CN107202775A - The detection method of Keratin 18 3A9 based on SPR - Google Patents
The detection method of Keratin 18 3A9 based on SPR Download PDFInfo
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- CN107202775A CN107202775A CN201710588379.6A CN201710588379A CN107202775A CN 107202775 A CN107202775 A CN 107202775A CN 201710588379 A CN201710588379 A CN 201710588379A CN 107202775 A CN107202775 A CN 107202775A
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The present invention relates to a kind of detection method of the Keratin 18 3A9 based on SPR, belong to keratin detection technique field.This method be after SPR chips are activated through EDC/NHS carboxylic group coupling BSA on amino after be used as sensing chip, when testing sample is by censorchip surface, detect blood to be detected with the light intensity change of molecular recognition sensitive chip interface total reflection light directly to determine the Keratin 18 3A9 contents in blood using surface plasma body resonant vibration detector.The detection method can real-time, quick, highly sensitive detection Keratin 18 3A9, substantially reduce colon cancer Susceptible population detection loss, the early screening of colorectal cancer is can be applied to, with certain clinical value.
Description
Technical field
The invention belongs to keratin detection technique field, and in particular to a kind of detection of the Keratin 18-3A9 based on SPR
Method.
Background technology
Although methods such as existing chemical method, chromatography, spectroscopic methodology, immunization, capillary electrophoresis and ion mobility spectrometries
It is applied in detection of biological samples, but chemical method sensitivity is low, chromatography, capillary electrophoresis and ion mobility spectrometry pre-treatment
Process time is longer, it is necessary to which dedicated technician and extensive expensive instrument, spectroscopic methodology sensitivity are low and pure to testing sample
Degree requires higher, and the immunization operating time is long.It is most widely colloid gold test paper, but it is with colon to be applied at present in detection
Cancer Susceptible population blood is detection object, and its sensitivity only has 300-1000ng/ml., can and this technology is with its high sensitivity
Realization is detected to cancer markers in the blood of colon cancer Susceptible population, so that the infringement to object privacy is avoided, and
Substantially increase operating efficiency.
Surface plasma body resonant vibration (Surface Plasmon Resonance, SPR) technology is to be based on physical optics phenomenon
A kind of analytical technology.When polarised light is impinged upon with resonance angle incidence on metal and dielectric interface, it is totally reflected, it is incident
The part energy of light is coupled with surface plasma wave, causes surface plasma body resonant vibration, is produced light intensity and is drastically reduced
Reflected light.It is usually that a kind of functional response thing (such as part) is fixed on sensing chip table when being determined using optical SPR technology
On the metal film of face, when adding testing sample (acceptor) and it is interacted with the functional response thing on sensing chip, cause
The variations in refractive index of system, so as to cause the change of SPR optical signallings.Due to the high identity of SPR technique, high sensitivity, fast
Speed, in situ (without mark), it is convenient, real-time the advantages of, SPR sensorgram technology has been widely used in life science, medicine
The fields such as thing screening, food analysis, environmental monitoring, agricultural production.At present, have no both at home and abroad by Keratin 18-3A9 detection with
The report that SPR technique is combined together.
The content of the invention
The invention aims to solve the deficiencies in the prior art, and provide a kind of Keratin 18-3A9 based on SPR
Detection method, the detection method can real-time, quick, highly sensitive detection Keratin 18-3A9, substantially reduce colon cancer easy
Feel the loss of crowd monitoring, the early screening of colorectal cancer is can be applied to, with certain clinical value.
The present invention is adopted the following technical scheme that：
The detection method of Keratin 18-3A9 based on SPR, comprises the following steps:
Step one：SPR chips are placed into as sensing chip in SPR instruments, instrument temperature is set as 20~30 DEG C,
SPR is the abbreviation of surface plasma resonance；
Step 2：200~300 μ LEDC and NHS mixing is passed through toward SPR chip surfaces with 10~30 μ L/min flow velocity
Liquid, activates the carboxylic group in the chip face, and the carboxylic group after the SPR chips are activated through EDC/NHS can be coupled the ammonia on BSA
Base；
Step 3：0.5 is passed through with 10~30 μ L/min flow velocity on SPR chips after step 2 activating surface carboxyl~
2mg/mLBSA-Methamphetamine solution, makes it be coupled on the carboxyl activated, it is desirable to which target response value (RU) reaches
2000~3000；
Step 4：With 10~30 μ L/min stream on SPR chips after step 3 BSA-Methamphetamine couplings
Speed is passed through 75~100 μ L ethanolamine hydrochloric salt solution, to close the unreacted carboxyl site activated；
Step 5：30~50 μ L gradients are passed through with 10~30 μ L/min flow velocity on SPR chips after step 4 processing
Keratin 18-3A9 the antibody of concentration, it is determined that suitable antibody concentration；
Step 6：A series of 30~50 μ L of various concentrations Keratin 18-3A9 standard items are determined with step 5 respectively
Concentration antibody mixing after, SPR chips are passed through with 10~30 μ L/min flow velocity successively, standard curve is set up；Wherein keratin
18-3A9 antigens and the concentration antibody that step 5 is determined are isometric；
Step 7：After the concentration antibody that 30~50 μ L detected sample and step 5 are determined is mixed in equal volume, with 10
~30 μ L/min flow velocity is passed through SPR chips, and recording responses signal substitutes into standard curve, tries to achieve Keratin 18-3A9 in blood
Content.
Further, SPR chips described in step one is that first the thick chromium of one layer of 2~3nm is plated on surface on the glass substrate
Layer, then in golden film thick layers of chrome upper surface one layer of 40~50nm of plating；Used after golden film Surface Creation sulfydryl alkanol at epoxychloropropane
Being handled after reason generation epoxy radicals, epoxy radicals covalent bond glucan with bromoacetic acid makes glucan carboxylated, so as to obtain carboxylated
Dextran surface.
Further, EDC described in step 2 and NHS mixed liquor is EDC and NHS isometric mixed solution, institute
EDC is stated for N- (3- dimethyl aminopropyls)-N '-ethyl carbodiimide, concentration is 0.2~0.4M；NHS is that N- hydroxysuccinimidyls acyl is sub-
Amine, concentration is 0.1~0.2M.
Further, BSA-Methamphetamine described in step 3 is complete for artificial synthesized Keratin 18-3A9
Holoantigen, i.e., be coupled Methamphetamine (Keratin 18-3A9) molecule on BSA (bovine serum albumin(BSA)) surface, commercially available
Buy acquisition；PH value is 4.5~5.5；
Further, the concentration of ethanolamine hydrochloric salt solution described in step 4 is 1~2M.
Further, the Keratin 18-3A9 antibody described in step 5 is to have to BSA-Methamphetamine
The mouse resource monoclonal antibody of specific recognition.
Further, in step 5 after the completion of each Keratin 18-3A9 antibody responses with 10~30 μ L/min flow velocity
NaOH solution is passed through, antigen-antibody is dissociated, completes to add the antibody of next concentration after chip regeneration；Described hydroxide
Sodium solution is 50~100mM.
Further, before step 6 is entered, the most suitable concentration antibody of multiple injection step 5 determination can be carried out, is tested
Confirm the repeatability tested.
Further, the testing sample in step 7 is blood or saliva.
Compared with prior art, its advantage is the present invention：
(1) detection method of the present invention is easy to operate, in real time, quickly, need to only mix antigen-antibody and be passed through chip, just
Can real-time monitored result；
(2) chip of the present invention prepares scheme maturation, has been commercialized, and purchase is convenient, and reusable up to a hundred
It is secondary, it is a kind of very economical detection method；
(3) sensitivity that detection method is reached is higher, and the time is shorter, is that other any technologies cannot all compare
's；
(4) because the sensitivity of colloid gold test paper only has 300-1000ng/mL, when measuring samples content is less than its sensitivity
When, colloid gold test paper will be unable to detection, and the present invention just solves this problem.
Brief description of the drawings
Fig. 1 is relation between various concentrations antibody and SPR response signals, wherein, antibody concentration be followed successively by 6.25 μ g/ml,
12.5 μ g/ml, 25 μ g/ml, 50 μ g/ml, 100 μ g/ml anti-keratins 18-3A9；
Fig. 2 is spr signal rapid drawdown after the regeneration schematic diagram of SPR chips, injection sodium hydroxide, and is gradually brought to just primordium
Wire state；
Fig. 3 is the SPR response diagrams for injecting 5 constant density antibody respectively；
The standard curve detected in Fig. 4 for Keratin 18-3A9.
Embodiment
With reference to embodiment, the present invention is described in further detail.
It will be understood to those of skill in the art that the following example is merely to illustrate the present invention, and it should not be regarded as limiting this hair
Bright scope.
The detection method of Keratin 18-3A9 based on SPR, comprises the following steps:
Step one：SPR chips are placed into as sensing chip in SPR instruments, instrument temperature is set as 20~30 DEG C, SP
R is the abbreviation of surface plasma resonance；
Step 2：200~300 μ LEDC and NHS mixing is passed through toward SPR chip surfaces with 10~30 μ L/min flow velocity
Liquid, activates the carboxylic group in the chip face, and the carboxylic group after the SPR chips are activated through EDC/NHS can be coupled the ammonia on BSA
Base；
Step 3：0.5 is passed through with 10~30 μ L/min flow velocity on SPR chips after step 2 activating surface carboxyl~
2mg/mLBSA-Methamphetamine solution, makes it be coupled on the carboxyl activated, it is desirable to which target response value (RU) reaches
2000~3000；
Step 4：With 10~30 μ L/min stream on SPR chips after step 3 BSA-Methamphetamine couplings
Speed is passed through 75~100 μ L ethanolamine hydrochloric salt solution, to close the unreacted carboxyl site activated；
Step 5：30~50 μ L gradients are passed through with 10~30 μ L/min flow velocity on SPR chips after step 4 processing
Keratin 18-3A9 the antibody of concentration, it is determined that suitable antibody concentration；
Step 6：A series of 30~50 μ L of various concentrations Keratin 18-3A9 standard items are determined with step 5 respectively
Concentration antibody mixing after, SPR chips are passed through with 10~30 μ L/min flow velocity successively, standard curve is set up；Wherein keratin
18-3A9 antigens and the concentration antibody that step 5 is determined are isometric；
Step 7：After the concentration antibody that 30~50 μ L detected sample and step 5 are determined is mixed in equal volume, with 10
~30 μ L/min flow velocity is passed through SPR chips, and recording responses signal substitutes into standard curve, tries to achieve Keratin 18-3A9 in blood
Content.
Further, SPR chips described in step one is that first the thick chromium of one layer of 2~3nm is plated on surface on the glass substrate
Layer, then in golden film thick layers of chrome upper surface one layer of 40~50nm of plating；Used after golden film Surface Creation sulfydryl alkanol at epoxychloropropane
Being handled after reason generation epoxy radicals, epoxy radicals covalent bond glucan with bromoacetic acid makes glucan carboxylated, so as to obtain carboxylated
Dextran surface.
Further, EDC described in step 2 and NHS mixed liquor is EDC and NHS isometric mixed solution, institute
EDC is stated for N- (3- dimethyl aminopropyls)-N '-ethyl carbodiimide, concentration is 0.2~0.4M；NHS is that N- hydroxysuccinimidyls acyl is sub-
Amine, concentration is 0.1~0.2M.
Further, BSA-Methamphetamine described in step 3 is complete for artificial synthesized Keratin 18-3A9
Holoantigen, i.e., be coupled Methamphetamine (Keratin 18-3A9) molecule on BSA (bovine serum albumin(BSA)) surface, commercially available
Buy acquisition；PH value is 4.5~5.5；
Further, the concentration of ethanolamine hydrochloric salt solution described in step 4 is 1~2M.
Further, the Keratin 18-3A9 antibody described in step 5 is to have to BSA-Methamphetamine
The mouse resource monoclonal antibody of specific recognition.
Further, in step 5 after the completion of each Keratin 18-3A9 antibody responses with 10~30 μ L/min flow velocity
NaOH solution is passed through, antigen-antibody is dissociated, completes to add the antibody of next concentration after chip regeneration；Described hydroxide
Sodium solution is 50~100mM.
Further, before step 6 is entered, the most suitable concentration antibody of multiple injection step (5) determination can be carried out,
The repeatability of confirmatory experiment.
The main agents information mentioned in following examples is shown in Table 1；Key instrument is shown in Table 2 with facility information.
Table 1
Table 2
The CM5 chips that the SPR chips that following examples are used produce for KEI companies, the surface of the chip is carboxymethyl
Change the amino on glucan, easily coupling BSA；Used SPR instruments are the ESPRIT that KEI companies produce；Colloid gold test paper is purchased
From Abon Biopharm (Hangzhou) Co., Ltd..
Embodiment 1
(1) SPR instruments are opened, SPR chip pockets is unlocked, SPR chips is placed into slot, chip pocket is locked；SPR
Instrument temperature is set as 25 DEG C；
(2) EDC and NHS that inject 250 μ L are passed through in equal volume than mixing toward in SPR chip surfaces with 20 μ L/min flow velocity
Liquid, activates the carboxylic group of the chip surface；Wherein EDC concentration is 0.4M, and NHS concentration is 0.1M；
(3) 1mg/mL pH5.0 BSA- is passed through on the SPR chips after activating surface carboxyl with 20 μ L/min flow velocity
Methamphetamine solution, makes it be coupled on the carboxyl activated, it is desirable to which target response value (RU) reaches 3000；
(4) 100 μ L 1M is passed through with 20 μ L/min flow velocity on coupling BSA-Methamphetamine SPR chips
Ethanolamine hydrochloric salt solution, to close the unreacted carboxyl site activated；
(5) 40 μ L 6.25 μ g/mL, 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL, 100 μ are passed through with 20 μ L/min flow velocity
Keratin 18-3A9 the antibody of g/mL gradient concentrations, as a result as shown in figure 1, as a result showing that 6.25 μ g/mL's and 12.5 μ g/mL is anti-
RU values under bulk concentration are too small, and mark song gradient is narrower, reduce sensitivity；RU under 50 μ g/mL and 100 μ g/mL antibody concentration
Value is higher, but can expend excessive antibody, is unfavorable under cost control, Integrated comparative, 25 μ g/mL antibody concentration is the most
Properly, it is 25 μ g/mL to determine the antibody concentration in follow-up test；
(6) 10 μ L50mM NaOH solution is passed through after the completion of each example reaction with 20 μ L/min flow velocity, resists antigen
Body is dissociated, and completes to add the antibody of next concentration after chip regeneration, as shown in Fig. 2 starting baseline is 1980RU, after regeneration
Baseline returns to 1978RU, and CV (coefficient of variation) is only 0.07%, shows that chip regeneration effect is preferable；
(7) it is passed through 25 μ g/mL μ L of antibody 40 totally 5 times with 20 μ L/min flow velocity, the repeatability of confirmatory experiment, as a result such as
Shown in Fig. 3；The RU of 5 repetitions is respectively 1039.5RU, 1065.0RU, 1062.3RU, 1047.1RU, 1006.2RU, and CV is only
2.26%, show the repeatability of experiment preferably；
(8) by a series of 40 μ L of various concentrations Keratin 18-3A9 standard items (125ng/ml, 62.5ng/mL,
31.25ng/mL、15.63ng/mL、7.81ng/mL、3.91ng/mL、1.95ng/mL、0.98ng/ml、0.49ng/ml、
0.24ng/ml, 0.12ng/ml, 0.06ng/ml, 0ng/mL) with 25 μ g/mL antibody it is isometric according to after mixing with 20 μ L/min's
Flow velocity is passed through 40 μ L to SPR chips and obtains SPR response signals successively, sets up standard curve, as a result as shown in figure 4,0.06~
In the range of 7.81ng/ml, Keratin 18-3A9 concentration and SPR response signals is linear, and minimal detectable concentration is in 0.06-
0.12ng/ml scopes, when Keratin 18-3A9 concentration is more than 7.81ng/ml or less than 0.06ng/ml, Keratin 18-3A9's
Concentration is no longer linear with spr signal.
(9) basic condition of people's (first) to be detected：Sex man, at the age 31, its blood is through colloid gold test paper testing result
Keratin 18-3A9 is positive.
After the blood 12000pm of first centrifugation 10min, take after 40 μ L sample mixes with isometric antibody, with 20 μ L/
Min flow velocity is passed through 30 μ L to SPR chips successively, recording responses signal, substitutes into standard curve, try to achieve Keratin 18 in blood-
3A9 contents are beyond mark song, more than 7.18ng/mL.
The authenticity of the lower the present embodiment testing result of checking：Extracted with Gas chromatographyMass spectrometry (GC/MS) and solid phase
Taking technology (SPE) to be combined, to measure in first blood Keratin 18-3A9 contents be 1042.12ng/mL, the knot measured with the present invention
Fruit is consistent.
Embodiment 2
(1) SPR instruments are opened, SPR chip pockets is unlocked, SPR chips is placed into slot, chip pocket is locked；SPR
Instrument temperature is set as 25 DEG C；
(2) the mixed of 100 μ L0.2MEDC and 100 μ L0.1M NHS is passed through toward SPR chip surfaces with about 10 μ L/min flow velocity
Liquid is closed, the carboxylic group of the chip surface is activated；
(3) 0.5mg/mLBSA- is passed through with 10 μ L/min flow velocity on the SPR chips after step (2) activating surface carboxyl
Methamphetamine solution (pH value is 4.5), makes it be coupled on the carboxyl activated, it is desirable to which target response value (RU) reaches
To about 2000；
(4) it is passed through on the SPR chips after step (3) BSA-Methamphetamine couplings with 10 μ L/min flow velocity
75 μ L2M ethanolamine hydrochloric salt solution, to close the unreacted carboxyl site activated；
(5) 30 μ L 6.25 μ g/mL, 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL, 100 μ are passed through with 10 μ L/min flow velocity
Keratin 18-3A9 the antibody of g/mL gradient concentrations, as a result as shown in figure 1, as a result showing that 6.25 μ g/mL's and 12.5 μ g/mL is anti-
RU values under bulk concentration are too small, and mark song gradient is narrower, reduce sensitivity；RU under 50 μ g/mL and 100 μ g/mL antibody concentration
Value is higher, but can expend excessive antibody, is unfavorable under cost control, Integrated comparative, 25 μ g/mL antibody concentration is the most
Properly, it is 25 μ g/mL to determine the antibody concentration in follow-up test；
(6) 10 μ L50mM NaOH solution is passed through after the completion of each example reaction with 10 μ L/min flow velocity, resists antigen
Body is dissociated, and completes to add the antibody of next concentration after chip regeneration, as shown in Fig. 2 starting baseline is 1980RU, after regeneration
Baseline returns to 1978RU, and CV (coefficient of variation) is only 0.07%, shows that chip regeneration effect is preferable；
(7) it is passed through 25 μ g/mL μ L of antibody 30 totally 5 times with 10 μ L/min flow velocity, the repeatability of confirmatory experiment, as a result such as
Shown in Fig. 3；The RU of 5 repetitions is respectively 1039.5RU, 1065.0RU, 1062.3RU, 1047.1RU, 1006.2RU, and CV is only
2.26%, show the repeatability of experiment preferably；
(8) by a series of 30 μ L of various concentrations Keratin 18-3A9 standard items (125ng/ml, 62.5ng/mL,
31.25ng/mL、15.63ng/mL、7.81ng/mL、3.91ng/mL、1.95ng/mL、0.98ng/ml、0.49ng/ml、
0.24ng/ml, 0.12ng/ml, 0.06ng/ml, 0ng/mL) with 25 μ g/mL antibody it is isometric according to after mixing with 20 μ L/min's
Flow velocity is passed through 30 μ L to SPR chips and obtains SPR response signals successively, sets up standard curve, as a result as shown in figure 4,0.06~
In the range of 7.81ng/ml, Keratin 18-3A9 concentration and SPR response signals is linear, and minimal detectable concentration is in 0.06-
0.12ng/ml scopes, when Keratin 18-3A9 concentration is more than 7.81ng/ml or less than 0.06ng/ml, Keratin 18-3A9's
Concentration is no longer linear with spr signal.
(9) basic condition of people's (second) to be detected：Sex man, at the age 43, its blood is through colloid gold test paper testing result
Keratin 18-3A9 is negative.
After the blood 12000pm centrifugations 10min of second, the body such as 30 μ L sample and the concentration antibody of step (5) determination is taken
After product mixing, 30 μ L to SPR chips are passed through with 10 μ L/min flow velocity, recording responses signal substitutes into standard curve, tries to achieve blood
Middle Keratin 18-3A9 contents exceed mark song, more than 7.81ng/mL.
The authenticity of the lower the present embodiment testing result of checking：Extracted with Gas chromatographyMass spectrometry (GC/MS) and solid phase
Taking technology (SPE) to be combined, to measure in second blood Keratin 18-3A9 contents be 142.47ng/mL, the result measured with the present invention
Unanimously.
Embodiment 3
(1) SPR instruments are opened, SPR chip pockets is unlocked, SPR chips is placed into slot, chip pocket is locked；SPR
Instrument temperature is set as 30 DEG C；
(2) the mixed of 150 μ L0.2M EDC and 150 μ L0.2MNHS is passed through toward SPR chip surfaces with about 30 μ L/min flow velocity
Liquid is closed, the carboxylic group of the chip surface is activated；
(3) 2mg/mLBSA- is passed through with 30 μ L/min flow velocity on the SPR chips after step (2) activating surface carboxyl
Methamphetamine solution (pH value is 5.5), makes it be coupled on the carboxyl activated, it is desirable to which target response value (RU) reaches
To about 3000；
(4) it is passed through on the SPR chips after step (3) BSA-Methamphetamine couplings with 30 μ L/min flow velocity
100 μ L2M ethanolamine hydrochloric salt solution, to close the unreacted carboxyl site activated；
(5) 50 μ L 6.25 μ g/mL, 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL, 100 μ are passed through with 30 μ L/min flow velocity
Keratin 18-3A9 the antibody of g/mL gradient concentrations, as a result as shown in figure 1, as a result showing that 6.25 μ g/mL's and 12.5 μ g/mL is anti-
RU values under bulk concentration are too small, and mark song gradient is narrower, reduce sensitivity；RU under 50 μ g/mL and 100 μ g/mL antibody concentration
Value is higher, but can expend excessive antibody, is unfavorable under cost control, Integrated comparative, 25 μ g/mL antibody concentration is the most
Properly, it is 25 μ g/mL to determine the antibody concentration in follow-up test；
(6) 10 μ L50mM NaOH solution is passed through after the completion of each example reaction with 30 μ L/min flow velocity, resists antigen
Body is dissociated, and completes to add the antibody of next concentration after chip regeneration, as shown in Fig. 2 starting baseline is 1980RU, after regeneration
Baseline returns to 1978RU, and CV (coefficient of variation) is only 0.07%, shows that chip regeneration effect is preferable；
(7) it is passed through 25 μ g/mL μ L of antibody 50 totally 5 times with 30 μ L/min flow velocity, the repeatability of confirmatory experiment, as a result such as
Shown in Fig. 3；The RU of 5 repetitions is respectively 1039.5RU, 1065.0RU, 1062.3RU, 1047.1RU, 1006.2RU, and CV is only
2.26%, show the repeatability of experiment preferably；
(8) by a series of 50 μ L of various concentrations Keratin 18-3A9 standard items (125ng/ml, 62.5ng/mL,
31.25ng/mL、15.63ng/mL、7.81ng/mL、3.91ng/mL、1.95ng/mL、0.98ng/ml、0.49ng/ml、
0.24ng/ml, 0.12ng/ml, 0.06ng/ml, 0ng/mL) with 25 μ g/mL antibody it is isometric according to after mixing with 20 μ L/min's
Flow velocity is passed through 40 μ L to SPR chips and obtains SPR response signals successively, sets up standard curve, as a result as shown in figure 4,0.06~
In the range of 7.81ng/ml, Keratin 18-3A9 concentration and SPR response signals is linear, and minimal detectable concentration is in 0.06-
0.12ng/ml scopes, when Keratin 18-3A9 concentration is more than 7.81ng/ml or less than 0.06ng/ml, Keratin 18-3A9's
Concentration is no longer linear with spr signal.
(9) basic condition of people (third) to be detected：Sex man, at the age 22, its blood is through colloid gold test paper testing result
Keratin 18-3A9 is negative.
After the blood 12000pm centrifugations 10min of second, the body such as 50 μ L sample and the concentration antibody of step (5) determination is taken
After product mixing, 50 μ L to SPR chips are passed through with 30 μ L/min flow velocity, recording responses signal substitutes into standard curve, tries to achieve blood
Middle Keratin 18-3A9 contents are 6.11ng/mL.
The authenticity of the lower the present embodiment testing result of checking：Extracted with Gas chromatographyMass spectrometry (GC/MS) and solid phase
Taking technology (SPE) to be combined, to measure in the third blood Keratin 18-3A9 contents be 6.02ng/mL, the result one measured with the present invention
Cause.
Embodiment 4
The basic condition of people's (fourth) to be detected：Sex man, age 21, its blood is angle egg through colloid gold test paper testing result
White 18-3A9 is positive.
Take after the blood sample of 40 μ L fourths mixes with isometric antibody, 30 μ L are passed through with 20 μ L/min flow velocity successively and arrived
SPR chips, recording responses signal substitutes into standard curve, and it is, beyond mark song, to be more than to try to achieve Keratin 18-3A9 contents in blood
7.18ng/mL。
The authenticity of the lower the present embodiment testing result of checking：Extracted with Gas chromatographyMass spectrometry (GC/MS) and solid phase
Taking technology (SPE) to be combined, to measure in fourth blood Keratin 18-3A9 contents be 3219.65ng/mL, the knot measured with the present invention
Fruit is consistent.
Embodiment 5
The basic condition of people's (penta) to be detected：Sex man, age 37, its blood is angle egg through colloid gold test paper testing result
White 18-3A9 is positive.
Take after 30 μ L penta blood sample mixes with isometric antibody, 30 μ L to SPR are passed through with 10 μ L/min flow velocity
Chip, recording responses signal substitutes into standard curve, tries to achieve Keratin 18-3A9 contents in blood and exceeds mark song, more than 7.81ng/
mL。
The authenticity of the lower the present embodiment testing result of checking：Extracted with Gas chromatographyMass spectrometry (GC/MS) and solid phase
Taking technology (SPE) to be combined, to measure in penta blood Keratin 18-3A9 contents be 2613.72ng/mL, the knot measured with the present invention
Fruit is consistent.
It should be understood by those skilled in the art that, the present invention is not limited to the above embodiments, above-described embodiment and explanation
Merely illustrating the principles of the invention described in book, without departing from the spirit and scope of the present invention, the present invention also have
Various changes and modifications, these changes and improvements all fall within the protetion scope of the claimed invention.The claimed scope of the invention
By appended claims and its equivalent thereof.
Claims (9)
1. the detection method of the Keratin 18-3A9 based on SPR, it is characterised in that comprise the following steps:
Step one：SPR chips are placed into as sensing chip in SPR instruments, instrument temperature is set as 20~30 DEG C；
Step 2：200~300 μ LEDC and NHS mixed liquor is passed through toward SPR chip surfaces with 10~30 μ L/min flow velocity, it is living
Change the carboxylic group in the chip face；
Step 3：0.5~2mg/ is passed through with 10~30 μ L/min flow velocity on SPR chips after step 2 activating surface carboxyl
MLBSA-Methamphetamine solution, makes it be coupled on the carboxyl activated, it is desirable to which target response value (RU) reaches 2000
~3000；
Step 4：It is logical with 10~30 μ L/min flow velocity on SPR chips after step 3 BSA-Methamphetamine couplings
Enter 75~100 μ L ethanolamine hydrochloric salt solution, to close the unreacted carboxyl site activated；
Step 5：30~50 μ L gradient concentrations are passed through with 10~30 μ L/min flow velocity on SPR chips after step 4 processing
Keratin 18-3A9 antibody, it is determined that suitable antibody concentration；
Step 6：A series of 30~50 μ L of various concentrations Keratin 18-3A9 standard items are dense with step 5 determination respectively
Spend after antibody mixing, SPR chips are passed through with 10~30 μ L/min flow velocity successively, standard curve is set up；Wherein Keratin 18-
3A9 antigens and the concentration antibody that step 5 is determined are isometric；
Step 7：After the concentration antibody that 30~50 μ L detected sample and step 5 are determined is mixed in equal volume, with 10~30 μ
L/min flow velocity is passed through SPR chips, and recording responses signal substitutes into standard curve, tries to achieve Keratin 18-3A9 contents in blood.
2. the detection method of the Keratin 18-3A9 according to claim 1 based on SPR, it is characterised in that in step one
The SPR chips are that first the thick layers of chrome of one layer of 2~3nm is plated on surface on the glass substrate, then plate in layers of chrome upper surface one layer 40~
Golden film thick 50nm；After golden film Surface Creation sulfydryl alkanol generation epoxy radicals, epoxy radicals covalent bond are handled with epoxychloropropane
Being handled after glucan with bromoacetic acid makes glucan carboxylated, so as to obtain the dextran surface of carboxylated.
3. the detection method of the Keratin 18-3A9 according to claim 1 based on SPR, it is characterised in that in step 2
The mixed liquor of the EDC and NHS be EDC and NHS isometric mixed solution, the EDC be N- (3- dimethyl aminopropyls)-
N '-ethyl carbodiimide, concentration is 0.2~0.4M；NHS is n-hydroxysuccinimide, and concentration is 0.1~0.2M.
4. the detection method of the Keratin 18-3A9 according to claim 1 based on SPR, it is characterised in that in step 3
The BSA-Methamphetamine is artificial synthesized Keratin 18-3A9 comlete antigens.
5. the detection method of the Keratin 18-3A9 according to claim 1 based on SPR, it is characterised in that in step 4
The concentration of the ethanolamine hydrochloric salt solution is 1~2M.
6. the detection method of the Keratin 18-3A9 according to claim 1 based on SPR, it is characterised in that in step 5
Described Keratin 18-3A9 antibody is the mouse resource monoclonal antibody to BSA-Methamphetamine with specific recognition.
7. the detection method of the Keratin 18-3A9 according to claim 1 based on SPR, it is characterised in that in step 5
NaOH solution is passed through with 10~30 μ L/min flow velocity after the completion of each Keratin 18-3A9 antibody responses, makes antigen-antibody solution
From completion chip adds the antibody of next concentration after regenerating；Described sodium hydroxide solution is 50~100mM.
8. the detection method of the Keratin 18-3A9 according to claim 1 based on SPR, it is characterised in that entering step
Before rapid six, the most suitable concentration antibody of multiple injection step 5 determination, the repeatability of confirmatory experiment can be carried out.
9. the detection method of the Keratin 18-3A9 according to claim 1 based on SPR, it is characterised in that in step 7
Testing sample be blood or saliva.
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