CN105785019B - A kind of detection method for prostate specific antigen - Google Patents
A kind of detection method for prostate specific antigen Download PDFInfo
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- CN105785019B CN105785019B CN201610156409.1A CN201610156409A CN105785019B CN 105785019 B CN105785019 B CN 105785019B CN 201610156409 A CN201610156409 A CN 201610156409A CN 105785019 B CN105785019 B CN 105785019B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57434—Specifically defined cancers of prostate
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
Abstract
The invention provides a kind of detection method for prostate specific antigen, this method is first combined prostate specific antigen with coated polyclonal antibody, then biotinylated monoclonal antibody is connected, reconnect the catalase C100 of marked by streptavidin, decomposing hydrogen dioxide solution is catalyzed by catalase, the fluorescent quenching of the cadmium telluride quantum dot to mercaptopropionic acid modification is reduced, according to the height of fluorescence intensity come the concentration of prostate specific antigen in judgement sample.This method is based on double antibodies sandwich Enzyme-multiplied immune technique, and employ biotin avidin system be used for react amplification.What is more important, due to present invention employs new antibody labeling enzyme (catalase C100) and more sensitive fluorogenic substrate (cadmium telluride quantum dot), and it have matched effective reaction condition, so that detection sensitivity is significantly improved, cost, lifting detection efficiency are reduced simultaneously, therefore there is good promotion prospect.
Description
Technical field
The present invention relates to Antigen Detection Techniques field, further to the Antigen Detection Techniques based on ELISA, and in particular to
A kind of detection method for prostate specific antigen.
Background technology
In recent years, with the improvement of people ' s living standards, the aggravation of the change of dietary structure and environmental pollution etc., forefront
The incidence of disease of gland cancer is in ascendant trend year by year.Prostate cancer exceed cutaneum carcinoma turn into the most common cancer of North America male, because
Position is ranked second in cancer lethal factor.Prostate specific antigen (prostate specific antigen, PSA) is from prostate
A kind of specificity glycoprotein for separating, purifying in tissue, has serine protease.PSA is generally acknowledged at present most valuable
The prostate cancer marker of value, it is the indispensable head of diagnosis of prostate cancer that specificity is high in clinical practice, sensitiveness is strong
TM detection methods are selected, the diagnosis, observation of curative effect and Prognosis scoveillance tool to prostate cancer are of great significance and acted on.Serum
Prostate specific antigen (PSA) normal value is general<4ng/mL, the PSA when prostate cancer occurs>10ng/mL, there is adjuvant clinical
The notable meaning of diagnosis.
Early detection PSA method mainly has Immune proliferation, immunoelectrophoresis, rocket electrophoresis etc., but because sensitivity is low, operation
Complexity etc. has all been eliminated.In recent years, the method for detection PSA a series of is established, and wherein polyclonal antibody radioimmunology is deposited
Sensitivity is low, poor repeatability, the range of linearity are narrow, time-consuming effort the shortcomings of, it is not easy to apply；Enzyme linked immunosorbent assay is by matrix
Specific effect, there is false negative or false positive in measurement result, and sensitivity is low；Automatic particulate ELISA measure
There is special instrument to complete, cumbersome, instrument is valuable, and the application of the detection method is still restricted；Chemistry hair
Light method depends on high-purity reagent and high precision instrument, and sample pretreatment process is cumbersome, therefore is also unsuitable for highly sensitive fast extensively
Speed detection.Disturbing factor has a great influence when PCR method determines, and methodology existing defects itself, measurement result is credible
Degree is inadequate, sensitivity deficiency.However, enzyme linked immunosorbent assay (ELISA) method because with it is quick, sensitive, special, accurate, can
It is quantitative, easy to operate, without valuable instrument and equipment, and, inspection especially suitable for batch samples less demanding to sample purity
The advantages that survey, it has also become the main method of PSA rapid screenings detection.It is universal for PSA ELISA detection method in the prior art
Antibody or antigen catalysis hydrogen peroxide generation hydroxy radical based on horseradish peroxidase-labeled, and then aoxidize colourless chemistry and show
Color substrate tetramethyl biphenyl diamines (TMB) forms blue product, then using terminate liquid (2M H2SO4) terminating reaction forms Huang
Color solution records absorbance at 450nm.Such method is because its colored intensity is relatively low, therefore detection sensitivity is relatively low,
Easily there is false negative result when object content is relatively low in testing sample, so as to which the requirement of practical application can not be met.
Therefore, in recent years, some novel signal transmission mechanisms substitute traditional ELISA signal transduction mechanisms and are used to improve tradition
For the method for ELISA sensitivity by wide coverage, such as resonate ELISA, chemiluminescence enzyme linked immunosorbent assay, fluorescent dye are enzyme-linked
Immunization, Fluorescence Increasing ELISA and fluorescent quenching ELISA etc..Wherein, based on the ELISA of fluorogenic substrate because
It is of great interest with higher sensitivity.It is reported that substituting traditional chromogenic substrate with fluorogenic substrate can incite somebody to action
Detection sensitivity improves at least 1-2 order of magnitude, but the validity of luminescence system is not only determined by substrate, also with detection pair
As, label enzyme, characterization processes etc. have substantial connection, therefore introduce new luminous embodiment in ELISA method and need to combine
Concrete condition carries out brand-new design.
The content of the invention
A kind of it is contemplated that technological deficiency for prior art, there is provided detection side for prostate specific antigen
Method, it is relatively low to solve the prostate specific antigen ELISA detection method precision of prior art.
Another technical problem to be solved by the present invention is that prior art is used for the ELISA method of prostate specific antigen detection
In, the enzyme sensitivity of labelled antibody is relatively low.
The invention solves another technical problem be prior art be used for prostate specific antigen detection ELISA method
In, Color Appearance System sensitivity is relatively low.
The invention solves another technical problem be when using catalase C100 as antibody labeling enzyme to prostate-specific
When antigen performs ELISA detections, concrete technology method is simultaneously indefinite.
The invention solves another technical problem be when using catalase C100 as antibody labeling enzyme, using hydrogen peroxide and
Mercaptopropionic acid modification cadmium telluride quantum dot as substrate to prostate specific antigen perform ELISA detection when, testing result and
The linear relationship of antigen concentration is bad.
The invention solves another technical problem be when using catalase C100 as antibody labeling enzyme, using hydrogen peroxide and
Mercaptopropionic acid modification cadmium telluride quantum dot as substrate to prostate specific antigen perform ELISA detect when, during wash
It is ineffective.
To realize above technical purpose, the present invention uses following technical scheme：
A kind of detection method for prostate specific antigen, this method belong to double antibodies sandwich ELISA, this method
It is the detection for prostate specific antigen, the enzyme that labelled antibody is used in this method is catalase C100.
Preferably, substrate includes the cadmium telluride quantum dot that hydrogen peroxide and mercaptopropionic acid are modified in this method.Wherein dioxygen
Water is as cadmium telluride quantum dot fluorescence quenching, and in course of reaction, hydrogen peroxide decomposes under catalase effect, is quenched so as to reduce fluorescence
The effect of going out, realize luminous.
Preferably, this method comprises the following steps：
1) it is coated with prostate specific antigen antibody；
2) take step 1) be coated with after antibody, mix with testing sample after in 35~39 DEG C of light protected environments react 40~
80min, washing；
3) biotinylated prostate specific antigen monoclonal antibody mixing is then added, in 35~39 DEG C of light protected environments
React 40~80min, washing；
4) then add marked by streptavidin catalase C100 mixing, in 35~39 DEG C of light protected environments react 40~
80min, washing；
5) the hydrogen peroxide solution mixing that concentration is 8~12 μm of ol/L is then added, is reacted in 35~39 DEG C of light protected environments
20~40min；
6) then add mercaptopropionic acid modification cadmium telluride quantum dot mixing, in room temperature light protected environment react 10~
20min；
7) detecting step 6) product fluorescence intensity.
The fluorescence intensity is used to react prostate specific antigen content in testing sample, using known in practical operation
Multigroup prostate specific antigen titer of concentration and distribution gradient draws fluorescence intensity-antigen scale standard by above method
Curve, the fluorescence intensity of testing sample is recycled to calculate the prostate specific antigen content of testing sample from standard curve.Tool
Body operating method can be according to any selection of general technology general knowledge of the art.Multigroup prostate of above-mentioned gradient distribution is special
Hapten titer, 10 can be selected respectively-14g/mL 10-13g/mL、10-12g/mL、10-11g/mL、10-10g/mL、10-9g/
mL、10-8g/mL。
In the optimal technical scheme, step 2) is used for the antigen antibody complex for obtaining solid phase；Step 3) realizes biotin
Connection between the prostate specific antigen monoclonal antibody of change and the antigen antibody complex of solid phase；Step 4) utilizes biology
Element-Streptavidin system realizes the connection with catalase C100；Step 5) enzymatic decomposing hydrogen dioxide solution；Step 6) realizes that colour developing is anti-
Should.In the optimal technical scheme：Each reagent can first can reuse before prior to more than equilibrium at room temperature 30min；Step
It is rapid 2) in the addition of testing sample be preferably 80~120 μ L/ holes, more optimizedly 100 μ L/ holes；It is biotinylated in step 3)
The addition of prostate specific antigen monoclonal antibody is preferably 80~120 μ L/ holes, more optimizedly 100 μ L/ holes；In step 4)
The catalase C100 of marked by streptavidin addition is preferably 80~120 μ L/ holes, more optimizedly 100 μ L/ holes；In step 5)
The addition of hydrogen peroxide solution is preferably 80~120 μ L/ holes, more optimizedly 100 μ L/ holes；Mercaptopropionic acid is modified in step 6)
The addition of cadmium telluride quantum dot is preferably 30~70 μ L/ holes, more optimizedly 50 μ L/ holes.
Preferably, step 1) specifically includes following operation：
A coating buffer, dilution) are used as using the carbonate buffer solution of 0.04~0.06mol/L, pH9.4~9.8 on ELISA Plate
Prostate specific antigen polyclonal antibody is to 9~11 μ g/mL；
B cleaning solution washing ELISA Plate is utilized after) removing the liquid on ELISA Plate, adds bovine serum albumin(BSA) confining liquid,
1~3h is closed in 35~39 DEG C, then discards confining liquid.
It can further perform following preferred：The concentration of the bovine serum albumin(BSA) is 0.3~0.7%, more optimizedly
0.5%；The addition of confining liquid is 320~360 μ L/ holes, and more excellent is 340 μ L/ holes；Discard the ELISA Plate after confining liquid in
Dry at room temperature, and after 2~6 DEG C of preservations.
Preferably, step A) in the addition of coating buffer be 80~120 μ g/ holes, 8~12h is stood after dilution and is performed again
Step B).
Preferably, step 3) the biotinylated prostate specific antigen monoclonal antibody is to make by the following method
Standby：It is molten to prepare the PBS containing 1~3mg/mL prostate specific antigens monoclonal antibody, 0.07~0.08mg/mL biotins
Liquid, 30~60min is reacted under the conditions of lucifuge, the concentration of the PBS solution is 0.005~0.02mol/L, and then dialysis removes
Biotin, that is, obtain the biotinylated prostate specific antigen monoclonal antibody.It can further perform following preferred：Institute
It is active esterification biotin to state biotin；The time of the dialysis is 66~78h, more optimizedly 72h.
Preferably, the catalase C100 of the step 4) marked by streptavidin is prepared by the following method：Prepare
PBS solution containing 0.5~2mg/mL Streptavidins, the concentration of the PBS solution is 0.005~0.02mol/L, is then added
Enter SM (PEG)24React 30~60min, then gel column purify, collect filtrate add thereto catalase C100 to final concentration 1~
3mg/mL, that is, obtain the catalase C100 of the marked by streptavidin.In the optimal technical scheme：Gel column purifying link is used
In the unnecessary crosslinking agent of removal；Wherein protein content can first be detected and mix the solution containing albumen by collecting obtained filtrate
Catalase C100 is added after conjunction.
Preferably, the cadmium telluride quantum dot of step 6) the mercaptopropionic acid modification is prepared by the following method：Match somebody with somebody
It is precursor to make the solution received containing 8~12mmol/L cadmium nitrates, 20~28mmol/L mercaptopropionic acids, 3~7mmol/L hydrogen tellurides
Solution, the pH of the precursor solution is 11~11.5, by the precursor solution heating water bath to 93~97 DEG C, that is, obtains the mercapto
The cadmium telluride quantum dot of base propionic acid modification.
Preferably, the detection of fluorescence intensity is realized using ELIASA in step 7), and excitation wavelength 310nm, hair
The a length of 590nm of ejected wave.
Preferable on the basis of any of the above technical scheme, the washing is rinsed or soaked using cleaning solution, described to wash
It is the PBST solution containing 0.3~0.7% (v/v) Tween-20 to wash liquid, the concentration of the PBST solution for 0.005~
0.02mol/L, the pH of the PBST solution is 7.0~7.5.
In above technical scheme, catalase (Catalase) is also known as catalase, the catalase used in the present invention
C100 refers exclusively to catalase produced by Sigma-Aldrich, model cat.No.C100.It is described biotinylated
Prostate specific antigen monoclonal antibody, refer to after biotin is covalently attached into prostate specific antigen monoclonal antibody molecule
Obtained compound.The catalase C100 of the marked by streptavidin, refer to carry out Streptavidin and catalase C100 covalently
With reference to the compound of gained.
In above technical scheme, ELISA Plate can select 96 hole black fluorescent ELISA Plates.96 hole black fluorescent ELISA Plates, it is raw
Thing element, anti-prostate-specific-antigen monoclonal antibody, anti-prostate-specific-antigen polyclonal antibody, Streptavidin, catalase
C100 can be bought in market.
Preferably, the washing is that the cleaning solution in 320~360 μ L/ holes is added into enzyme mark hole, washing 3~4 times, often
10~30s of minor tick.
Preferably, the testing sample is serum：(1) the prostate specific antigen standard items bought are used into whole blood respectively
Thin to release, concentration determines according to required actually detected limit；(2) the prostate specific antigen whole serum sample diluted is put into 4
DEG C refrigerator is standby.
The present invention is detected using enzyme-linked immunologic adsorption test method.For detecting the novel fluorescence of prostate specific antigen
The principle of ELISA detection method is：Prostate specific antigen is combined with coated polyclonal antibody, then plus biotinylation
Monoclonal antibody, along with SA CAT (the catalase C100 of marked by streptavidin, similarly hereinafter), pass through catalase and be catalyzed dioxygen moisture
Solution, the fluorescent quenching of the cadmium telluride quantum dot to mercaptopropionic acid modification is reduced, according to the height of fluorescence intensity come in judgement sample
The concentration of prostate specific antigen.If the prostate specific antigen concentration in sample is high, fluorescence intensity is high；Conversely, then fluorescence
Low intensity.That is the concentration of the height of fluorescence intensity and the bacterium in sample is into positive correlation.This method can be directly used for detecting serum
Prostate specific antigen.The detection method of the present invention is easy to operate, and detection is sensitive, accurate, quick, suitable for batch samples
Detection.
Had the advantages that using technical solution of the present invention：
1st, the inventive method uses the horseradish peroxidase in more sensitive new catalase substitution conventional ELISA method
Enzyme, cost can be greatlyd save.
2nd, the inventive method uses more sensitive new fluorogenic substrate (cadmium telluride quantum dot that mercaptopropionic acid is modified)
Substitute the chemical colour reaction substrate in conventional ELISA method, its detection sensitivity can be greatly improved, relative to traditional ELISA extremely
The 2-3 orders of magnitude are improved less.
Brief description of the drawings
Fig. 1 is detection method and the conventional detection method using horseradish peroxidase as antibody labeling thing enzyme
Principle comparison diagram.
Fig. 2 is percentage in the embodiment of the present invention 1-prostate specific antigen concentration standard curve figure.
Fig. 3 is percentage in the embodiment of the present invention 2-prostate specific antigen concentration standard curve figure.
Embodiment
The embodiment of the present invention will be described in detail below.In order to avoid excessive unnecessary details,
It is will not be described in detail in following examples to belonging to known structure or function.
Approximating language used in following examples can be used for quantitative expression, show do not changing the feelings of basic function
Under condition quantity can be allowed to have certain variation.Therefore, it is accurate that the numerical value corrected with the language such as " about ", " left and right " is not limited to this
Numerical value is in itself.In certain embodiments, the numerical value for " about " representing to allow its amendment is in the scope of positive and negative 10 (10%)
Interior change, such as, " about 100 " represent can be any numerical value between 90 to 110.In addition, " the about first numerical value arrives
In the statement of second value ", the first and second numerical value two values are at about corrected.In some cases, approximating language
May be relevant with the precision of measuring instrument.
In addition to being defined, technology used and scientific terminology have and art technology people of the present invention in following examples
The identical meanings that member is commonly understood by.
Test reagent consumptive material used, is routine biochemistry reagent unless otherwise specified in following examples；The experiment
Method, it is conventional method unless otherwise specified；Quantitative test in following examples, it is respectively provided with and repeats to test three times, as a result
Average；% in following examples, it is weight/mass percentage composition unless otherwise instructed.
In following examples, the prostate specific antigen monoclonal antibody and polyclonal antibody of Anti-Biotin are bought in nothing
The Sino-German Bai Er Bioisystech Co., Ltd of tin；Described biotin (Cat.No.B5161), Streptavidin
(Cat.No.S4762), catalase (cat.No.C100) and substrate solution A hydrogen peroxide (35%, cat.No.349887) are bought in U.S.
Sigma-Aldrich companies of state.Described catalase C100 refers to catalase (cat.No.C100).
(the novel fluorescence ELISA detection method of present invention detection prostate specific antigen is before detecting in serum for embodiment 1
Application in row gland specific antigen content)
When novel fluorescence ELISA detection method of the present invention is used to detect the prostate specific antigen content in serum, pass through
Following steps are implemented：Sample pre-treatments, detected with detection method, analysis result.
(1) sample pre-treatments
(1) the prostate specific antigen standard items bought are diluted with whole serum respectively, concentration is according to required actual inspection
Limit is surveyed to determine；(2) it is standby that the prostate specific antigen whole serum sample diluted is put into 4 DEG C of refrigerators.(2) with detection side of the invention
Method detect prostate specific antigen content in above-mentioned sample
The ELISA Plate for being coated with anti-prostate-specific-antigen content polyclonal antibody is taken, adds the μ L/ holes of standard items/sample 100
Into corresponding micropore；Take the ELISA Plate for being coated with anti-prostate-specific-antigen polyclonal antibody, this 100 μ L/ hole of sample-adding to pair
In the micropore answered, gently vibration mixes, and reacts 60min with being placed after cover plate membrane cover plate in 37 DEG C of light protected environments；Carefully open lid
Plate film, liquid in hole is dried, with the μ L/ holes of wash operating solution 340, fully washing 4 times, per minor tick 10s, patted dry with blotting paper
(bubble not being eliminated after patting dry can be poked with original pipette tips), adds biotinylation prostate specific antigen Dan Ke
The grand μ L/ holes of antibody 100, gently vibration mix, and react 60min with being placed after cover plate membrane cover plate in 37 DEG C of light protected environments；Carefully take off
Uncap plate film, liquid in hole is dried, with the μ L/ holes of wash operating solution 340, fully washing 3 times, per minor tick 10s, use blotting paper
Pat dry (bubble not being eliminated after patting dry can be poked with original pipette tips), add SA CAT, gently vibration mixes, with lid
Placed after plate membrane cover plate in 37 DEG C of light protected environments and react 60min；Cover plate film carefully is opened, liquid in hole is dried, uses washers
Make the μ L/ holes of liquid 340, fully washing 3 times, per minor tick 10s, pat dry that (bubble not being eliminated after patting dry is available not with blotting paper
Used pipette tips are poked).Substrate solution A hydrogen peroxide (10 μm of ol/L) dilution, 100 μ L/ holes are added, gently vibration is mixed, used
30min is reacted in the rearmounted 37 DEG C of light protected environments of cover plate membrane cover plate；Add the cadmium telluride quantum of fluorogenic substrate liquid B mercaptopropionic acids modification
Point substrate solution 50 μ L/ holes, gently vibration mix, and with 15min is reacted in the rearmounted 37 DEG C of light protected environments of cover plate membrane cover plate, set fluorescence
ELIASA (U.S. Thermo Varioskan Flash all-wave lengths multi-function microplate reader) is 310nm in excitation wavelength, transmitted wave
Detect, determined per hole fluorescent value (data are please run through in 5min) at a length of 590nm；With the fluorescent value and standard of standard items test
The log concentration value of product draws standard curve, and reference standard curve calculates the content of prostate specific antigen in sample.
The preparation of the coated 96 hole black fluorescent ELISA Plate of anti-prostate-specific-antigen polyclonal antibody is to use
0.05mol/L pH 9.6 carbonate (CBS) buffer solution is as coating buffer, by (the purchase of prostate specific antigen polyclonal antibody
In Wuxi Zhongde Bore Bioisystech Co., Ltd) 10 μ g/mL are interpreted into, 100 μ L/ holes, 4 DEG C stand overnight, and take out ELISA Plate and get rid of
Liquid in board falling, with the μ L/ holes of concentrated cleaning solution 340 after dilution, board-washing is secondary, 30s/ times；Then 0.5% bovine serum albumin is added
(BSA, buying in Sigma-Aldrich, cat.No.A4737) is closed in vain, 340 μ L/ holes, 37 DEG C of placement 2h, discards
Confining liquid, (25 DEG C) dry between the ELISA Plate after patting dry places constant temperature；Inspect by random samples it is qualified after by rearmounted 4 DEG C of ELISA Plate vacuum sealing
Preserve.Described prostate specific antigen debita spissitudo gradient is respectively 10-14g/mL 10-13g/mL、10-12g/mL、10-11g/
mL、10-10g/mL、10-9g/mL、10-8g/mL。
The biotinylated prostate specific antigen monoclonal antibody obtains in the following manner：1mg prostates is special
Hapten monoclonal antibody is diluted with PBS (pH 8.6,0.01mol/L), is added 50 μ L and is vivaciously esterified biotin (0.76mg/mL)
Make the final concentration of 2mg/mL of antibody, room temperature lucifuge reaction 45min.Reaction product is dialysed in 0.01mol/L PBS solution
72h, remove free biotin；After dialysis terminates, sample is freeze-dried to obtain biotinylated prostate specific antigen list
Clonal antibody, packing, -20 DEG C of preservations.
The SA@CAT are obtained in the following manner：SA is dissolved as 1mg/mL with PBS (pH 8.6,0.01mol/L), then
Add 10 μ L SM (PEG)24(Thermo：22104,82.8mg/mL) 45min is reacted, reaction uses gel column (Thermo after terminating：
43230) isolate and purify, unnecessary crosslinking agent of going out；The liquid of collection is determined with Nanodrop, will have being mixed with solution for albumen
Close, be then added in 5mg catalase solution (final concentration 2mg/mL).The sample of acquisition is freeze-dried, packing, -20 DEG C of preservations.
The preparation of the biotinylation prostate specific antigen monoclonal antibody working solution：Using active esterification biotin with
Prostate specific antigen monoclonal antibody is coupled what is obtained, and 1 is diluted to PBS (0.01M, pH7.4):230.
The preparation of the SA@CAT working solutions：Be coupled what is obtained using Streptavidin and catalase, with PBS (0.01M,
PH7.4) it is diluted to 1:300.
The preparation of the substrate solution A hydrogen peroxide：10 μm of ol/L are diluted to PBS (0.01mol/L, pH7.4).The sulfydryl
The preparation of the cadmium telluride quantum dot fluorogenic substrate liquid of propionic acid modification：Method, 1 is diluted to PBS (0.01mol/L, pH7.4):
400。
The concentrated cleaning solution is 10 times of concentrated cleaning solutions, and it includes 0.5% Tween-20,0.01mol/L PBST, pH
It is worth between scope 7.0-7.5.
(3) analysis result
With the bacterium solution 10 of 6 prostate specific antigen various concentrations of above-mentioned preparation-13g/mL、10-12g/mL、10-11g/
mL、10-10g/mL、10-9g/mL、10-8g/mL.It is 310nm in excitation wavelength, launch wavelength is fluorescence intensity at 590nm.
It is quenched the calculating of percentage fluorescence rate, percentage fluorescence rate equal to first standard (0 mark is quenched in standard items or sample
It is accurate) fluorescence intensity level subtract standard items or sample fluorescence intensity level average value (diplopore), then except in first standard (0
Standard), i.e. percentage fluorescence rate (%)=(F0-F)/F0× 100%, wherein F0For the fluorescence intensity of first standard (0 standard)
Value, F are the average value (diplopore) of the fluorescence intensity level of standard items or sample.
So that percentage fluorescence rate is quenched as ordinate, it is bent that standard is drawn with prostate specific antigen concentration (g/mL) abscissa
Line, obtain linear equation.Standard curve is y=8.8983Log (x)+19.601, R2=0.9971, see accompanying drawing 2.This method
Minimum detection limit is defined as 10-14G/mL fluorescent value adds three standard deviations.Lowest detection is calculated to obtain by the standard curve
Line is 10-13g/mL.When carrying out actual sample detection, by the fluorescent value ((F of sample0-F)/F0× 100%) it is bent to substitute into standard for value
In line, the concentration of sample corresponding to reading from standard curve, it is prostate spy in sample to be multiplied by its corresponding extension rate
The actual concentrations of hapten.
Embodiment 2 (is resisted using prostate-specific of the horseradish peroxidase as antibody labeling enzyme, using TMB as chromogenic substrate
Former ELISA detection method)
When traditional ELISA detection method is used to detect prostate specific antigen content in serum, implemented by following steps：
Sample pre-treatments, detected with traditional ELISA detection method, analysis result.
(1) sample pre-treatments
(1) the prostate specific antigen standard items bought are diluted with whole serum respectively, concentration is according to required actual inspection
Limit is surveyed to determine；(2) it is standby that the prostate specific antigen whole serum sample diluted is put into 4 DEG C of refrigerators.((2) are examined with traditional ELISA
Survey method detect prostate specific antigen content in above-mentioned sample
The ELISA Plate for being coated with anti-prostate-specific-antigen content polyclonal antibody is taken, adds the μ L/ holes of standard items/sample 100
Into corresponding micropore；Take the ELISA Plate for being coated with anti-prostate-specific-antigen polyclonal antibody, this 100 μ L/ hole of sample-adding to pair
In the micropore answered, gently vibration mixes, and reacts 60min with being placed after cover plate membrane cover plate in 37 DEG C of light protected environments；Carefully open lid
Plate film, liquid in hole is dried, with the μ L/ holes of wash operating solution 340, fully washing 4 times, per minor tick 10s, patted dry with blotting paper
(bubble not being eliminated after patting dry can be poked with original pipette tips), adds biotinylation prostate specific antigen Dan Ke
The grand μ L/ holes of antibody 100, gently vibration mix, and react 60min with being placed after cover plate membrane cover plate in 37 DEG C of light protected environments；Carefully take off
Uncap plate film, liquid in hole is dried, with the μ L/ holes of wash operating solution 340, fully washing 3 times, per minor tick 10s, use blotting paper
Pat dry (bubble not being eliminated after patting dry can be poked with original pipette tips), add SA HRP, gently vibration mixes, with lid
Placed after plate membrane cover plate in 37 DEG C of light protected environments and react 60min；Cover plate film carefully is opened, liquid in hole is dried, uses washers
Make the μ L/ holes of liquid 340, fully washing 3 times, per minor tick 10s, pat dry that (bubble not being eliminated after patting dry is available not with blotting paper
Used pipette tips are poked)；TMB nitrite ions, 100 μ L/ holes are added, gently vibration is mixed, kept away for rearmounted 37 DEG C with cover plate membrane cover plate
15min is reacted in luminous environment；Terminate liquid 50 μ L/ holes are added, gently vibration mixes, and setting ELIASA is at 450nm or dual wavelength
Detect, determined per hole absorbance (data are please run through in 5min) at 450nm；Contrast the absorbance of testing sample and standard items
It is worth size, the residual quantity of the prostate specific antigen in quantitative analysis testing sample.
(3) analysis result
With 6 prostate specific antigen various concentrations 10 of above-mentioned preparation-13g/mL、10-12g/mL、10-11g/mL、10- 10g/mL、10-9g/mL、10-8g/mL.The percent absorption of the calculating of percentage absorptance, standard items or sample be equal to standard items or
The photon absorbing intensity average value (diplopore) of sample subtracts the photon absorbing intensity value of first standard (0 standard), then except in standard items or sample
This photon absorbing intensity average value (diplopore), i.e. percentage absorptance (%)=(B-B0)/B × 100%, wherein B are standard items or sample
This photon absorbing intensity average value (diplopore), B0For the photon absorbing intensity value of first standard (0 standard).
It is that abscissa draws standard with prostate specific antigen concentration (g/mL) using standard items percentage absorptance as ordinate
Curve, obtain linear equation.Standard curve is y=-11.58Log (x)+85.315, R2=0.9748, see accompanying drawing 3.This method
Lowest detection line be defined as percentage absorptance more than 10-14G/mL absorptance adds three standard deviations.Pass through the standard
Curve calculate lowest detection is limited to 10-13g/mL.When carrying out actual sample detection, by the percentage fluorescence rate of sample ((B-
B0)/B × 100%) value substituted into standard curve, read from standard curve corresponding to sample concentration, be multiplied by dilute corresponding to it
Release the actual concentrations that multiple is prostate specific antigen in sample.
By the contrast of embodiment 1 and embodiment 2 it can be found that the new E LISA methods sensitivity of the present invention is up to classical
1000 times of ELISA method, while also demonstrate that the new E LISA methods of the present invention are applicable to any biography of high-sensitivity detection
The material that system ELISA method can detect.
Embodiment 3
A kind of detection method for prostate specific antigen, this method belong to double antibodies sandwich ELISA, this method
It is the detection for prostate specific antigen, the enzyme that labelled antibody is used in this method is catalase C100.
On the basis of above technical scheme, meet following condition：
Substrate includes the cadmium telluride quantum dot that hydrogen peroxide and mercaptopropionic acid are modified in this method.
Specific detection method comprises the following steps：
1) it is coated with prostate specific antigen antibody；
2) antibody after step 1) coating is taken, mixes after reacting 40min in 35 DEG C of light protected environments, washes with testing sample
Wash；
3) biotinylated prostate specific antigen monoclonal antibody mixing is then added, is reacted in 35 DEG C of light protected environments
40min, washing；
4) the catalase C100 mixing of marked by streptavidin is then added, 40min is reacted in 35 DEG C of light protected environments, washes
Wash；
5) the hydrogen peroxide solution mixing that concentration is 8 μm of ol/L is then added, reacts 20min in 35 DEG C of light protected environments；
6) the cadmium telluride quantum dot mixing of mercaptopropionic acid modification is then added, reacts 10min in 35 DEG C of light protected environments；
7) detecting step 6) product fluorescence intensity.
Wherein step 1) specifically includes following operation：
A) using 0.04mol/L, pH9.4 carbonate buffer solution as coating buffer on ELISA Plate, prostate-specific is diluted
For antigen polyclonal antibody to 9 μ g/mL, the addition of coating buffer is 80 μ g/ holes, and 8h is stood after dilution；
B cleaning solution washing ELISA Plate is utilized after) removing the liquid on ELISA Plate, adds bovine serum albumin(BSA) confining liquid,
1h is closed in 35 DEG C, then discards confining liquid.
Step 3) the biotinylated prostate specific antigen monoclonal antibody is prepared by the following method：Prepare
Containing 1mg/mL prostate specific antigens monoclonal antibody, the PBS solution of 0.07mg/mL biotins, reacted under the conditions of lucifuge
30min, the concentration of the PBS solution is 0.005mol/L, then dialysis remove biotin, that is, obtain it is described it is biotinylated before
Row gland specific antigen monoclonal antibody.
The catalase C100 of the step 4) marked by streptavidin is prepared by the following method：Preparation contains 0.5mg/
The PBS solution of mL Streptavidins, the concentration of the PBS solution is 0.005mol/L, then adds SM (PEG)24Reaction
30min, then gel column purifying, collect filtrate and add catalase C100 to final concentration 1mg/mL thereto, that is, obtain the strepto-
The catalase C100 of Avidin mark.
The cadmium telluride quantum dot of step 6) the mercaptopropionic acid modification is prepared by the following method：Preparation contains
The solution that 8mmol/L cadmium nitrates, 20mmol/L mercaptopropionic acids, 3mmol/L hydrogen tellurides are received is precursor solution, the precursor solution
PH be 11, by the precursor solution heating water bath to 93 DEG C, that is, obtain the cadmium telluride quantum dot of mercaptopropionic acid modification.
The detection of fluorescence intensity is realized using ELIASA in step 7), excitation wavelength 310nm, and launch wavelength is
590nm。
The washing is rinsed or soaked using cleaning solution, and the cleaning solution contains 0.3% (v/v) Tween-20
PBST solution, the concentration of the PBST solution is 0.005mol/L, and the pH of the PBST solution is 7.0.
Embodiment 4
A kind of detection method for prostate specific antigen, this method belong to double antibodies sandwich ELISA, this method
It is the detection for prostate specific antigen, the enzyme that labelled antibody is used in this method is catalase C100.
On the basis of above technical scheme, meet following condition：
Substrate includes the cadmium telluride quantum dot that hydrogen peroxide and mercaptopropionic acid are modified in this method.
Specific detection method comprises the following steps：
1) it is coated with prostate specific antigen antibody；
2) antibody after step 1) coating is taken, mixes after reacting 80min in 39 DEG C of light protected environments, washes with testing sample
Wash；
3) biotinylated prostate specific antigen monoclonal antibody mixing is then added, is reacted in 39 DEG C of light protected environments
80min, washing；
4) the catalase C100 mixing of marked by streptavidin is then added, 80min is reacted in 39 DEG C of light protected environments, washes
Wash；
5) the hydrogen peroxide solution mixing that concentration is 12 μm of ol/L is then added, reacts 40min in 39 DEG C of light protected environments；
6) the cadmium telluride quantum dot mixing of mercaptopropionic acid modification is then added, reacts 20min in 39 DEG C of light protected environments；
7) detecting step 6) product fluorescence intensity.
Wherein step 1) specifically includes following operation：
A) using 0.06mol/L, pH9.8 carbonate buffer solution as coating buffer on ELISA Plate, prostate-specific is diluted
For antigen polyclonal antibody to 11 μ g/mL, the addition of coating buffer is 120 μ g/ holes, and 12h is stood after dilution；
B cleaning solution washing ELISA Plate is utilized after) removing the liquid on ELISA Plate, adds bovine serum albumin(BSA) confining liquid,
3h is closed in 39 DEG C, then discards confining liquid.
Step 3) the biotinylated prostate specific antigen monoclonal antibody is prepared by the following method：Prepare
Containing 3mg/mL prostate specific antigens monoclonal antibody, the PBS solution of 0.08mg/mL biotins, reacted under the conditions of lucifuge
60min, the concentration of the PBS solution is 0.02mol/L, then dialysis remove biotin, that is, obtain it is described it is biotinylated before
Row gland specific antigen monoclonal antibody.
The catalase C100 of the step 4) marked by streptavidin is prepared by the following method：Preparation contains 2mg/mL
The PBS solution of Streptavidin, the concentration of the PBS solution is 0.02mol/L, then adds SM (PEG)2460min is reacted, and
Gel column purifies afterwards, collects filtrate and adds catalase C100 to final concentration 3mg/mL thereto, that is, obtains the Streptavidin mark
The catalase C100 of note.
The cadmium telluride quantum dot of step 6) the mercaptopropionic acid modification is prepared by the following method：Preparation contains
The solution that 12mmol/L cadmium nitrates, 28mmol/L mercaptopropionic acids, 7mmol/L hydrogen tellurides are received is precursor solution, and the precursor is molten
The pH of liquid is 11.5, by the precursor solution heating water bath to 97 DEG C, that is, obtains the cadmium telluride quantum of the mercaptopropionic acid modification
Point.
The detection of fluorescence intensity is realized using ELIASA in step 7), excitation wavelength 310nm, and launch wavelength is
590nm。
The washing is rinsed or soaked using cleaning solution, and the cleaning solution contains 0.7% (v/v) Tween-20
PBST solution, the concentration of the PBST solution is 0.02mol/L, and the pH of the PBST solution is 7.5.
Embodiment 5
A kind of detection method for prostate specific antigen, this method belong to double antibodies sandwich ELISA, this method
It is the detection for prostate specific antigen, the enzyme that labelled antibody is used in this method is catalase C100.
On the basis of above technical scheme, meet following condition：
Substrate includes the cadmium telluride quantum dot that hydrogen peroxide and mercaptopropionic acid are modified in this method.
Specific detection method comprises the following steps：
1) it is coated with prostate specific antigen antibody；
2) antibody after step 1) coating is taken, mixes after reacting 60min in 37 DEG C of light protected environments, washes with testing sample
Wash；
3) biotinylated prostate specific antigen monoclonal antibody mixing is then added, is reacted in 37 DEG C of light protected environments
60min, washing；
4) the catalase C100 mixing of marked by streptavidin is then added, 60min is reacted in 37 DEG C of light protected environments, washes
Wash；
5) the hydrogen peroxide solution mixing that concentration is 10 μm of ol/L is then added, reacts 30min in 37 DEG C of light protected environments；
6) the cadmium telluride quantum dot mixing of mercaptopropionic acid modification is then added, reacts 15min in 37 DEG C of light protected environments；
7) detecting step 6) product fluorescence intensity.
Embodiment 6
A kind of detection method for prostate specific antigen, this method belong to double antibodies sandwich ELISA, this method
It is the detection for prostate specific antigen, the enzyme that labelled antibody is used in this method is catalase C100, substrate bag in this method
Include hydrogen peroxide and the cadmium telluride quantum dot of mercaptopropionic acid modification.
Embodiment 7
A kind of detection method for prostate specific antigen, this method belong to double antibodies sandwich ELISA, this method
It is the detection for prostate specific antigen, the enzyme that labelled antibody is used in this method is catalase C100.
Embodiments of the invention are described in detail above, but the content is only presently preferred embodiments of the present invention,
It is not intended to limit the invention.All all any modification, equivalent and improvement done in the application range of the present invention etc., all should
Within protection scope of the present invention.
Claims (8)
1. a kind of detection method of non-diagnostic purpose for prostate specific antigen, this method belong to double antibodies sandwich enzyme linked immunological
Method, this method are the detections for prostate specific antigen, and the enzyme that labelled antibody is used in this method is catalase C100, this method
Middle substrate includes the cadmium telluride quantum dot that hydrogen peroxide and mercaptopropionic acid are modified.
2. the detection method of the non-diagnostic purpose according to claim 1 for prostate specific antigen, it is characterised in that
Comprise the following steps：
1) it is coated with prostate specific antigen antibody；
2) antibody after step 1) coating is taken, is mixed with testing sample after 40~80min of reaction in 35~39 DEG C of light protected environments,
Washing；
3) biotinylated prostate specific antigen monoclonal antibody mixing is then added, is reacted in 35~39 DEG C of light protected environments
40~80min, washing；
4) the catalase C100 mixing of marked by streptavidin is then added, 40~80min is reacted in 35~39 DEG C of light protected environments,
Washing；
5) the hydrogen peroxide solution mixing that concentration is 8~12 μm of ol/L is then added, react 20 in 35~39 DEG C of light protected environments~
40min；
6) the cadmium telluride quantum dot mixing of mercaptopropionic acid modification is then added, 10~20min is reacted in room temperature light protected environment；
7) detecting step 6) product fluorescence intensity.
3. the detection method of the non-diagnostic purpose according to claim 2 for prostate specific antigen, it is characterised in that
Step 1) specifically includes following operation：
A coating buffer, dilution forefront) are used as using the carbonate buffer solution of 0.04~0.06mol/L, pH9.4~9.8 on ELISA Plate
Gland specific antigen polyclonal antibody is to 9~11 μ g/mL；
B cleaning solution washing ELISA Plate is utilized after) removing the liquid on ELISA Plate, bovine serum albumin(BSA) confining liquid is added, in 35
~39 DEG C of 1~3h of closing, then discard confining liquid.
4. the detection method of the non-diagnostic purpose according to claim 3 for prostate specific antigen, it is characterised in that
Step A) in the addition of coating buffer be 80~120 μ L/ holes, 8~12h is stood after dilution and performs step B again).
5. the detection method of the non-diagnostic purpose according to claim 2 for prostate specific antigen, it is characterised in that
Step 3) the biotinylated prostate specific antigen monoclonal antibody is prepared by the following method：Prepare containing 1~
3mg/mL prostate specific antigens monoclonal antibody, the PBS solution of 0.07~0.08mg/mL biotins, it is anti-under the conditions of lucifuge
30~60min is answered, the concentration of the PBS solution is 0.005~0.02mol/L, and then dialysis removes biotin, that is, is obtained described
Biotinylated prostate specific antigen monoclonal antibody.
6. the detection method of the non-diagnostic purpose according to claim 2 for prostate specific antigen, it is characterised in that
The catalase C100 of the step 4) marked by streptavidin is prepared by the following method：Preparation contains 0.5~2mg/mL chains
The PBS solution of mould Avidin, the concentration of the PBS solution is 0.005~0.02mol/L, then adds SM (PEG)24Reaction 30
~60min, then gel column purifying, collect filtrate and add catalase C100 to 1~3mg/mL of final concentration thereto, that is, obtain described
The catalase C100 of marked by streptavidin.
7. the detection method of the non-diagnostic purpose according to claim 2 for prostate specific antigen, it is characterised in that
The cadmium telluride quantum dot of step 6) the mercaptopropionic acid modification is prepared by the following method：Preparation contains 8~12mmol/L
Cadmium nitrate, 20~28mmol/L mercaptopropionic acids, the solution of 3~7mmol/L sodium hydrogen tellurides are precursor solution, the precursor solution
PH be 11~11.5, by the precursor solution heating water bath to 93~97 DEG C, that is, obtain the telluride of mercaptopropionic acid modification
Cadmium quantum dot.
8. the detection method of the non-diagnostic purpose according to claim 2 for prostate specific antigen, it is characterised in that
The detection of fluorescence intensity is realized using ELIASA in step 7), excitation wavelength 310nm, launch wavelength 590nm.
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