CN206002550U - A kind of proportioning device of human neutrophil apolipoprotein homodimer - Google Patents

Abstract

A kind of proportioning device of human neutrophil apolipoprotein homodimer, belongs to technical field of immunological detection.It is made up of the container of the container of 5 times of sample diluting liquids of storage, the container of 25 times of washing liquids of storage, the container of storage HNL homodimer standard substance mother solution, coated 96 hole elisa Plates of anti-HNL antibody, the container of storage enzyme mark anti-HNL antibody-solutions, U-shaped bottom 96 orifice plate, shrouding film, the container of storage enzyme substrate solution and storage terminate liquid.It is same antibody that ELISA Plate is coated anti-HNL antibody and enzyme mark anti-HNL antibody, thus it is quantitative simply to achieve HNL homodimer.Additionally, incubation only needs two steps and cleaning only to need a step, thus significantly shorten detection time during this device uses.Therefore, to have the advantages that to prepare simple, easy to use and testing time short for this device.This device can be used for the detection of disease (as acute bacterial infection) and the monitoring with HNL homodimer as mark, therefore has huge laboratory research and clinical value.

Description

A kind of proportioning device of human neutrophil apolipoprotein homodimer

Technical field

This utility model belongs to technical field of immunological detection and in particular to a kind of human neutrophil apolipoprotein (HNL) the fast quantification device of homodimer.

Background technology

It is thin that human neutrophil lipocalin protein (human neutrophil lipocalin, HNL) is also referred to as neutral grain Born of the same parents' gelatinase associated lipocalin (neutrophil gelatinase associated lipocalin, NGAL), and Claim Lipocalin-2 (Lipocalin-2), be a kind of lipocalin protein (J.Biol.Chem.1993,268:10425- 10432；Scand.J.Clin.Lab.Invest.1994,54:365-376).HNL is with monomer, homodimer and heterodimeric The different kinds of molecules forms such as body (monomer and MMP9 molecule are formed) exist, and different molecular form HNL is relevant from different pathological processes. As monomer HNL may have of a relatively high dependency (Clin.Chim.Acta.2009,403 with acute injury of kidney: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 of China:46-47), heterodimer HNL may with swollen Tumor aggressive is relevant.Cai Linjun et al. research finds homodimer HNL reflection urinary tract infection situation (Clin.J.Am.Soc.Nephrol.2010)；Additionally, Venge Per and Xu S.Y. et al. research finds homodimer HNL To differentiation diagnosing acute bacterium infection and virus infected with very high specificity and sensitivity (J.Immunol.Methods 2015,424:85-90；J.Clin.Lab.Invest.1995,55:125-131).Other molecular forms HNL relatively, homology two Aggressiveness has higher specificity and sensitivity to diagnosing acute bacterium infection it may be possible to because participate in acute bacterial infection process The HNL of neutrophilic granulocyte secretion is mainly homodimer (Clin.Chim.Acta.2009,403:121-125).

Existing multiple HNL assay methods are open at present, such as RIA (international monopoly WO/1995/029404A1 and Chinese ZL 200980155194 etc.), ELISA (European patent EP 2225268 and Chinese patent ZL200980155194 etc.), Western- Blotting, latex immunoturbidimetry (Chinese patent ZL 201410431182), chemiluminescence immunoassay (Chinese patent Application number 201510184818), immuno-chromatographic test paper strip technology (Chinese patent ZL201220323587, ZL201420423962, ZL201220392399, ZL201220497401, ZL201220323586 and ZL201220497401) etc..Any of the above HNL is fixed Amount method has respective pluses and minuses and corresponding uses, but in addition to Western-blotting can distinguish different molecular form HNL, above-mentioned HNL quantitative approach is all unable to specific quantification homodimer HNL, thus affecting this albumen as some diseases such as acute bacterial The diagnosis capability of infection.Although, Western-blotting can distinguish different molecular form HNL, due to this technology itself The drawbacks limit practical application of the method.As limited and accurate in complex operation, test period length, each analysis sample size Really difficult quantitation etc..

To sum up, current laboratory research and clinical practice possible in the future are badly in need of setting up quick, specific quantification homology two The device of aggressiveness HNL and correlation method.

Utility model content

In order to realize the specific quantification of human neutrophil Apolipoprotein H NL homodimer molecule, this utility model Design and have developed a kind of fast quantification device of the HNL homodimer based on sandwich ELISA techniques.This device has system Preparation Method simply (only needs a kind of anti-NHL antibody), using quick (incubation process only has two steps), test limit low (0.1ng/mL, energy Meet the detection of HNL in body fluid sample) etc. excellent properties, in the disease early diagnosiss laboratory research such as acute bacterial infection with face There is very big application potential in bed inspection field.

In order to realize above-mentioned target, this utility model adopts the following technical scheme that：

A kind of proportioning device of human neutrophil apolipoprotein (HNL) homodimer it is characterised in that：By storage 5 The container of times sample diluting liquid, the container of 25 times of washing liquids of storage, the container of storage HNL homodimer standard substance mother solution, anti-HNL Coated 96 hole elisa Plates of antibody, the container of storage enzyme mark anti-HNL antibody-solutions, U-shaped bottom 96 orifice plate, shrouding film, storage enzyme bottom The container composition of the container of thing solution and storage terminate liquid, coated 96 hole elisa Plates of described anti-HNL antibody and the anti-HNL of enzyme mark resist The anti-HNL antibody that body is used is identical；Described anti-HNL antibody is monoclonal antibody, and described anti-HNL monoclonal antibody is business Anti- HNL monoclonal antibody (ab63929 the or P＆M Venge Diagnostics Development as Abcam company of product HNL mab 697 of company etc.) or pass through conventional method with the anti-HNL of HNL monomer or the preparation of HNL homodimer immune animal Monoclonal antibody or the anti-HNL monoclonal antibody of gene engineering method preparation, the antigen that described anti-HNL monoclonal antibody is identified Epi-position only one of which on monomer HNL；The enzyme of described enzyme mark anti-HNL antibody institute labelling can be horseradish peroxidase, alkalescence 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 During individual use, incubation process only needs twice, and described incubation process first time twice refers to be added to anti-for enzyme mark HNL antibody Pre-prepd standard concentration gradient solution and the sample after dilution is rapidly joined after coated 96 hole elisa Plates of anti-HNL antibody Product liquid is incubated 20～60min, the described process of incubation twice refer to for the second time be incubated for the first time after by coated for anti-HNL antibody 96 holes ELISA Plate adds enzyme substrate solution after the cleaning of washing liquid working solution and is incubated after 10～20min and adds terminate liquid；Described accurate in advance Standby standard concentration gradient solution refers to that HNL homodimer standard substance mother solution is made after diluting through 2 times of methods, described through dilution Sample liquid afterwards refers to the body fluid sample after diluting through sample diluting liquid working solution, and described body fluid sample includes urine, serum, blood Slurry, cerebrospinal fluid, saliva etc..

5 times of sample diluting liquid deionized waters are diluted 5 times of acquisition sample diluting liquid working solutions；By 25 times of washing liquids spend from Sub- water dilutes 25 times and makes washing liquid working solution.

This utility model device composition, method of preparation and use are as follows：

1) this utility model device composition

Table 1 forms for this utility model device, Fig. 1 and Fig. 2 is the schematic diagram of each ingredient of this device.

Table 1：This utility model device forms

Sequence number	Title	Quantity
			1	Coated 96 hole elisa Plates 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	Terminate liquid	1 bottle of 12mL
8	Conventional U-shaped bottom 96 orifice plate	1 piece
			9	Shrouding film	1

2) preparation method

A) preparation of coated 96 hole elisa Plates of anti-HNL antibody

(1) anti-HNL antibody is coated 96 hole elisa Plates

By anti-HNL antibody 50mmol/L Na₂CO₃-NaHCO₃, the sodium carbonate buffer of pH 9.6 is diluted to 1～5 μ g/ ML makes antibody and is coated liquid；To the every hole of 96 hole elisa Plates add 100 μ L antibody be coated liquid, 37 DEG C incubation 2h or incubated at room 4h or 4 DEG C overnight.

(2) close ELISA Plate

Abandon antibody and be coated liquid, state then up and add 200～300 μ L confining liquids in the every hole of 96 hole elisa Plates (confining liquid is 50mmol/L Na containing 2%BSA (wt/vol)₂CO₃-NaHCO₃, the sodium carbonate buffer of pH 9.6)；37 DEG C of incubation 2h or room 4h or 4 DEG C of temperature incubation is overnight.

(3) fixing ELISA Plate

Abandon confining liquid, (fixative is containing 50% then to add 200～300 μ L fixatives in the every hole of 96 hole elisa Plates (wt/vol) sucrose, 50% (wt/vol) trehalose or 25% (wt/vol) sucrose and 25% (wt/vol) trehalose 50mmol/L Na₂CO₃-NaHCO₃, the sodium carbonate buffer of pH 9.6, room temperature fixes 30min～2h.

(4) seal ELISA Plate

Abandon fixative, room temperature is dried or 25～30 DEG C of vacuum drying；ELISA Plate and bagged dryer are put into tinfoil paper paper bag Interior and then evacuation, 4 DEG C of preservations；

The anti-HNL antibody coated elisa plate prepared by above method, through 37 DEG C of accelerated tests, can preserve 32 days.Root According to A Luoniwusi formula (Arrhenius equation), the ELISA Plate of this method preparation can preserve sky under the conditions of 4 DEG C Number=32 × 48 days=1536 days.

B) preparation of 5 times of sample diluting liquids

It is containing 1%BSA (wt/vol), 0.5%Tween-20 (vol/vol), 0.25% cetyl trimethyl bromination Ammonium (CTAB) (wt/vol) and 0.1%NaN₃(wt/vol) phosphate radical molar concentration is the PBS buffering of 50mmol/L, pH 7.4 Liquid.

C) preparation of standard substance mother solution

It is containing 0.1%BSA (wt/vol), 0.1%Tween-20 (vol/vol), 0.05% cetyl trimethyl bromine Change ammonium (CTAB) (wt/vol), 0.02%NaN₃(wt/vol) the phosphate radical molar concentration of and 12.8 μ g/L homodimer HNL PBS for 10mmol/L, pH 7.4.

D) preparation of enzyme mark anti-HNL antibody-solutions

Enzyme mark anti-HNL antibody is will by conventional method (ab102850, ab102890, ab102887 of Abcam etc.) The enzyme molecule covalent labeling such as horseradish peroxidase (HRP) or alkali phosphatase (ALP) is on anti-HNL antibody.

Enzyme mark anti-HNL antibody-solutions are containing 1%BSA (wt/vol), 0.1%NaN₃(wt/vol) and 0.2～2 μ g/mL The phosphate radical molar concentration of enzyme mark anti-HNL antibody is the PBS of 50mmol/L, pH7.4.

E) preparation of 25 times of washing liquids

It is that the PBS that the phosphate radical molar concentration containing 2.5%Tween-20 (vol/vol) is 250mmol/L, pH 7.4 delays Rush liquid.

F) preparation of enzyme substrate solution and terminate liquid

The enzyme of corresponding enzyme mark anti-HNL antibody institute labelling adopts different enzyme substrate solutions and terminate liquid.This system is Disclosed conventional method.As table 2.

Table 2：The composition of different system enzyme labelled antibodies, enzyme substrate solution and corresponding terminate liquid

3) using method

The specifically used step of device is as follows：

(1) take out solution, coated 96 hole elisa Plates of antibody of above-mentioned preparation etc., 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 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 to dilute (blood Cleer and peaceful blood plasma generally 50 times of dilution, urine generally dilute 25 times, saliva and usual 5～10 times of dilutions of cerebrospinal fluid), after being diluted Sample liquid；

(4) homodimer HNL standard substance mother solution sample diluting liquid working solution is carried out 2 times of gradient dilutions, make mark Quasi- product Concentraton gradient solution；

(5) sample liquid after above-mentioned 100 μ L standard concentration gradient solutions and dilution is added to U-shaped bottom 96 orifice plate；

(6) anti-for 50 μ L enzyme marks HNL antibody-solutions are added to coated 96 hole elisa Plates of anti-HNL antibody；

(7) the standard concentration gradient in the every hole of U-shaped bottom 96 orifice plate taking 50 μ L step (5) to prepare with multichannel pipettor is molten Sample liquid after liquid and dilution adds in 96 hole elisa Plates in step (6)；

(8) use shrouding membrane cover to live 96 hole elisa Plates, 96 hole elisa Plates are positioned on ELISA Plate oscillator, 150～ 180rpm, incubated at room 20～60min；

(9) topple over the material in 96 hole elisa Plates, with washing liquid working solution, ELISA Plate is carried out with 3～5 cleanings；

(10) add enzyme substrate solution 100 μ L in the every hole of 96 hole elisa Plates, under the conditions of lucifuge, be incubated 10～20min；

(11) add terminate liquid 100 μ L in the every hole of 96 hole elisa Plates, gently mix, using conventional light splitting in 30min Light absorption value at photometric determination 450nm.

(12) with the concentration value of standard concentration gradient solution for transverse and longitudinal mark, at standard concentration gradient solution 450nm Light absorption value make standard curve for vertical coordinate, calculate sample further according to the light absorption value at the 450nm of standard curve and sample liquid The concentration of middle homodimer HNL.

This utility model is tested (ELISA) technology according to sandwich ELISA and is had high flux, high specificity and general And rate high the advantages of, manufactured a kind of the quickly fixed of human neutrophil apolipoprotein homodimer (HNL) based on ELISA Amount device.Due to employing technique scheme, this utility model compared with prior art has prominent at following two aspects Substantive distinguishing features and significantly improving.

First, this utility model is the feature being formed by two identical monomer HNL according to HNL homodimer, with list Body HNL compares, and HNL homodimer has two sets of identical epitopes, and each HNL homodimer can be with two complete phases Same antibodies, and a monomer HNL can only be with same antibodies；In addition, HNL heterodimer is a HNL dividing Son is combined with MMP9 molecule covalent, and also only one of which identical epitope can only be with one point for therefore one HNL heterodimer The same antibodies of son.So this utility model adopts identical solid-phase coating antibody and enzyme labelled antibody to build HNL ELISA Detection means, this device specific detection goes out HNL homodimer, and does not then detect (figure for HNL monomer and heterodimer 3).This utility model can be used for the detection of disease (as acute bacterial infection) and the monitoring with HNL homodimer as mark, Therefore there is huge laboratory research and clinical value.

Second it is contemplated that conventional ELISA operating procedure is various, and test period is longer, and needs fast during clinical practice application Speed obtains the concentration information of target molecule, and therefore this utility model by being coated concentration, reaction reagent formula, enzyme to insolubilized antibody The many factors such as labeling antibody concentration, incubation time are optimized, thus establishing two-step method to detect the sandwich of HNL homodimer ELISA method, incubation during entirely using only needs two steps and cleaning only to need a step.Thus there is preparation letter using this device Single, using quick the advantages of, this device is using only needing the 1/4 to 1/3 of the conventional ELISA testing time.This is of the present utility model Another creative invention.

Brief description

Fig. 1：For a kind of this utility model human neutrophil apolipoprotein (HNL) homodimer proportioning device anti- Coated 96 hole elisa Plates of HNL antibody, conventional U-shaped bottom 96 orifice plate and shrouding film schematic diagram.20 represent anti-HNL antibody coated 96 Hole elisa Plates, are made up of 21 (96 orifice plate supports) and 22 (being coated the hole of anti-HNL antibody), altogether 12 row, each column 8 hole, Kong Weiping Bottom, is the place of sandwich ELISA reaction；30 represent conventional U-shaped bottom 96 orifice plate, and wherein 31 is 96 orifice plate supports, and 32 is U-shaped hole, 12 row altogether, each column 8 hole, hole is U-shaped bottom, for the preparation of the sample liquid after standard concentration gradient solution and dilution；40 are Shrouding film, has viscosity, can be pasted onto 20 surfaces, for preventing the evaporation of liquid inside during 20 incubation.

Fig. 2 is needed for a kind of proportioning device of this utility model human neutrophil apolipoprotein (HNL) homodimer Various solution storage solution schematic diagrams.51 containers representing 5 times of sample diluting liquids of storage, are transparent plastic bottle；52 representatives The container of 25 times of washing liquids of storage, is transparent plastic bottle；53 containers representing storage enzyme mark anti-HNL antibody-solutions, are transparent Plastic bottle；54 containers representing storage HNL homodimer standard substance mother solution, are transparent plastic bottle；55 and 56 represent storage The container of enzyme substrate solution, is opaque plastic bottle, and the number of 55 and 56 containers is true according to the composition of table 2 enzyme substrate solution Fixed；57 is the container of storage terminate liquid, is transparent plastic bottle.

Fig. 3 is that a kind of proportioning device of this utility model human neutrophil apolipoprotein (HNL) homodimer can be special The schematic diagram of different in nature fast quantification HNL.Wherein 24 represent a hole for coated 96 hole elisa Plates of antibody, and 25 represent and are used for It is coated the anti-HNL antibody of 96 hole elisa Plates, 26 represent enzyme mark anti-HNL antibody, 27 represent HNL homodimer, 28 to represent HNL mono- Body, 29 represent the heterodimer that HNL and MMP-9 is formed；Anti- HNL antibody described in 25 and 26 is same antibody, and mono- in HNL The corresponding epi-position of only one of which on body；HNL monomer 28, HNL homodimer 27 and heterodimer 29 can be coated on 24 On anti-HNL antibody 25 combine, but due to 28 and 29 only one of which can with 25 specific binding epi-position, therefore can not with 26 knot Close, and HNL homodimer 27 be because have two identical epi-positions, therefore except one with 25 are combined in addition to, can also with resist HNL enzyme labelled antibody 26 specifically binds, thus 26 produce color products using the enzyme catalysiss enzyme substrate solution of institute's labelling thereon, enters And utilize spectrophotometer to obtain the absorbance of product under specific wavelength.

Fig. 4 is a kind of mark of the proportioning device of this utility model human neutrophil apolipoprotein (HNL) homodimer Directrix curve.

Specific embodiment

This utility model will be further illustrated with embodiment below.This utility model embodiment is for of the present utility model It is explained further rather than to restriction of the present utility model.According to design of the present utility model and the concrete variation that carries out of essence and Retouching belongs to the scope that this utility model claims.

Embodiment 1：A kind of composition of the proportioning device 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	Coated 96 hole elisa Plates 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 H2SO4Terminate liquid	1 bottle (12mL)
			8	Conventional U-shaped bottom 96 orifice plate	1 piece (96 hole)
9	Shrouding film	1
			10	Operation instructions	1

Embodiment 2：A kind of composition of the proportioning device 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	Coated 96 hole elisa Plates 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 U-shaped bottom 96 orifice plate	1 piece (96 hole)
			9	Shrouding film	1
10	Operation instructions	1

A kind of anti-HNL antibody of the proportioning device of human neutrophil apolipoprotein homodimer of embodiment 3 is coated The preparation of 96 hole elisa Plates

Anti- HNL antibody coated 96 taking coated 96 hole elisa Plates of anti-HNL antibody that embodiment 1 is comprised as a example to be described The preparation process of hole elisa Plates.Comprise the following steps that：

(1) anti-HNL antibody is coated 96 hole elisa Plates

Anti- HNL monoclonal antibody (ab23477 of Abcam company) is used 50mmol/LNa₂CO₃-NaHCO₃, pH's 9.6 Sodium carbonate buffer is diluted to 1 μ g/mL to be made antibody and is coated liquid；To 96 hole elisa Plates (Nuncflat-bottom 96well plate) every hole adds 100 μ L antibody and is coated liquid；4 DEG C overnight.

(2) close ELISA Plate

Abandon antibody and be coated liquid；(confining liquid is containing 2%BSA (wt/vol) to add 300 μ L confining liquids to the every hole of 96 hole elisa Plates 50mmol/L Na₂CO₃-NaHCO₃, the sodium carbonate buffer of pH 9.6)；37 DEG C of incubation 2h.

(3) fixing ELISA Plate

Abandon fixative；(fixative is the sucrose containing 50% (wt/vol) to add 300 μ L fixatives to the every hole of 96 hole elisa Plates 50mmol/L Na₂CO₃-NaHCO₃, the sodium carbonate buffer of pH=9.6；Room temperature fixes 1h；

(4) seal ELISA Plate

Abandon fixative liquid, 25 DEG C of vacuum drying；It is in bag and then evacuation that ELISA Plate and bagged dryer are put into tinfoil paper, 4 DEG C of preservations.

Embodiment 4：A kind of enzyme mark anti-HNL antibody of the proportioning device of human neutrophil apolipoprotein homodimer Preparation

The present embodiment to illustrate the preparation step of enzyme mark anti-HNL antibody taking the HRP- anti-HNL antibody that embodiment 1 is comprised as a example Suddenly.The present embodiment adopts Over-voltage protection that with anti-HNL monoclonal antibody (ab23477 of Abcam company), HRP is covalently attached system Become enzyme mark anti-HNL antibody.Comprise the following steps that：

(1) various buffer are prepared

A.0.1mol/L sodium metaperiodate (NaIO₄) solution:Weigh 241mg NaIO₄It is dissolved in 10mL distilled water.

B.1mmol/L, Sodium Acetate Trihydrate (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, plus distilled water is to 2000mL.

C.0.2mol/L, pH9.5 carbonate buffer solution：Na₂CO₃0.32 gram, NaHCO₃0.586 gram, plus distilled water is extremely 50mL.

D.0.01mol/L, pH9.5 carbonate buffer solution：0.2mol/L, pH9.5 carbonate buffer solution makees 20 with distilled water Times dilute, 0.01mol/L, pH9.5 carbonate buffer solution.

E.4mg/mL sodium borohydride (NaBH₄) solution：NaBH is weighed during use₄4mg is dissolved in 1mL distilled water.

F.0.1mol/L, the PBS of pH7.4：NaCl 8g、KCl 0.2g、Na₂HPO₄.12H₂O 3.58g、KH₂PO₄ 0.24g, distillation water dissolution are settled to 100 milliliters.

G. enzyme labelled antibody preserves buffer：The PBS distilled water diluting 10 of 0.1mol/L, pH7.4 is obtained again The PBS of 0.01mol/L, pH7.4, add 0.2%BSA (wt/vol), 0.1%Tween-20 (vol/vol) and 0.02%NaN₃(wt/vol).

(2) enzyme labelled antibody preparation

A. weigh 5mg HRP (Sigma company, V900503) to be dissolved in 1mL distilled water.

B. add the 0.1mol/L NaIO that 0.2mL newly joins₄Solution, lucifuge stirring 20min under room temperature.

C. above-mentioned solution is loaded in bag filter, with the sodium-acetate buffer dialysis of 1mmol/L, pH=4.4,4 DEG C overnight.

D. plus 20 μ l 0.2mol/L, pH=9.5 carbonate buffer solution, make the pH of above hydroformylation HRP be increased to 9.5, stand Add in 1mL, 5mg/mL anti-HNL monoclonal antibody (ab23477 of Abcam company) 0.01M carbonate buffer solution, room temperature is kept away Light is gently mixed 2h.

Plus the 4mg/mL NaBH that newly joins of 0.1mL e.₄Liquid, mixes, then puts 4 DEG C, 2h.

F. above-mentioned solution liquid is loaded in bag filter, with the dialysis of 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. calculate enzyme labelled antibody concentration：HRP- anti-HNL amount of antibody (mg/mL)=(OD280nm-OD403nm × 0.3) × 0.62.

I. anti-for HRP- HNL antibody is configured to 1mg/mL, subpackage is saved in -20～-80 DEG C of environment；During use, HRP- is resisted HNL antibody sample diluting liquid is diluted to 0.5 μ g/mL.

Embodiment 5：A kind of use of the proportioning device of human neutrophil apolipoprotein homodimer

The specifically used step of this device now taking the use of embodiment 1 device is as a example described.

(1) take out solution, coated 96 hole elisa Plates of antibody of above-mentioned preparation 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 diluting liquid Working solution and washing liquid working solution.

(3) biological sample such as urine is diluted 25 times with sample diluting liquid working solution, 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 bottom 96 hole Plate.

(6) anti-for 50 μ L HRP- HNL antibody is added to 96 hole elisa Plates.

(7) the standard substance gradient in U-shaped bottom 96 orifice plate taking 50 μ L step (5) to prepare with multichannel pipettor, corresponding comparison In solution and sample 96 hole elisa Plates to step (6).

(8) use shrouding membrane cover to live 96 hole elisa Plates, ELISA Plate is positioned on ELISA Plate oscillator, 180rpm, room temperature is incubated Educate 30min.

(9) topple over the material in ELISA Plate, with washing liquid working solution, ELISA Plate is carried out with 3 cleanings, after last cleaning ELISA Plate is tipped upside down on 10s on absorbent filter.

(10) before cleaning terminates, tmb substrate solution A and tmb substrate solution B are mixed by reverse reagent bottle respectively, Equal-volume solution A and solution B is taken to mix and make tmb substrate working solution.

(11) add 100 μ L tmb substrate working solutions to 96 hole elisa Plates, cover shrouding film and masking foil, be incubated 15min；

(12) 100 μ L, 2mol/L H are added₂SO₄Terminate liquid, gently mix 30S, using conventional light splitting light in 30min Degree meter with 540nm wavelength be with reference to, read 450nm wavelength under light absorption value.

(13) with concentration as abscissa, light absorption value be vertical coordinate draw standard curve, according to standard curve Trendline formula Calculate HNL concentration in sample.

Suction under the corresponding 450nm wavelength of each concentration homodimer HNL standard substance that table 5 records for embodiment 5 device Shading value, draws reference standard curve (as shown in Figure 4) according to table 5

Table 5：Absorbance at the corresponding 450nm wavelength of variable concentrations homodimer HNL standard concentration gradient solution Value

Homodimer HNL concentration (ng/mL)	O.D.1	O.D.2	Meansigma methodss
				0	0.012	0.009	0.0105
0.10	0.038	0.041	0.0395
				0.20	0.069	0.069	0.069
0.40	0.14	0.132	0.136
				0.80	0.318	0.324	0.321
1.60	0.621	0.603	0.612
				3.20	1.265	1.234	1.2495
6.40	2.353	2.401	2.377

By table 5 can draw this device quantitation homodimer concentration can as little as 0.1ng/mL, can draw in conjunction with Fig. 4 The range of linearity of this device quantitation homodimer is 0.1ng.mL～6.4ng/mL.

Batch interior difference CV meansigma methodss of the present embodiment device are 4.31% (scope 0.54%～7.40%, n=12), between batch Difference CV meansigma methodss are 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, list Body HNL and MMP9/HNL, obtains the measured value concentration (C of the homodimer HNL in urine solution using the present embodiment₂)；Theoretical Value concentration is C₃.According to formula (1) response rate 1 (%)=C₃÷C₂× 100% and formula (2) response rate 2 (%)=(C₂-C₀)÷ C1 × 100% is calculated the response rate (table 6) in urine sample for the above molecule.

Table 6：HNL homodimer, the HNL monomer and MMP9/HNL response rate in urine

HN homodimer L in table 6 data display this device energy specific quantification urine specimen, and to HNL monomer and HNL heterodimer then hardly detects.Therefore deduce that this method has very high specificity and sensitivity.

Claims (2)

1. a kind of human neutrophil apolipoprotein heterodimer HNL/MMP-9 based on Enzyme-linked Immunosorbent Assay technology quantitatively fills Put it is characterised in that：By the container (51) of 5 times of sample diluting liquids of storage, the container (52) of 25 times of washing liquids of storage, storage HNL/ The coated 96 hole enzymes of the container (54,55,56,57,58,59,60) of MMP-9 standard concentration gradient lyophilized powder, anti-mm P-9 antibody The container (53) of target (20) and storage enzyme mark anti-HNL antibody-solutions or coated 96 hole elisa Plates of anti-HNL antibody (20) and storage Deposit the container (53) of enzyme mark anti-mm P-9 antibody-solutions, U-shaped bottom 96 orifice plate (30), shrouding film (40), the appearance of storage enzyme substrate solution Container (63) composition of device (61,62) and storage terminate liquid, described HNL/MMP-9 standard substance behaviour source HNL/MMP-9；Described enzyme The enzyme of labeling antibody institute labelling is horseradish peroxidase or alkali phosphatase；Described standard concentration gradient solution refers to HNL/ MMP-9 standard concentration gradient lyophilized powder adds 200 μ L sample diluent working solutions to make；Described sample solution refers to through sample Body fluid sample after the dilution of diluent working solution, described body fluid sample is urine, serum, blood plasma, cerebrospinal fluid or saliva；Described sample Product diluent working solution is that 5 times of sample diluting liquid deionized waters are diluted 5 times of acquisitions.

2. a kind of human neutrophil apolipoprotein heterodimeric based on Enzyme-linked Immunosorbent Assay technology as claimed in claim 1 Body HNL/MMP-9 proportioning device it is characterised in that：Anti-mm P-9 antibody is the anti-mm P-9 antibody of commercialization or passes through conventional method With the antibody of MMP-9 immune animal preparation or the antibody of gene engineering method preparation, described antibody can be monoclonal antibody or many Clonal antibody；Anti- HNL antibody be the anti-HNL antibody of commercialization or the antibody prepared with HNL immune animal by conventional method or The anti-HNL antibody of gene engineering method preparation, described antibody can be monoclonal antibody or polyclonal antibody.