of evaluation experimental the present embodiment evaluation and have been respectively kit disclosed in the present embodiment 1 and use normal
The nanometer selenium kit of the detection HE4 and CA125 of rule method preparation.
General reagent box and the difference of reagent box preparation method disclosed in embodiment 1 be, the formula of sample pad treatment fluid
Difference, other preparation steps are all the same:
Sample pad treatment fluid disclosed in the embodiment of the present invention 1 by phosphate buffer, bovine serum albumin(BSA), sucrose and
Tween-20 composition, wherein the concentration of phosphate buffer is 0.01mol/L, pH 7.2, sodium chloride concentration 300mM, ox
The volume final concentration of 0.05% of sero-abluminous final concentration of 10%, the Tween-20 of volume, the mass concentration of sucrose are
7%, Na in phosphate buffer2HPO4·12H2The mass content of O is 0.3%, NaH2PO4·2H2The mass content of O is
0.06%；
The formula for the sample pad treatment fluid that general reagent box uses are as follows: by phosphate buffer, bovine serum albumin(BSA), chlorination
Sodium, sucrose and Tween-20 composition, wherein the concentration of phosphate buffer is 0.01mol/L, pH 7.2, and sodium chloride concentration is
The volume final concentration of 0.05% of 150mM, final concentration of 10%, the Tween-20 of the volume of bovine serum albumin(BSA), the quality of sucrose
Concentration is 7%, Na in phosphate buffer2HPO4·12H2The mass content of O is 0.3%, NaH2PO4·2H2The mass content of O
It is 0.06%.
Firstly, the detection of two kinds of kits is used for negative serum respectively, secondly, being with the concentration that negative serum is prepared
Kit of the HE4 standard solution that the CA125 standard solution and concentration of 250U/mL is 10ng/mL for the embodiment of the present invention 1
Detection.The above-mentioned sample solution of 80 μ L is added dropwise in kit and is detected, specific testing result is as shown in Figure 6.
Fig. 6 the results show that 1 for general reagent box be directed to CA125 negative serum testing result, be shown as the vacation of CA125
Positive and HE4 is negative, illustrates to will appear the detection of normal blood using the kit of common sample pad treatment fluid processing
CA125 false positive results lead to the mistaken diagnosis of normal population；2 be the positive test symbol of general reagent box, it can be clearly seen that
T1, T2 detection line and the colour developing of C nature controlling line are good；3 be the testing result of the normal blood of the kit of the present embodiment 1, is shown as
The detection of CA125 and HE4 is negative, illustrates in kit preparation process, and kit sample pad passes through sample disclosed by the invention
After padding treatment fluid processing, the problem of CA125 that normal population blood occurs detects false positive can be obviously eliminated, is greatly reduced
Misdiagnosis rate；4 be the testing result of 1 kit of embodiment, and T1, T2 detection line and the colour developing of C nature controlling line are good, illustrate of the invention real
Sensitivity and the color developing effect for applying 1 kit of example are unaffected.
Conclusion: carbohydrate antigen (CA125) nanometer is combined with people's epididymal secretory protein 4 (HE4) of double antibody sandwich method preparation
The sensitivity of selenium early detection kit is 2.5ng/mL+35U/mL (i.e. 100pmol/L+35U/mL), is reached and better than China
Healthy women ovarian cancer diagnosis general reference value, plays fabulous warning function and specificity, stability are all preferable.Nanometer selenium mark
Remember that antibody usage amount is less, more saves the use of raw material, reduce costs；The sensitivity of nanometer selenium kit is better than nanometer
Gold.
And people's epididymal secretory protein 4 (HE4) combination of sugar prepared using preparation method of the invention
Chain antigen (CA125) nanometer selenium early detection kit detection false positive rate is low, reduces commonsense method preparation
The misdiagnosis rate of kit.
The kit high specificity, high sensitivity, false positive rate are low, detection speed is fast, easy to operate, carrying is easy, nothing
Need special installation, it is low in cost, clearly easily distinguished without professional training, result, be suitable for detection and family's self-test by bed.
Claims (10)
1. a kind of nanometer selenium kit of quickly detection HE4 and CA125, the kit by sample pad (1), bonding pad (3),
Reacting pad (2), water absorption pad (4) and PVC bottom plate (5) composition, the sample pad (1), bonding pad (3), reacting pad (2) and water absorption pad
(4) it is successively overlapped on PVC bottom plate (5), it is characterised in that:
The detection for being provided on the reacting pad (2) and being coated with the detection line T1 of anti-CA 125 antibody, be coated with anti-HE4 antibody
Line T2, the nature controlling line C for being coated with sheep anti-mouse igg.
2. the nanometer selenium kit of quickly detection HE4 and CA125 as described in claim 1 a kind of, it is characterised in that described
Sample pad (1) is handled through sample pad treatment fluid, and sample pad treatment fluid is by phosphate buffer, sucrose, neutral salt, bovine serum albumin
White and tween composition.
3. the nanometer selenium kit of quickly detection HE4 and CA125 as claimed in claim 2 a kind of, it is characterised in that described
Neutral salt is selected from one of sodium chloride, potassium chloride, calcium chloride, barium chloride, calcium sulfate, potassium nitrate.
4. the nanometer selenium kit of quickly detection HE4 and CA125 as claimed in claim 3 a kind of, which is characterized in that the sample
It is sodium chloride or potassium chloride, concentration 300mM that product, which pad neutral salt contained by treatment fluid,.
5. nanometer selenium kit as claimed in claim 4, which is characterized in that the neutral salt in the sample pad treatment fluid is
Sodium chloride, concentration 300mM.
6. the nanometer selenium kit of quickly detection HE4 and CA125 as described in claim 1 a kind of, it is characterised in that described
The compound of nanometer selenium and anti-HE4 antibody and anti-CA 125 antibody coupling suspends solidification on bonding pad (3) through working solution, described
Working solution is made of phosphate buffer, neutral salt, bovine serum albumin(BSA), carbohydrate and tween, the neutral salt be selected from sodium chloride,
One of potassium chloride, calcium chloride, barium chloride, calcium sulfate, potassium nitrate；The carbohydrate in monosaccharide and disaccharide or polysaccharide one
Kind.
7. nanometer selenium kit as claimed in claim 6, which is characterized in that the carbohydrate in the working solution is sucrose and seaweed
The concentration of the phosphate buffer of sugar, the treatment fluid and working solution is 0.01mol/L, pH 7.2.
8. the nanometer selenium kit of quickly detection HE4 and CA125 as claimed in claim 6 a kind of, which is characterized in that in described
Property salinity be 150-500mM.
9. a kind of preparation method of the nanometer selenium kit of quickly detection HE4 and CA125, comprising the following steps:
(1) preparation of nanometer selenium
Ultrapure water is taken, PEG is added, stirring obtains PEG solution；SDS is dissolved in PEG solution, stirs, obtains template solution；By sub- selenium
Acid is dissolved in template solution, and stirring obtains selenous acid solution；Vitamin C is dissolved in selenous acid template solution, is stirred to get receiving
Rice selenium；
(2) antibody marks
The solution of potassium carbonate that 10 μ L concentration are 1mol/L is added in the nanometer selenium for taking 1mL step (1) to be prepared, regulation system
PH value is 7.5；The Anti-HE4-Ab1 of the 2mg/mL of 2 μ L is added, mixed at room temperature 30min keeps Anti-HE4-Ab1 sufficient
It is integrated on nanometer Se particle, completes the label of Anti-HE4-Ab1；
The solution of potassium carbonate that 12 μ L concentration are 1mol/L is added in the nanometer selenium for taking 1mL step (1) to be prepared, regulation system
PH value is 8.0；The Anti-CA125-Ab1 of the 2mg/mL of 3 μ L is added, mixed at room temperature 30min fills Anti-CA125-Ab1
That divides is integrated on nanometer Se particle, completes the label of Anti-CA125-Ab1；
(3) it closes
Ox blood is added in the Anti-HE4-Ab1 for Anti-CA125-Ab1 and the nanometer selenium label that nanometer selenium marks in step (2)
Pure protein solution, controls the quality final concentration of 1% of bovine serum albumin(BSA), and room temperature mixes 30min；
(4) it is resuspended
Nanometer selenium labelled antibody after closing is centrifuged 10min at 10000rpm, discards supernatant, the working solution of 120 μ L of precipitating
It is resuspended；
The working solution is made of phosphate buffer, sodium chloride, bovine serum albumin(BSA), sucrose, trehalose and Tween-20,
Middle phosphate buffering liquid concentration is 0.01mol/L, pH 7.2；Sodium chloride concentration is 150mM, and protein amount is dense eventually
Degree is 2%, sucrose quality final concentration of 3%, final concentration of 3%, the Tween-20 volume final concentration of 0.1% of trehalose quality；
In phosphate buffer, Na2HPO4·12H2O mass content is 0.3%, NaH2PO4·2H2O mass content is 0.06%；
(5) preparation of sample pad
GF-08 glass fibre membrane is cut into 220mm × 100mm, is immersed in 5min in sample pad treatment fluid, then at 40 DEG C
Dry 12h, takes out up to sample pad, is put into spare in valve bag in air dry oven；
Sample pad treatment fluid is made of phosphate buffer, sodium chloride, bovine serum albumin(BSA), sucrose and Tween-20, wherein phosphorus
The concentration of phthalate buffer is 0.01mol/L, and the volume of pH 7.2, sodium chloride concentration 300mM, bovine serum albumin(BSA) are dense eventually
Degree is the volume final concentration of 0.05% of 10%, Tween-20, and the mass concentration of sucrose is 7%, in phosphate buffer
Na2HPO4·12H2The mass content of O is 0.3%, NaH2PO4·2H2The mass content of O is 0.06%；
(6) preparation of bonding pad
The Anti-HE4-Ab1 and Anti-CA125-Ab1 of step (2) nanometer selenium label are uniformly layered on GF-06 glass respectively
It is dry on tunica fibrosa, it is solidificated in the nanometer Se particle of labelled antibody on glass fibre membrane up to bonding pad, taking-up is put into self-styled
Bag is added desiccant, is sealed；
(7) preparation of reacting pad
Nitrocellulose filter is attached in PVC board, then uses 0.01mol/L, the PBS of pH7.2 that Anti-CA125-Ab2 is diluted
It is 2mg/mL to concentration, as T1 detection line coating antigen；It is 1mg/mL that Anti-HE4-Ab2, which is diluted to concentration, is detected as T2
Line coating antigen；Sheep anti-mouse igg is diluted to 2mg/mL, as nature controlling line C coating antigen；Film instrument is drawn with metal spraying, and film is drawn with 1 μ L/cm,
40 DEG C of forced air drying 3h are placed in up to reacting pad, taking-up is put into valve bag, spare；
(8) kit assembles
Bonding pad is attached to the front end of reacting pad, then sample pad is attached to the front end of bonding pad, finally pastes the front end of blotting paper
In the rear end of reacting pad, the other end is attached to the end of PVC board, that is, completes the assembling of test strips, and it is quick can to obtain HE4 joint CA125
Detection kit.
10. a kind of sample pad treatment fluid, which is characterized in that the sample pad treatment fluid is by phosphate buffer, sucrose, 300mM
Sodium chloride, bovine serum albumin(BSA) and tween composition.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910003827.0A CN109444435A (en) | 2019-01-03 | 2019-01-03 | A kind of nanometer selenium kit of quick detection HE4 and CA125 |
| CN201910675929.7A CN110221084B (en) | 2019-01-03 | 2019-07-25 | Nano-selenium kit for rapidly detecting HE4 and CA125 |
| CN201910675948.XA CN110275028B (en) | 2019-01-03 | 2019-07-25 | Colloidal gold-labeled anti-HE4 antibody kit and preparation method thereof |
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| CN201910003827.0A CN109444435A (en) | 2019-01-03 | 2019-01-03 | A kind of nanometer selenium kit of quick detection HE4 and CA125 |
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| CN201910675929.7A Active CN110221084B (en) | 2019-01-03 | 2019-07-25 | Nano-selenium kit for rapidly detecting HE4 and CA125 |
| CN201910675948.XA Active CN110275028B (en) | 2019-01-03 | 2019-07-25 | Colloidal gold-labeled anti-HE4 antibody kit and preparation method thereof |
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| CN201910675948.XA Active CN110275028B (en) | 2019-01-03 | 2019-07-25 | Colloidal gold-labeled anti-HE4 antibody kit and preparation method thereof |
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| WO2016161126A1 (en) * | 2015-04-02 | 2016-10-06 | Provista Diagnostics, Inc. | Biomarkers for detection of ovarian cancer |
| CN204719056U (en) * | 2015-05-15 | 2015-10-21 | 德康润生物科技（天津）有限公司 | A kind of fast joint detects the kit of HE4 and CA125 |
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| CN110221084B (en) | 2022-07-26 |
| CN110275028B (en) | 2022-07-26 |
| CN110275028A (en) | 2019-09-24 |
| CN110221084A (en) | 2019-09-10 |
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