CN105974128A - Quantifying device for human neutrophil lipophorin homodimers - Google Patents
Quantifying device for human neutrophil lipophorin homodimers Download PDFInfo
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- CN105974128A CN105974128A CN201610404148.0A CN201610404148A CN105974128A CN 105974128 A CN105974128 A CN 105974128A CN 201610404148 A CN201610404148 A CN 201610404148A CN 105974128 A CN105974128 A CN 105974128A
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—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 diseases not provided for elsewhere
Abstract
The invention discloses a quantifying device for human neutrophil lipophorin (HNL) homodimers, and belongs to the technical field of immunological detection. The quantifying device is composed of 5-time sample diluent, 25-time washing liquid, HNL homodimer standard substance mother liquid, an anti-HNL-antibody-coated 96-pore elisa plate, an elisa anti-HNL antibody, a U-shaped 96-pore plate, a seal plate film, an enzyme substrate solution and a stop solution. According to the quantifying device, an elisa-plate-coated anti-HNL antibody is same as the elisa anti-HNL antibody, and therefore the HNL homodimer concentration is easily quantified. In addition, in the using process of the device, incubating is conducted by two steps, cleaning is conducted by one step, and therefore the detection time is significantly shortened. Therefore, the quantifying device has the advantages that preparation is easy, use is convenient, and the testing time is short. The quantifying device can be used for detection and monitoring on diseases (such as acute bacterial infection) taking the HNL homodimers as markers, and therefore the great laboratory research and clinical application value is achieved.
Description
Technical field
The invention belongs to technical field of immunological detection, be specifically related to a kind of human neutrophil apolipoprotein (HNL)
The fast quantification device of homodimer.
Background technology
The most neutral grain of human neutrophil lipocalin protein (human neutrophil lipocalin, HNL)
Cell gelatinase associated lipocalin (neutrophil gelatinase associated lipocalin,
NGAL), also known as Lipocalin-2 (Lipocalin-2), it is a kind of lipocalin protein
(J.Biol.Chem.1993,268:10425-10432；Scand.J.Clin.Lab.Invest.1994,54:365-
376).HNL is with different kinds of molecules such as monomer, homodimer and heterodimers (monomer is formed with MMP9 molecule)
Form exists, and different molecular form HNL is relevant from different pathological processes.As monomer HNL may be with acute injury of kidney
There is of a relatively high dependency (Clin.Chim.Acta.2009,403:121-125；Lancet 2005,365:
1231-1238；Biomed.Res.Int.2015,2009,186:48-51；Am.J.Nephrol.2004,24:
307-315；The practical medicine 2015,24:46-47 of China), heterodimer HNL may be relevant with tumor invasiveness.
Cai Linjun et al. research finds that homodimer HNL reflects urinary tract infection situation
(Clin.J.Am.Soc.Nephrol.2010)；Additionally, Venge Per and Xu S.Y. et al. research finds homology two
Aggressiveness HNL has the highest specificity and sensitivity (J.Immunol. to distinguishing the infection of diagnosing acute antibacterial with virus infection
Methods 2015,424:85-90；J.Clin.Lab.Invest.1995,55:125-131).Other points relatively
Sub-form HNL, diagnosing acute antibacterial is infected by homodimer higher specificity and sensitivity, it may be possible to because
The HNL of the neutrophilic granulocyte secretion participating in acute bacterial infection process is mainly homodimer
(Clin.Chim.Acta.2009,403:121-125)。
The most existing multiple HNL assay method is open, such as RIA (international monopoly WO/1995/029404A1 and China
ZL 200980155194 etc.), ELISA (European patent EP 2225268 and Chinese patent ZL200980155194
Deng), Western-blotting, latex immunoturbidimetry (Chinese patent ZL 201410431182), chemiluminescence
Immunoassay (Chinese Patent Application No. 201510184818), immuno-chromatographic test paper strip technology (Chinese patent
ZL201220323587、ZL201420423962、ZL201220392399、ZL201220497401、
ZL201220323586 and ZL201220497401) etc..Any of the above HNL quantitative approach has respective pluses and minuses and phase
Application, but in addition to Western-blotting can distinguish different molecular form HNL, above-mentioned HNL quantitative approach is equal
Can not specific quantification homodimer HNL, thus affect this albumen examining as some diseases such as acute bacterial infection
Cutting capacity.Although, Western-blotting can distinguish different molecular form HNL, but due to this technology itself
The drawbacks limit actual application of the method.As operate complexity, test period length, every time analyze sample size limited with
And accurate quantitative analysis difficulty etc..
To sum up, current laboratory research and the most possible clinical practice are badly in need of setting up quick, specific quantification homology two
The device of aggressiveness HNL and correlation method.
Summary of the invention
In order to realize the specific quantification of human neutrophil Apolipoprotein H NL homodimer molecule, the present invention designs
And have developed the fast quantification device of a kind of HNL homodimer based on sandwich ELISA techniques.This device has
Preparation method simple (only needing a kind of anti-NHL antibody), use quick (process of hatching only has two steps), detection limit low by (0.1
Ng/mL, can meet the detection of HNL in body fluid sample) etc. excellent properties, examine in early days in the disease such as acute bacterial infection
There is very big application potential in disconnected laboratory research and Clinical Laboratory field.
In order to realize above-mentioned target, the present invention adopts the following technical scheme that:
A kind of proportioning device of human neutrophil apolipoprotein (HNL) homodimer, it is characterised in that: by 5
Times sample diluting liquid, 25 times of washing liquids, homodimer HNL standard substance mother solution, the coated 96 hole enzymes of anti-HNL antibody
Target, enzyme mark anti-HNL antibody-solutions, orifice plate of the U-shaped end 96, shrouding film, enzyme substrate solution and stop buffer composition,
The anti-HNL antibody that described anti-HNL antibody coated 96 hole ELISA Plate HNL anti-with enzyme mark antibody is used is identical；
Described anti-HNL antibody is monoclonal antibody, and described anti-HNL monoclonal antibody is the anti-HNL monoclonal antibody of commercialization
(the HNL mab such as ab63929 or the P&M Venge Diagnostics Development company of Abcam company
697 etc.) the anti-HNL monoclonal or by conventional method prepared with HNL monomer or HNL homodimer immune animal
Anti-HNL monoclonal antibody prepared by antibody or gene engineering method, the antigen that described anti-HNL monoclonal antibody is identified
Epi-position is only one of which on monomer HNL；The enzyme of described enzyme mark anti-HNL antibody institute labelling can be horseradish peroxidase,
Alkali phosphatase etc..
Described proportioning device realizes the fast quantification of HNL homodimer through two-step method, and described two-step method refers to that device is whole
Process of hatching during individual use only needs twice, and process of hatching for described twice refers to add anti-for enzyme mark HNL antibody for the first time
Enter and rapidly join pre-prepd standard concentration gradient solution and warp after the coated 96 hole ELISA Plate of anti-HNL antibody
Sample liquid after dilution hatches 20～60min, and hatching for described twice will be anti-after process second time refers to hatch for the first time
The coated 96 hole ELISA Plate of HNL antibody add enzyme substrate solution after washing liquid working solution cleans and hatch 10～20min
Rear addition stop buffer；Described pre-prepd standard concentration gradient solution refers to HNL homodimer standard substance mother solution
Making after 2 times of method dilutions, described sample liquid after dilution refers to the body fluid after sample diluting liquid working solution dilutes
Sample, described body fluid sample includes urine, serum, blood plasma, cerebrospinal fluid, saliva etc..
5 times of sample diluting liquids are diluted 5 times with deionized water and obtains sample diluting liquid working solution；25 times of washing liquids are used
Deionized water dilutes 25 times and makes washing liquid working solution.
Apparatus of the present invention composition, method of preparation and use are as follows:
1) apparatus of the present invention composition
Table 1: form for apparatus of the present invention
| Sequence number | Title | Quantity |
| 1 | The coated 96 hole ELISA Plate of anti-HNL antibody | 1 piece |
| 2 | 5 times of sample diluting liquids | 1 bottle (80mL) |
| 3 | HNL homodimer standard substance mother solution | 1 pipe (300 μ L) |
| 4 | Enzyme mark anti-HNL antibody-solutions | 1 pipe (5.5mL) |
| 5 | 25 times of washing liquids | 1 bottle of 35mL |
| 6 | Enzyme substrate solution | 1 set |
| 7 | Stop buffer | 1 bottle of 12mL |
| 8 | Conventional orifice plate of the U-shaped end 96 | 1 piece |
| 9 | Shrouding film | 1 |
2) preparation method
A) preparation of the coated 96 hole ELISA Plate of anti-HNL antibody
(1) anti-HNL antibody is coated 96 hole ELISA Plate
By anti-HNL antibody 50mmol/L Na2CO3-NaHCO3, the sodium carbonate buffer dilution of pH 9.6
Make antibody to 1～5 μ g/mL and be coated liquid；Add 100 μ L antibody to the 96 every holes of hole ELISA Plate to be coated
Liquid, hatches 2h or incubated at room 4h or 4 DEG C overnight for 37 DEG C.
(2) sealase target
Abandon antibody and be coated liquid, state then up and the 96 every holes of hole ELISA Plate add 200～300 μ L closings
(confining liquid is the 50mmol/L Na containing 2%BSA (wt/vol) to liquid2CO3-NaHCO3, pH's 9.6
Sodium carbonate buffer)；Hatch 2h or incubated at room 4h or 4 DEG C overnight for 37 DEG C.
(3) immobilized enzyme target
Abandon confining liquid, in the 96 every holes of hole ELISA Plate, then add 200～300 μ L fixatives (fixing
Liquid is containing 50% (wt/vol) sucrose, 50% (wt/vol) trehalose or 25% (wt/vol) sucrose and 25%
(wt/vol) the 50mmol/L Na of trehalose2CO3-NaHCO3, the sodium carbonate buffer of pH 9.6, room temperature
Fixing 30min～2h.
(4) ELISA Plate is sealed
Abandoning fixative, room temperature is dried or 25～30 DEG C of vacuum drying；ELISA Plate and bagged dryer are put into
Then evacuation, 4 DEG C of preservations in tinfoil paper paper bag；
The anti-HNL antibody coated elisa plate prepared by above method is through 37 DEG C of accelerated tests, permissible
Preserve 32 days.According to A Luoniwusi formula (Arrhenius equation), enzyme prepared by this method
Target can preserve natural law=32 × 48 day=1536 day under the conditions of 4 DEG C.
B) preparation of 5 times of sample diluting liquids
For containing 1%BSA (wt/vol), 0.5%Tween-20 (vol/vol), 0.25% hexadecane
Base trimethylammonium bromide (CTAB) (wt/vol) and 0.1%NaN3(wt/vol) phosphate radical mole is dense
Degree is the PBS of 50mmol/L, pH 7.4.
C) preparation of standard substance mother solution
For containing 0.1%BSA (wt/vol), 0.1%Tween-20 (vol/vol), 0.05% 16
Alkyl trimethyl ammonium bromide (CTAB) (wt/vol), 0.02%NaN3(wt/vol) and 12.8 μ g/L
The PBS that phosphate radical molar concentration is 10mmol/L, pH 7.4 of homodimer HNL.
D) preparation of enzyme mark anti-HNL antibody-solutions
Enzyme mark anti-HNL antibody be by conventional method (ab102850 of Abcam, ab102890,
Ab102887 etc.) by enzyme molecule covalent such as horseradish peroxidase (HRP) or alkali phosphatases (ALP)
It is marked on anti-HNL antibody.
Enzyme mark anti-HNL antibody-solutions is containing 1%BSA (wt/vol), 0.1%NaN3(wt/vol) and
The phosphate radical molar concentration of 0.2～2 μ g/mL enzyme mark anti-HNL antibody is 50mmol/L, pH7.4
PBS.
E) preparation of 25 times of washing liquids
For the phosphate radical molar concentration containing 2.5%Tween-20 (vol/vol) be 250mmol/L,
The PBS of pH 7.4.
F) enzyme substrate solution and the preparation of stop buffer
The enzyme of corresponding enzyme mark anti-HNL antibody institute labelling uses different enzyme substrate solutions and stop buffer.This system is
The disclosedest conventional method.Such as table 2.
Table 2: different system enzyme labelled antibodies, enzyme substrate solution and the composition of corresponding stop buffer
3) using method
The specifically used step of device is as follows:
(1) take out the solution of above-mentioned preparation, the coated 96 hole ELISA Plate of antibody etc., be positioned over room temperature ring
Border；
(2) 5 times of sample diluting liquids and 25 times of washing liquid distilled water are diluted 5 times and 25 times respectively, system
Become sample diluting liquid working solution and washing liquid working solution；
(3) biological sample (serum, blood plasma, urine, saliva or cerebrospinal fluid etc.) is used sample diluting liquid
Working solution dilution (serum and blood plasma generally dilute 50 times, urine generally dilute 25 times, saliva
Liquid and the usual 5～10 times of dilutions of cerebrospinal fluid), the sample liquid after being diluted；
(4) homodimer HNL standard substance mother solution sample diluting liquid working solution is carried out 2 times of gradients dilute
Release, make standard concentration gradient solution；
(5) sample liquid after above-mentioned 100 μ L standard concentration gradient solutions and dilution is added to the U-shaped end 96
Orifice plate；
(6) 50 μ L enzyme mark anti-HNL antibody-solutions are added to the coated 96 hole ELISA Plate of anti-HNL antibody；
(7) standard in the every hole of orifice plate, the U-shaped end 96 prepared by 50 μ L step (5) is taken with multichannel pipettor
Sample liquid after product Concentraton gradient solution and dilution adds to 96 hole ELISA Plate in step (6)
On；
(8) live 96 hole ELISA Plate with shrouding membrane cover, 96 hole ELISA Plate be positioned on ELISA Plate oscillator,
150～180rpm, incubated at room 20～60min；
(9) topple over the material in 96 hole ELISA Plate, with washing liquid working solution, ELISA Plate is carried out 3～5 times clearly
Wash；
(10) in the 96 every holes of hole ELISA Plate add enzyme substrate solution 100 μ L, hatch under the conditions of lucifuge 10～
20min；
(11) in the 96 every holes of hole ELISA Plate, add stop buffer 100 μ L, mix gently, utilize in 30min
The conventional light absorption value at spectrophotometric determination 450nm.
(12) with the concentration value of standard concentration gradient solution for transverse and longitudinal mark, with standard concentration gradient solution
Light absorption value at 450nm is that vertical coordinate makees standard curve, further according to standard curve and sample liquid
450nm at light absorption value calculate the concentration of homodimer HNL in sample.
The present invention has high flux, high specificity according to sandwich ELISA test (ELISA) technology and popularizes
Rate advantages of higher, has manufactured a kind of human neutrophil apolipoprotein homodimer (HNL) based on ELISA
Fast quantification device.Owing to have employed technique scheme, the present invention compared with prior art has at following two aspects
Prominent substantive distinguishing features and significantly progress.
First, the present invention is the feature formed by two identical monomer HNL according to HNL homodimer, with monomer
HNL compares, and HNL homodimer has the epitope that two sets are identical, and each HNL homodimer can be with two
Identical antibodies, and a monomer HNL can only be with a same antibodies；It addition, the different dimerization of HNL
Body is that a HNL molecule is combined with MMP9 molecule covalent, and therefore a HNL heterodimer also only one of which is identical
Epitope can only as a molecule antibodies.So the present invention uses identical solid-phase coating antibody and enzyme
Labeling antibody builds HNL ELISA and detects device, and this device specific detection goes out HNL homodimer, and for HNL
Monomer and heterodimer do not detect (Fig. 1).The present invention can be used for the disease with HNL homodimer as mark
The detection of (such as acute bacterial infection) and monitoring, therefore have huge laboratory research and clinical value.
Second, it is contemplated that conventional ELISA operating procedure is various, and test period is longer, and needs during clinical practice application
Quickly obtain the concentration information of target molecule, therefore the present invention by insolubilized antibody is coated concentration, reaction reagent formula,
The many factors such as enzyme labelled antibody concentration, incubation time are optimized, thus establish two-step method detection HNL homologous dimerization
The sandwich ELISA method of body, hatches during whole use and only needs two steps and cleaning only to need a step.So that filling with this
Put and have that preparation is simple, use the advantages such as quick, this device use only need the conventional ELISA testing time 1/4 to
1/3.This is another creative invention of the present invention.
Accompanying drawing explanation
Fig. 1 is that the proportioning device of one human neutrophil apolipoprotein (HNL) homodimer of the present invention can be special
The schematic diagram of property fast quantification HNL.Wherein 24 representatives are for a hole of the coated 96 hole ELISA Plate of antibody, and 25
Representing the anti-HNL antibody for being coated 96 hole ELISA Plate, 26 represent enzyme mark anti-HNL antibody, and 27 represent HNL homology
Dimer, 28 represent HNL monomer, and 29 represent the heterodimer that HNL Yu MMP-9 is formed；Described in 25 and 26
Anti-HNL antibody be same antibody, and the epi-position that only one of which is corresponding on HNL monomer；HNL monomer 28, HNL
Homodimer 27 and heterodimer 29 can be combined with the anti-HNL antibody 25 being coated on 24, but due to
28 and 29 only one of which can be specific binding with 25 epi-position, therefore can not be combined with 26, and HNL homologous dimerization
Body 27 is because having two identical epi-positions, therefore except one in addition to 25 are combined, it is also possible to resist with anti-HNL enzyme mark
Body 26 is specific binding, thus 26 utilize the enzyme catalysis enzyme substrate solution of institute's labelling on it to produce color products, and then
Spectrophotometer is utilized to obtain the absorbance of product under specific wavelength.
Fig. 2 is the standard of the proportioning device of one human neutrophil apolipoprotein (HNL) homodimer of the present invention
Curve.
Detailed description of the invention
The present invention will be further illustrated below by embodiment.The embodiment of the present invention is for being explained further of the present invention
It it not limitation of the invention.Concrete variation and retouching that design according to the present invention and essence are carried out belong to the present invention
Claimed scope.
Embodiment 1: the composition of the proportioning device of a kind of human neutrophil apolipoprotein homodimer
The present embodiment device comprises following reagent and material (being shown in Table 3).
Table 3: the composition of embodiment 1 device
| Sequence number | Title | Quantity |
| 1 | The coated 96 hole ELISA Plate of anti-HNL antibody | 1 piece (96 hole) |
| 2 | 5 times of sample diluting liquids | 1 bottle (80mL) |
| 3 | HNL homodimer standard substance mother solution | 1 pipe (300 μ L) |
| 4 | Enzyme mark anti-HNL antibody (HRP-anti-HNL antibody) | 1 pipe (5.5mL) |
| 5 | 25 times of washing liquids | 1 bottle of 35mL |
| 6-A | Tmb substrate solution A | 1 bottle (6mL) |
| 6-B | Tmb substrate solution B | 1 bottle (6mL) |
| 7 | 2mol/L H2SO4Stop buffer | 1 bottle (12mL) |
| 8 | Conventional orifice plate of the U-shaped end 96 | 1 piece (96 hole) |
| 9 | Shrouding film | 1 |
| 10 | Operation instructions | 1 |
Embodiment 2: the composition of the proportioning device of a kind of human neutrophil apolipoprotein homodimer
The present embodiment device comprises following reagent and material (being shown in Table 4).
Table 4: the composition of embodiment 2 device
| Sequence number | Title | Quantity |
| 1 | The coated 96 hole ELISA Plate of anti-HNL antibody | 1 piece (96 hole) |
| 2 | 5 times of sample diluting liquids | 1 bottle (80mL) |
| 3 | HNL homodimer standard substance mother solution | 1 pipe (300 μ L) |
| 4 | Enzyme mark anti-HNL antibody (ALP-anti-HNL antibody) | 1 pipe (5.5mL) |
| 5 | 25 times of washing liquids | 1 bottle (35mL) |
| 6-A | Alkaline phosphatase enzyme buffer solution | 1 bottle (15mL) |
| 6-B | NBT solution | 1 pipe (200 μ L) |
| 6-C | BCIP solution | 1 pipe (100 μ L) |
| 7 | 3mol/L NaOH | 1 bottle of 12mL |
| 8 | Conventional orifice plate of the U-shaped end 96 | 1 piece (96 hole) |
| 9 | Shrouding film | 1 |
| 10 | Operation instructions | 1 |
The anti-HNL antibody coated 96 of the proportioning device of 3 one kinds of human neutrophil apolipoprotein homodimers of embodiment
The preparation of hole ELISA Plate
As a example by the coated 96 hole ELISA Plate of anti-HNL antibody that embodiment 1 is comprised, illustrate that anti-HNL antibody is coated
The preparation process of 96 hole ELISA Plate.Specifically comprise the following steps that
(1) anti-HNL antibody is coated 96 hole ELISA Plate
Anti-HNL monoclonal antibody (ab23477 of Abcam company) is used 50mmol/L
Na2CO3-NaHCO3, the sodium carbonate buffer of pH 9.6 is diluted to 1 μ g/mL and makes antibody and be coated liquid；To
96 hole ELISA Plate (NuncFlat-bottom 96well plate) every hole adds 100 μ L
Antibody is coated liquid；4 DEG C overnight.
(2) sealase target
Abandon antibody and be coated liquid；Add 300 μ L confining liquids (confining liquid is for containing 2%BSA to the 96 every holes of hole ELISA Plate
(wt/vol) 50mmol/L Na2CO3-NaHCO3, the sodium carbonate buffer of pH 9.6)；Incubate for 37 DEG C
Educate 2h.
(3) immobilized enzyme target
Abandon fixative；Add 300 μ L fixatives (fixative is for containing 50% to the 96 every holes of hole ELISA Plate
(wt/vol) the 50mmol/L Na of sucrose2CO3-NaHCO3, the sodium carbonate buffer of pH=9.6；Room
The fixing 1h of temperature；
(4) ELISA Plate is sealed
Abandon fixative liquid, 25 DEG C of vacuum drying；ELISA Plate and bagged dryer are put into tinfoil paper is bag
Then evacuation, 4 DEG C of preservations.
Embodiment 4: the system of the enzyme mark anti-HNL antibody of the proportioning device of a kind of human neutrophil apolipoprotein homodimer
Standby
The present embodiment illustrates the system of enzyme mark anti-HNL antibody as a example by the HRP-anti-HNL antibody that embodiment 1 is comprised
Standby step.The present embodiment uses Over-voltage protection by HRP and anti-HNL monoclonal antibody (ab23477 of Abcam company)
Covalently bound make enzyme mark anti-HNL antibody.Specifically comprise the following steps that
(1) various buffer is prepared
A.0.1mol/L sodium metaperiodate (NaIO4) solution: weigh 241mg NaIO4It is dissolved in 10mL distilled water.
B.1mmol/L, sodium acetate (NaAc) buffer of pH=4.4: take 0.2mol/L NaAc (1.361 grams
/ 50mL) 3.7mL, 0.2mol/L acetic acid (HAc) solution (0.601mL/50mL) 6.3mL, add
Distilled water is to 2000mL.
C.0.2mol/L, pH9.5 carbonate buffer solution: Na2CO30.32 gram, NaHCO30.586 gram, add
Distilled water is to 50mL.
D.0.01mol/L, pH9.5 carbonate buffer solution: 0.2mol/L, pH9.5 carbonate buffer solution steams
Distilled water make 20 times of dilutions, 0.01mol/L, pH9.5 carbonate buffer solution.
E.4mg/mL sodium borohydride (NaBH4) solution: weigh NaBH during use44mg is dissolved in 1mL distilled water
In.
F.0.1mol/L, the PBS of pH7.4: NaCl 8g, KCl 0.2g, Na2HPO4.12H2O 3.58
g、KH2PO40.24g, distilled water dissolve and are settled to 100 milliliters.
G. enzyme labelled antibody preserves buffer: by the PBS of 0.1mol/L, pH7.4 distilled water diluting 10
Obtain the PBS of 0.01mol/L, pH7.4 again, add 0.2%BSA (wt/vol), 0.1%
Tween-20 (vol/vol) and 0.02%NaN3(wt/vol)。
(2) prepared by enzyme labelled antibody
A. weigh 5mg HRP (Sigma company, V900503) to be dissolved in 1mL distilled water.
B. the 0.1mol/L NaIO that 0.2mL newly joins is added4Solution, lucifuge stirring 20min under room temperature.
C. above-mentioned solution is loaded in bag filter, dialyses with the sodium-acetate buffer of 1mmol/L, pH=4.4,
4 DEG C overnight.
D. add 0.2mol/L, pH=9.5 carbonate buffer solution of 20 μ l, make the pH liter of above hydroformylation HRP
High to 9.5, add 1mL, 5mg/mL anti-HNL monoclonal antibody (ab23477 of Abcam company) immediately
In 0.01M carbonate buffer solution, room temperature lucifuge is gently mixed 2h.
E. the 4mg/mL NaBH that 0.1mL newly joins is added4Liquid, mixing, then put 4 DEG C, 2h.
F. above-mentioned solution liquid is loaded in bag filter, dialyse by the PBS solution of 0.1mol/L, 4 DEG C, overnight.
G. the antibody in bag filter is diluted in and preserves buffer containing enzyme labelled antibody.
H. enzyme labelled antibody concentration: HRP-anti-HNL amount of antibody (mg/mL)=(OD280nm-OD403nm × 0.3) is calculated
×0.62。
I. anti-for HRP-HNL antibody being configured to 1mg/mL, subpackage is saved in-20～-80 DEG C of environment；Will during use
HRP-anti-HNL antibody sample diluting liquid is diluted to 0.5 μ g/mL.
Embodiment 5: the use of the proportioning device of a kind of human neutrophil apolipoprotein homodimer
The specifically used step of this device is now described as a example by the use of embodiment 1 device.
(1) take out the solution of above-mentioned preparation, the coated 96 hole ELISA Plate of antibody etc., be positioned over room temperature environment balance；
(2) 5 times of sample diluting liquids and 25 times of washing liquid distilled water are diluted 5 times and 25 times respectively, make sample
Diluent working solution and washing liquid working solution.
(3) biological sample such as urine sample diluting liquid working solution is diluted 25 times, the sample liquid after being diluted.
(4) homodimer HNL standard substance mother solution sample diluting liquid working solution is carried out 2 times of method gradient dilutions,
Make standard concentration gradient solution.
(5) by each 100 μ L of sample liquid after above-mentioned gradient concentration standard concentration gradient solution and dilution to U-shaped
The end 96 orifice plate.
(6) 50 μ L HRP-anti-HNL antibody are added to 96 hole ELISA Plate.
(7) with multichannel pipettor take the standard substance gradient in orifice plate of the U-shaped end 96 prepared by 50 μ L step (5),
Corresponding contrast solution and sample are in 96 hole ELISA Plate in step (6).
(8) live 96 hole ELISA Plate with shrouding membrane cover, ELISA Plate be positioned on ELISA Plate oscillator, 180rpm,
Incubated at room 30min.
(9) topple over the material in ELISA Plate, with washing liquid working solution, ELISA Plate is carried out 3 times and clean, the most clearly
After washing, ELISA Plate is tipped upside down on 10s on absorbent filter.
(10) before cleaning terminates, by tmb substrate solution A and tmb substrate solution B respectively by reverse reagent bottle
Mixing, takes equal-volume solution A and tmb substrate working solution is made in solution B mixing.
(11) add 100 μ L tmb substrate working solutions to 96 hole ELISA Plate, cover shrouding film and masking foil, hatch
15min；
(12) 100 μ L, 2mol/L H are added2SO4Stop buffer, mix gently in 30S, 30min and utilize
The spectrophotometer of routine is reference, the light absorption value read under 450nm wavelength with 540nm wavelength.
(13) with concentration as abscissa, light absorption value draw standard curve for vertical coordinate, public according to standard curve Trendline
Formula calculates HNL concentration in sample.
Under the 450nm wavelength that each concentration homodimer HNL standard substance that table 5 records for embodiment 5 device are corresponding
Absorbance, draw reference standard curve (Fig. 2) according to table 5
Table 5: the suction at 450nm wavelength that variable concentrations homodimer HNL standard concentration gradient solution is corresponding
Shading value
By table 5 can draw the quantitative homodimer of this device concentration can as little as 0.1ng/mL, permissible in conjunction with Fig. 1
The range of linearity drawing the quantitative homodimer of this device is 0.1ng.mL～6.4ng/mL.
Batch interior difference CV meansigma methods of the present embodiment device is 4.31% (scope 0.54%～7.40%, n=12), difference between batch
Different CV meansigma methods is 7.96% (scope is 1.69%～12.50%, n=3).
By to known homologous dimerization bulk concentration (C0) urine in add concentration known (C1) homodimer HNL,
Monomer HNL and MMP9/HNL, utilizes the present embodiment to obtain the measured value concentration of the homodimer HNL in urine solution
(C2)；Theoretical value concentration is C3.According to formula (1) response rate 1 (%)=C3÷C2× 100% and formula (2) reclaim
Rate 2 (%)=(C2-C0) ÷ C1 × 100% is calculated the above molecule response rate (table 6) in urine sample.
Table 6:HNL homodimer, HNL monomer and the MMP9/HNL response rate in urine
Table 6 data show the HN homodimer L in this device energy specific quantification urine specimen, and mono-to HNL
Body and HNL heterodimer detect the most hardly.Therefore deduce that this method has the highest specificity and sensitive
Degree.
Claims (8)
1. the proportioning device of a human neutrophil Apolipoprotein H NL homodimer, it is characterised in that: by 5
Times sample diluting liquid, 25 times of washing liquids, HNL homodimer standard substance mother solution, anti-HNL antibody coated 96
Hole ELISA Plate, enzyme mark anti-HNL antibody-solutions, orifice plate of the U-shaped end 96, shrouding film, enzyme substrate solution and termination
Liquid forms, the anti-HNL that the coated 96 hole ELISA Plate of described anti-HNL antibody and enzyme mark anti-HNL antibody are used
Antibody is identical, for monoclonal antibody；The enzyme of described enzyme mark anti-HNL antibody is horseradish peroxidase or alkali
Acid phosphatase；Described proportioning device realizes the fast quantification of HNL homodimer, described two-step method through two-step method
Hatching process during referring to the whole use of device only to need twice, process of hatching for described twice refers to for the first time by enzyme
Mark after anti-HNL antibody joins the coated 96 hole ELISA Plate of anti-HNL antibody and rapidly join pre-prepd standard
Product Concentraton gradient solution and the sample liquid after dilution hatch 20～60min, hatch process second described twice
Secondary refer to hatch for the first time after coated for anti-HNL antibody 96 hole ELISA Plate are added after washing liquid working solution cleans again
Enter addition stop buffer after enzyme substrate solution hatches 10～20min；Described pre-prepd standard concentration gradient
Solution refers to that HNL homodimer standard substance mother solution is made after 2 times of method dilutions, described sample after dilution
Liquid refer to through sample diluting liquid working solution dilute after body fluid sample, described body fluid sample include urine, serum,
Blood plasma, cerebrospinal fluid or saliva；
5 times of sample diluting liquids are diluted 5 times with deionized water and obtains sample diluting liquid working solution；25 times are washed
Liquid deionized water dilutes 25 times and makes washing liquid working solution.
The quantitative dress of a kind of human neutrophil Apolipoprotein H NL homodimer the most as claimed in claim 1
Put, it is characterised in that: described anti-HNL monoclonal antibody is the anti-HNL monoclonal antibody of commercialization or often passes through
Anti-HNL monoclonal antibody that rule method is prepared with HNL monomer or HNL homodimer immune animal or gene work
Anti-HNL monoclonal antibody prepared by Cheng Fangfa, the epitope that described anti-HNL monoclonal antibody is identified is at list
Only one of which on body HNL.
The proportioning device of a kind of human neutrophil Apolipoprotein H NL homodimer the most as claimed in claim 1,
It is characterized in that: the coated 96 hole ELISA Plate of anti-HNL antibody are to be prepared by following steps,
(1) anti-HNL antibody is coated 96 hole ELISA Plate
By anti-HNL antibody 50mmol/L Na2CO3-NaHCO3, the sodium carbonate buffer of pH 9.6 be diluted to 1～
5 μ g/mL make antibody and are coated liquid；In the 96 every holes of hole ELISA Plate, add 100 μ L antibody be coated liquid, 37 DEG C
Hatch 2h, incubated at room 4h or 4 DEG C overnight；
(2) sealase target
Abandon antibody and be coated liquid, state addition 200～300 μ L confining liquids in the 96 every holes of hole ELISA Plate then up,
Confining liquid is the 50mmol/L Na containing 2%BSA (wt/vol)2CO3-NaHCO3, the sodium carbonate of pH 9.6 delays
Rush liquid；Hatch 2h, incubated at room 4h or 4 DEG C overnight for 37 DEG C；
(3) immobilized enzyme target
Abandoning confining liquid, then add 200～300 μ L fixatives in the 96 every holes of hole ELISA Plate, fixative is
Containing 50% (wt/vol) sucrose, 50% (wt/vol) trehalose or 25% (wt/vol) sucrose and 25% (wt/vol)
The 50mmol/L Na of trehalose2CO3-NaHCO3, the sodium carbonate buffer of pH 9.6, room temperature fix 30min～
2h；
(4) ELISA Plate is sealed
Abandoning fixative, room temperature is dried or 25～30 DEG C of vacuum drying；ELISA Plate and bagged dryer are put into stannum
Then evacuation, 4 DEG C of preservations in foil paper bag.
The proportioning device of a kind of human neutrophil Apolipoprotein H NL homodimer the most as claimed in claim 1,
It is characterized in that: 5 times of sample diluting liquids for containing 1%BSA (wt/vol), 0.5%Tween-20 (vol/vol),
0.25% cetyl trimethylammonium bromide (CTAB) (wt/vol) and 0.1%NaN3(wt/vol) phosphoric acid
Root molar concentration is the PBS of 50mmol/L, pH 7.4.
The proportioning device of a kind of human neutrophil Apolipoprotein H NL homodimer the most as claimed in claim 1,
It is characterized in that: standard substance mother solution for containing 0.1%BSA (wt/vol), 0.1%Tween-20 (vol/vol),
0.05% cetyl trimethylammonium bromide (CTAB) (wt/vol), 0.02%NaN3(wt/vol) and 12.8
The PBS that phosphate radical molar concentration is 10mmol/L, pH 7.4 of μ g/L HNL homodimer.
The proportioning device of a kind of human neutrophil Apolipoprotein H NL homodimer the most as claimed in claim 1,
It is characterized in that: enzyme mark anti-HNL antibody is by horseradish peroxidase enzyme HRP or alkaline phosphatase covalent labeling
On anti-HNL antibody, enzyme mark anti-HNL antibody-solutions is containing 1%BSA (wt/vol), 0.1%NaN3(wt/vol)
The PBS that phosphate radical molar concentration is 50mmol/L, pH7.4 with 0.2～2 μ g/mL enzyme mark anti-HNL antibody
Buffer.
The proportioning device of a kind of human neutrophil Apolipoprotein H NL homodimer the most as claimed in claim 1,
It is characterized in that: 25 times of washing liquids are that the phosphate radical molar concentration containing 2.5%Tween-20 (vol/vol) is
The PBS of 250mmol/L, pH 7.4.
8. a kind of human neutrophil Apolipoprotein H NL homodimer as described in claim 1～7 any one
Proportioning device, it is characterised in that: specifically used step is as follows,
(1) take out each solution and the coated 96 hole ELISA Plate of antibody, be positioned over room temperature environment；
(2) 5 times of sample diluting liquids and 25 times of washing liquid distilled water are diluted 5 times and 25 times respectively, make sample
Product diluent working solution and washing liquid working solution；
(3) biological sample sample diluting liquid working solution is diluted, the sample liquid after being diluted；
(4) homodimer HNL standard substance mother solution sample diluting liquid working solution is carried out 2 times of gradient dilutions,
Make standard concentration gradient solution；
(5) sample liquid after above-mentioned 100 μ L standard concentration gradient solutions and dilution is added to orifice plate of the U-shaped end 96；
(6) 50 μ L enzyme mark anti-HNL antibody-solutions are added to the coated 96 hole ELISA Plate of anti-HNL antibody；
(7) standard concentration in the every hole of orifice plate, the U-shaped end 96 prepared by 50 μ L step (5) is taken with multichannel pipettor
Sample liquid after gradient solution and dilution adds in 96 hole ELISA Plate in step (6)；
(8) live 96 hole ELISA Plate with shrouding membrane cover, 96 hole ELISA Plate be positioned on ELISA Plate oscillator, 150～
180rpm, incubated at room 20～60min；
(9) topple over the material in 96 hole ELISA Plate, with washing liquid working solution, ELISA Plate is carried out 3～5 times and clean；
(10) in the 96 every holes of hole ELISA Plate, add enzyme substrate solution 100 μ L, under the conditions of lucifuge, hatch 10～20
min；
(11) in the 96 every holes of hole ELISA Plate, add stop buffer 100 μ L, mix gently, in 30min, utilize routine
Spectrophotometric determination 450nm at light absorption value；
(12) with the concentration value of standard concentration gradient solution for transverse and longitudinal mark, with standard concentration gradient solution 450
Light absorption value at nm is that vertical coordinate makees standard curve, further according to standard curve and the 450nm of sample liquid
The light absorption value at place calculates the concentration of homodimer HNL in sample.
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