CN105911274A - Immunochromatography device for synchronously and quantitatively detecting different molecular forms of human neutrophil lipocalin (HNL) and preparation method thereof - Google Patents

Abstract

An immunochromatographic device for synchronously and quantitatively detecting different molecular forms of human neutrophil lipocalin (HNL) and a preparation method thereof, belonging to the field of laboratory medicine. It is composed of sample diluent lyophilized powder, standard product lyophilized powder and immunochromatography test strip; the immunochromatography test strip consists of a liner, two layers of blood filter membrane, sample flow pad, UCP binding pad, analysis membrane and absorbent pads. The UCP binding pad is coated with UCP-labeled anti-three molecular forms of HNL antibodies; the analysis membrane is provided with three parallel detection areas T1, T2, T3 and a quality control area C, and the three detection areas are sequentially Anti-matrix metalloproteinase-9 antibody, anti-monomeric HNL antibody and anti-homodimeric HNL antibody; quality control area C is coated with anti-UCP antibody-derived animal immunoglobulin antibody. The invention enables the same sample to jointly detect three molecular forms of HNL on the same test strip, which has the advantages of rapidity, quantification, convenience, etc., and has great scientific research and clinical application prospects.

Description

A Simultaneous Quantitative Detection of Different Molecular Forms of Human Neutrophil Lipocalin Immunochromatography device and preparation method thereof

technical field

The invention belongs to the technical field of clinical laboratory medicine, and in particular relates to an immunochromatographic device for synchronously and quantitatively detecting different molecular forms of human neutrophil lipocalin (HNL) and a preparation method thereof.

Background technique

Human neutrophil lipocalin (human neutrophil lipocalin, HNL) was originally a glycoprotein discovered in neutrophil granules by Xu S. et al. (Scand.J.Clin.Lab Invest 54,365-376) , a member of the lipocalin superfamily, has a similar spatial structure to other lipocalins. The protein is also called neutrophil gelatinase associated lipocalin (neutrophil gelatinase associated lipocalin, NGAL), also known as human lipocalin 2 (lipocalin 2) and so on. Cai Linjun et al. (Clin.J.Am.Soc.Nephrol.2010,5:2229-35) found that urinary HNL exists in various molecular forms, including monomer (about 25kDa), homodimer (about 45kDa) and A heterodimer (about 135kDa) formed with gelatinase (gelatinase). In addition to the ability of neutrophils to secrete HNL, HNL is expressed in multiple tissues of the human body under physiological or pathological conditions (such as inflammation, infection, tumor, ischemia, etc.). In addition, oxidative stress and some cytokines (such as IL-1β, TNF-α) can induce epithelial cell lines to express HNL in vitro (Clin.J.Am.Soc.Nephrol.2010,5:2229-35; J.Immunol. 2003, 171:6630-9).

In recent years, HNL has attracted more and more attention as a biomarker for the early diagnosis of kidney injury, tumor, acute bacterial infection and inflammation. Early diagnosis of HNL is caused by antineoplastic drugs cisplatin (Am.J.Nephrol.2004,24:307-15), ischemia (J.Am.Soc.Nephrol.2004,15:3073-82), sepsis ( Chinese Practical Medicine 2015,24:46-47), IgA nephropathy (Clin.Immunol.2007,123:227-34; Chinese Journal of Integrated Traditional Chinese and Western Medicine Nephrology 2013,3:223-226), systemic lupus erythematosus (Arthritis Rheum. 2006,54:2577-84), cardiac surgery (Lancet 2005,365:1231-8; Clin.Chim.Acta.2009,403:121-5; Journal of Southeast University (Medical Edition) 2012,1:67-71 ) etc., showed good early prediction ability. In addition, studies have shown that HNL has a role in ovarian cancer (Int.J.Cancer 2007,120:2426-2434; Journal of Practical Oncology 2010,5:539-542.), pancreatic cancer (Br.J.Cancer 2008,98:1540 -1547.), gastric cancer (Anat.Rec.2010,293:1855-1863.), colorectal cancer (Genet.Mol.Res.2014,13:7102-7112; Journal of Oncology 2009,2:115-119. ), breast cancer (Biomed.Rep.2013,1:479-483; China Practical Medicine 2011,20:43-45.) and other cancer biomarker potential. Venge P. et al reported the correlation between HNL and acute infection (Scand.J.Clin.Lab Invest 2011,55:125-131; Clin.Chem.Lab Med.2011,49:999-1003; Int.Urol. Nephrol. 2014, 46:2243-2249). With the deepening of research, the function of HNL as a biomarker for disease diagnosis has attracted more and more attention.

At present, there are many research reports on HNL as a biomarker for early diagnosis of acute kidney injury. Compared with the current clinically used serum creatinine (sCr) index, many research results show that HNL can predict whether a patient will develop AKI 1 to 2 days in advance, which has won valuable time for clinical intervention in the treatment of AKI in advance. In the future, HNL It is possible to become a point of care testing (POCT) project for AKI clinical practice. Therefore, it is urgent to develop a portable, rapid, easy-to-operate and quantitative HNL detection technology. At present, the quantitative methods of HNL in biological fluid samples are mainly based on the immunoassay technology of antigen-antibody specific reaction, such as RIA (International Patent WO/1995/029404A1 and Chinese ZL 200980155194), ELISA (European Patent EP2225268 and Chinese Patent ZL200980155194), Western-blotting , latex immunoturbidimetric method (Chinese patents ZL201410431182 and ZL201210394943), chemiluminescence immunoassay (Chinese patent application numbers 201510184818 and 201410197672), etc. The above methods have their own advantages, but they also have disadvantages, especially when HNL is used as a POCT item for detection. For example, RIA has problems such as too long test time, requires a special radiation protection site, and is easy to cause environmental pollution; ELISA has a low degree of automation, long experimental operation time, requires professional operation, and is expensive. Western-blotting operation time is long and complicated, and the measurement precision is low; the linear range of chemiluminescence method is small, and the detection cost is high; latex immunoturbidimetric method has low specificity, poor stability and uniformity of reagents, narrow linear range of detection and testing A long time. It can be seen that the above-mentioned HNL detection method cannot meet the detection requirements of HNL POCT items at present. In recent years, in order to meet the demand for rapid quantification of HNL, various immunochromatographic techniques have been used for the quantitative detection of HNL (Chinese patents ZL201220323587, ZL201420423962, ZL201220392399, ZL201220323586 and ZL201220497401). Immunochromatography is a rapid diagnostic method that is often used in POCT projects. The basic principle of immunochromatography is to pre-immobilize specific antibodies or antigens in the detection area of the detection membrane (such as nitrocellulose membrane) and dry it. When one end of the dried membrane is immersed in the sample, due to capillary phenomenon (capillary action), ), the sample will move forward along the membrane, and when it moves to the area where the antibody or antigen is immobilized, the corresponding antigen or antibody in the sample will react specifically with the antibody or antigen, through various techniques such as fluorescent labeling Technology, immune colloidal gold technology, immunoenzyme staining technology, quantum dot labeling technology and up-conversion luminescence technology and other methods make the detection area display a certain signal, so as to achieve specific qualitative or quantitative detection of specific antigens or antibodies. Due to different chromogenic techniques, immunochromatography can be divided into immunocolloidal gold chromatography, immunoenzyme-labeled chromatography, and immunofluorescence chromatography. Compared with the above-mentioned other HNL quantitative techniques, the HNL immunochromatographic technique has the advantages of convenient operation and rapid detection, but its disadvantages are also obvious. Fluorescent immunochromatography is prone to decay or quenching of fluorescent dyes, which affects product stability; the disadvantage of fluorescent latex particles is that fluorescent dyes are physically doped and prone to leakage. At the same time, the surface modification methods of polymer latex particles are limited and most of them are hydrophobic. , which is prone to non-specific adsorption. The immunocolloidal gold technology is combined by physical adsorption, and the antigen or antibody is easy to fall off the surface of the gold nanoparticle, resulting in instability of the marker, thereby affecting the detection performance.

In recent years, immunochromatographic up-converting phosphor technology (UPT) using the above-mentioned up-converting phosphor (UCP) as a display agent is a research and development hotspot in this field. UCP consists of three parts: host matrix, absorber and emitter, and is usually composed of rare earth metal elements doped in the crystal lattice. UCP has the phenomenon of upconversion luminescence, and emits visible light under the excitation of infrared light. Due to its excellent properties such as no background interference, good stability, no quenching, environmental friendliness and good biocompatibility, UCP has been used as a tracer in a variety of POCT detection systems. Chinese patent (ZL201220497401) discloses an upconversion luminescent rapid quantitative device for HNL detection. This technology partially overcomes the shortcomings of current HNL detection technology but cannot distinguish and measure different molecular forms of HNL, thus limiting the clinical application potential of this method.

Although the above HNL quantitative methods have their own advantages, they all have a serious deficiency, that is, they cannot effectively distinguish and simultaneously measure different molecular forms of HNL. The molecular form of HNL has a significant correlation with the disease, and different molecular forms of HNL indicate different pathological processes. Therefore, the technique of distinguishing and measuring different molecular forms of HNL has important laboratory research and future clinical application value. At present, there is no public disclosure of combined simultaneous quantitative and rapid detection of HNL.

Contents of the invention

In order to overcome the problems existing in the prior art, the object of the present invention is to provide an immunochromatographic device and a preparation method thereof for synchronous joint quantitative detection of different molecular forms of human neutrophil lipocalin (HNL). Up-converting phosphor technology (up-converting phosphor technology, UPT) and immunochromatography technology. Compared with the prior art, the biggest advantage of the invention is that three molecular forms HNL of monomer, homodimer and heterodimer can be quantified rapidly and simultaneously. The device has the advantages of simple sample pretreatment, less sample consumption, convenient and rapid detection process, high sensitivity and specificity, and the like.

In order to achieve the above goals, the present invention adopts the following technical solutions: an immunochromatographic device for simultaneous quantitative detection of human neutrophil lipocalin (HNL) in different molecular forms, composed of sample diluent freeze-dried powder, standard freeze-dried Composed of dry powder and immunochromatographic test strips; the sample diluent freeze-dried powder is 20mL, pH=7.2～7.5, containing 150～250mmol/L NaCl, 0.25～0.5% (vol/vol) Tween-20 , 0.5%～2% (wt/vol) BSA 50～100mM HEPES buffer solution is mechanically or magnetically stirred sufficiently, adopts 0.22μm or 0.45μm filter to carry out filter sterilization and obtain after lyophilization; The standard freeze-dried powder is 400 μg/mL monomer, homodimer or heterodimer three molecular forms of HNL are obtained by freeze-drying 10 μL of PBS buffer solution of pH=7.2～7.4, 10 mmol/L; Plate, two layers of blood filtration membrane, sample flow pad, UCP binding pad, analysis membrane and absorption pad; the two layers of blood filtration membrane are divided into pre-filtration blood membrane and post-filtration Pads, UCP binding pads, analysis membranes and absorbent pads are lapped and pasted on the liner from front to back in sequence, assembled by conventional methods or packaged with plastic shells; the blood filtration membrane is treated with fMLP ( N-formyl-L-methionyl-L-leucyl-L-phenylalanine) solution and 7.0～28.5U/mL heparin sodium solution are soaked and then dried; the UCP binding pad is coated UCP-labeled anti-three molecular forms of HNL antibody capable of simultaneously recognizing monomer, homodimer and heterodimer three molecular forms of human neutrophil lipocalin (HNL), the UCP particle size is 400 ~600nm; the analysis membrane is provided with three parallel detection areas (T1, T2 and T3) and a quality control area (C), and the three detection areas are coated with anti-matrix metalloproteinase-9 (MMP- 9) Antibodies, anti-monomeric HNL antibodies and anti-homodimeric HNL antibodies, the antigens targeted by the antibodies are human HNL; the quality control area (C) is coated with anti-UCP labeled antibody-derived animal immunoglobulin antibodies; the The liner is used to support the assembly of two layers of blood filtration membrane, sample flow pad, UCP binding pad, analysis membrane and absorption pad. The present invention is used for the analysis of whole blood samples, the sample flow pad is used to connect the blood filtration membrane and the UCP binding pad, the UCP binding pad is used to bind UCP labeled antibodies, and the analysis membrane is used for the detection area and quality control The area is set so as to realize the quantification of HNL, and the absorbent pad is used to collect the sample solution after analysis.

The specific preparation method is as follows:

1) Preparation of reagents

a) Sample diluent lyophilized powder

20mL, pH = 7.2-7.5, containing 150-250mmol/L NaCl, 0.25-0.5% (vol/vol) Tween-20, 0.5%-2% (wt/vol) BSA 50-100mM HEPES buffer by mechanical Or after magnetic stirring is sufficient, the sample diluent is sterilized by filtration with a 0.22 μm or 0.45 μm filter, freeze-dried and stored at room temperature.

Sample diluent lyophilized powder was added to 20mL deionized water to fully dissolve before use to obtain sample diluent.

b) standard freeze-dried powder

400 μg/mL monomer, homodimer or heterodimer three molecular forms of HNL, pH = 7.2-7.4, 10 μL lyophilized 10 mmol/L PBS buffer solution;

Add 10 μL of deionized water to fully dissolve the lyophilized powder of the standard before use to obtain the mother solution of the standard.

c) HNL and MMP-9 proteins

HNL and MMP-9 proteins are prepared using conventional techniques in the art and/or obtained through commercial channels. Monomeric HNL is obtained by expressing and purifying the corresponding host cells using prokaryotic expression vectors and eukaryotic expression vectors (Biophys. Res. Commun. 1994, 202:1468-1475; SRP6465 of Sigma Company). Homodimer HNL and heterodimer HNL are separated from human blood. Specifically, granulocytes are obtained after natural sedimentation of blood buffy coat to remove red blood cells. Granulocytes are broken by nitrogen cavitation to obtain granulocyte granules, and then acid lysed. Separation and purification are carried out after the particles (Scand.J.Clin.Lab.Invest.1994,54(5):365-376; J.Biol.Chem.268(14):10425-10432.). MMP-9 protein is MMP-9 obtained by expression and purification in corresponding host cells using prokaryotic expression vectors and eukaryotic expression vectors (Protein Expr.Purif.2010,72:87-94; Protein Expr.Purif.2016 , 126:42-48; R&D M8945, etc.).

d) Antibodies against specific molecular forms of HNL

Anti-HNL antibodies (including anti-monomeric HNL antibodies, anti-homodimeric HNL antibodies and anti-heterodimeric HNL antibodies) are commercial antibodies or anti-HNL antibodies prepared by conventional antibody preparation techniques using HNL as an antigen (such as Abcam ab63929 of P&M Venge Diagnostics Development Company or HNL mab 697 of P&M Venge Diagnostics Development Company, etc.); the above antibodies were screened by monomeric HNL, homodimeric HNL or heterodimeric HNL antigens to obtain antibodies against specific molecular forms of anti-HNL.

e) MMP-9 antibody and antibody for quality control area coating

The MMP-9 antibody and the antibody used for coating the quality control region were prepared using conventional techniques in the art and/or obtained commercially (HPA001238, AMAB90805, etc. from Sigma). MMP-9 antibody is a polyclonal antibody or monoclonal antibody prepared with human MMP-9 as an antigen; the quality control area is coated with an antibody against UCP-labeled antibody source animal immunoglobulin, such as rabbit anti-mouse IgG antibody or goat anti-mouse IgG antibody or other animal-derived anti-mouse IgG antibody or anti-rabbit IgG, depending on the type of UCP-labeled antibody.

f) Preparation of antibody solution

The antibody solution (including anti-HNL antibody solution or anti-MMP9 antibody solution or the antibody solution of anti-UCP labeled antibody source animal immunoglobulin in the quality control area) is prepared by dissolving the antibody in the antibody buffer; the antibody concentration in the antibody solution is 1mg/ mL; Antibody buffer solution is 100mM Tris-HCL solution containing 1% (vol/vol) methanol, pH=8.0, or 10mmol/L PBS buffer solution with pH=7.2～7.4.

g) Preparation of UCP-labeled antibody solution

UCP is a laboratory-synthesized or commercially available crystalline material composed of rare earth metal elements with an average particle size of 400-600 nm, coated with silica and modified by surface functionalization (Anal. Biochem. 2001, 293 (1) :22-30; USpatant 1997,5:674-698); UCP is covalently linked to anti-HNL antibody to form a UCP-labeled antibody.

The specific preparation steps of the UCP-labeled antibody solution are as follows:

(a) Take 1 mL of UCP (NaYF4:Er, Yb or other UCP material) suspension (5 mg/mL) with an average diameter of 400-600 nm and add it into a centrifuge tube and centrifuge at 13000 rpm for 10 min. Discard the supernatant;

(b) Add 1mL DMSO-MES buffer solution (containing 33% (V/V) DMSO, 10mM MES aqueous solution) to the obtained precipitate, mix slightly and perform ultrasonic treatment (ultrasonic conditions: 20kHz, 320W, each ultrasonic 15s , stop for 15s, a total of 3 times);

(c) Centrifuge at 13000rpm for 10min, discard the supernatant;

(d) repeat steps (b) and (c) twice;

(e) Add 0.8mL DMSO-MES buffer solution to the obtained precipitate, mix slightly, and then perform ultrasonic treatment (ultrasound conditions: 20kHz, 320W, each ultrasonic 15s, stop for 15s, a total of 3 times);

(f) Add newly prepared 40 μL, 30 mM carboxyl activation reagent EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 40 μL, 30 mM protein cross-linking agent Sulfo -NHS (N-hydroxyl sulfosuccinimide), then vortexed to mix;

(g) Ultrasonic treatment in an ice bath (ultrasound conditions: 20kHz, 320W, each ultrasonic 15s, stop 15s, a total of 60 times);

(h) Centrifuge at 13000rpm for 10min, discard the supernatant;

(i) Add 1.2mL DMSO-MES buffer solution (33% (V/V) DMSO, 10mM MES aqueous solution) and mix slightly, then perform ultrasonic treatment (ultrasonic conditions: 20kHz, 320W, each ultrasonic 15s, stop 15s , a total of 3 times);

(j) centrifuge at 13000rpm for 10min, discard the supernatant;

(k) repeat steps (i) and (j) 3 times;

(l) Add 1mL DMSO-MES buffer solution (containing 33% (V/V) DMSO, 10mM MES aqueous solution) and mix slightly, then perform ultrasonic treatment (ultrasonic conditions: 20kHz, 320W, each ultrasonic 15s, stop 15s, 3 times in total);

(m) Add 0.5 mL, 1 mg/mL anti-HNL antibody solution to the above UCP suspension, invert and mix well and incubate at room temperature for 3 h, and ensure that UCP does not precipitate during the incubation process;

(n) After adding 150 μL of 2M glycine buffer solution with pH=11, perform ultrasonic treatment and mix well (ultrasound conditions: 20 kHz, 320 W, each ultrasonic 15 s, stop 15 s, a total of 3 times);

(o) Centrifuge at 13000rpm for 10min, discard the supernatant;

(p) Add 5mL of UCP-labeled antibody preservation solution (25mmol/L glycerol, 0.02% (vol/vol) Triton and 0.1% (wt/vol) NaN ₃ aqueous solution), carry out ultrasonic treatment and mix (ultrasonic condition: 20kHz , 320W, each ultrasound 15s, stop 15s, a total of 3 times);

(q) Repeat steps (o) and (p) once overnight;

(r) centrifuge at 13000rpm for 10min, discard the supernatant;

(s) Add an aqueous solution containing 25 mmol/L glycerol, 0.02% (vol/vol) Triton-100 and 0.1% (wt/vol) NaN ₃ to the obtained precipitate, and mix to obtain a UCP-labeled antibody solution.

2) Preparation of immunochromatographic test strips

a) blood filter membrane

The blood filtration membrane material is glass cellulose membrane or membrane material with similar properties. The upper and lower layers are staggered and overlapped with each other. After soaking in fMLP solution with a concentration of 0.0-10 μmol/L and heparin sodium solution with a concentration of 7.0-28.5 U/mL, dry it for later use. .

b) Sample Flow Pad

The material of the sample flow pad is highly hydrophilic glass fiber or a membrane material with similar properties, which is used to connect the blood filtration membrane and the UCP binding pad.

c) UCP binding pad

The UCP binding pad is a nitrocellulose membrane or a membrane material with similar properties, and the UCP-labeled antibody solution with a concentration of 1 mg/mL is evenly added to the UCP binding pad in an amount of 0.25 to 0.5 mL/cm ² , at 30 ° C to 35 ° C dry.

d) Analytical membrane T/C zone preparation

The analysis membrane material is a nitrocellulose membrane or a membrane material with similar properties; the anti-matrix metalloproteinase-9 (MMP-9 ) antibody solution (T1), anti-monomeric HNL antibody solution (T2), anti-homodimer HNL antibody solution (T3) and anti-UCP labeled antibody source animal immunoglobulin antibody solution (C), the antibody concentration used is 1mg /mL, the final spraying amount is 0.025-0.05mL/mm, which is equivalent to spraying 25ng-50ng antibody per mm T/C line; the distance between each line is set according to the detection window of the UCP reader, and it is dried for later use.

e) Absorbent pads

The absorbent pad is absorbent filter paper or a material with a similar function.

f) liner

The material of the liner is a self-adhesive polyvinyl chloride rubber sheet or a material with similar functions.

g) Test strip assembly

Assemble the liner, two-layer blood filtration membrane filter, UCP binding pad, sample flow pad, analysis membrane and absorption pad according to the conventional method, and further can be packaged with a plastic shell.

3) Use of the device

First, add 10 μL of deionized water to fully dissolve the lyophilized powder of the standard to prepare the standard mother solution, and then use the sample diluent to perform a 2-fold gradient dilution of the standard mother solution to obtain a gradient concentration solution of the standard with different concentrations. Take 50-150 μL Add the gradient concentration solution of the standard substance into the sample hole of the immunochromatography device placed horizontally (the sample hole is the pre-filtration membrane or the position corresponding to the gap reserved in the plastic shell on the pre-filtration membrane); within 15 to 20 minutes Use the UPT reader to scan and read the corresponding T1, T2 and T3 bands, and draw a standard curve according to the standard product peak area readings (AUC);

Then, after diluting the serum, plasma, urine sample or whole blood sample to be tested by 5-200 times with the sample diluent, take 50-150 μL and add it to the sample hole of the immunochromatography device placed horizontally; within 15-20 minutes, use UPT The reader scans and reads the corresponding T1, T2, and T3 bands, and then quantifies different molecular forms of NGAL in different samples according to the drawn standard curve.

Description of drawings

1 is a schematic cross-sectional view of a UCP immunochromatographic test strip of an immunochromatographic device for simultaneous quantitative detection of human neutrophil lipocalin (HNL) in different molecular forms according to the present invention. Among them, 1 and 2 are pre- and post-filtering membranes respectively, 3 is sample flow pad, 4 is UCP binding pad, 5 is analysis membrane, 6 is absorption pad, 7 is liner, 8 is UCP binding pad inner bag Anti-three molecular forms of HNL antibody labeled with UCP, 9 is the detection area T1 (coated with anti-MMP-9 antibody), 10 is the detection area T2 (coated with anti-monomeric HNL antibody), 11 is the detection area T3 (coated with Anti-homodimeric HNL antibody), 12 is the quality control area C (coated with anti-UCP labeled antibody source animal immunoglobulin antibody). Each part overlaps (2-5mm) from end to end according to the order shown in Figure 1 and then sticks to the liner.

FIG. 2 is a schematic structural view of the analytical membrane described in Example 2. FIG. The analysis membrane is dumbbell-shaped (dumbbell-shaped analysis membrane can improve the detection sensitivity), the width of the middle part is 4mm, and the width of both ends is 5mm; the two ends are isosceles trapezoid, and the height of the trapezoid is 2mm; among them, T1, T2 and T3 are the parts of the detection area , C is the quality control area; T1 is 10mm away from the front end of the analytical membrane, T2 is 15mm away from the front end of the analytical membrane, T3 is 20mm away from the front end of the analytical membrane, and C is 25mm away from the front end of the analytical membrane; T1, T2, T3 and C are respectively coated with anti-MMP-9 antibody, anti-monomeric HNL antibody, anti-homodimeric HNL antibody, and anti-mouse IgG antibody.

Fig. 3 is the standard curve of three molecular forms of HNL corresponding to UCP peak area (AUC) drawn by the method of Example 3. The standard curve is drawn according to the numbers in Table 1, wherein the abscissa is the concentration of different molecular forms of HNL, and the ordinate is the peak area; the left figure in Figure 3 is based on the relationship between the concentration of heterodimer HNL in Table 1 and T1 (1 ^st ) and T1 (2 ^nd ) the standard curve drawn by the average value, the figure in Figure 3 is the standard curve drawn according to the average value of the monomer HNL concentration in Table 1 and T2 (1 ^st ) and T2 (2 ^nd ), the right figure in Figure 3 is based on Table 1 The standard curve drawn by the average value of homodimer HNL concentration and T3 (1 ^st ) and T3 (2 ^nd ); the mathematical formula listed in the figure is the formula corresponding to the trend line for the calculation of various HNL concentrations in actual samples; in the figure ^The R2 values listed are the square of the correlation coefficients corresponding to the trend lines of the standard curves.

Fig. 4 is the concentration of heterodimer, monomer and homodimer HNL in the urine samples of healthy controls, virus infection and bacterial infection patients in Example 4. Figure 4 is based on the area under the peak (AUC) data in Table 2 and using the trend line formula of the standard curve in Figure 3 to calculate the concentration of various molecular forms of HNL; 4. Figure 4 is a Box-whisker diagram drawn by the medical statistical software MedCalc (version 13.0.0.0.0), where each "○", "△" and "■" represent a sample.

detailed description

The present invention will be further illustrated below with examples. The embodiments of the present invention are used to further explain the present invention rather than limit the present invention. The specific improvements made according to the essence of the present invention all belong to the protection scope of the present invention.

Example 1: Composition and storage of an immunochromatographic device for simultaneous quantitative detection of different molecular forms of human neutrophil lipocalin (HNL)

1) The composition of the device: 1 bottle of sample diluent freeze-dried powder, several UCP immunochromatography test strips, and 1 set of standard solution freeze-dried powder;

2) Storage conditions of the device: storage at room temperature.

Example 2: Preparation of a UPT immunochromatography test strip for simultaneous quantitative detection of different molecular forms of human neutrophil lipocalin (HNL) immunochromatography device

1) Solution preparation

a) Preparation of fMLP solution

Firstly, fMLP (purchased from Sigma) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 50 mM stock solution, and then the 50 mM fMLP stock solution was diluted into a 5 μmol/L fMLP solution with 50 mM HEPES solution at pH=7.4.

b) Preparation of heparin sodium solution

Heparin sodium (purchased from Sigma) was dissolved in 50 mM HEPES (pH 7.4) solution to prepare a 14 U/mL heparin sodium solution.

c) Antibody solution preparation

Anti-MMP-9 polyclonal antibody (purchased from abcam company, ab5707), anti-three molecular forms of human HNL monoclonal antibody (monoclonal antibody prepared by immunizing mice with human homodimer HNL as antigen, can bind to three molecular forms Form HNL carries out antigen-antibody reaction, and this antibody is denoted as anti-HNL Ab 1), anti-monomeric HNL antibody (purchased from abcam, ab125075, this antibody is denoted as anti-HNL Ab2), anti-homodimer antibody (identified in human Dimer HNL is a monoclonal antibody prepared by antigen-immunized mice, which has no cross-reaction with monomer and heterodimer HNL, and the antibody is denoted as anti-HNL Ab 3); the above anti-MMP-9 antibody, anti-HNL Ab2 and anti-HNL Ab3 were respectively dissolved in pH=8.0 and 100mM Tris-HCL solutions containing 1% (vol/vol) methanol, and the antibody concentration was 1mg/mL; anti-HNL Ab1 was dissolved in PBS buffer at pH=7.4 In , the concentration is 1mg/mL.

d) Preparation of UCP-labeled antibody solution

(a) Take 1 mL of UCP (NaYF4:Er, Yb) suspension (5mg/mL) with an average diameter of 400nm and silanization and carboxylation modification on the surface, add it to a centrifuge tube and centrifuge at 13000rpm for 10min, discard the supernatant;

(b) Add 1mL DMSO-MES buffer solution (containing 33% (V/V) DMSO, 10mM MES aqueous solution) to the obtained precipitate, mix slightly and perform ultrasonic treatment (ultrasonic conditions: 20kHz, 320W, each ultrasonic 15s , stop for 15s, a total of 3 times);

(c) Centrifuge at 13000rpm for 10min, discard the supernatant;

(d) repeat steps (b) and (c) twice;

(e) Add 0.8mL DMSO-MES buffer solution to the obtained precipitate, mix slightly, and then perform ultrasonic treatment (ultrasound conditions: 20kHz, 320W, each ultrasonic 15s, stop for 15s, a total of 3 times);

(f) Add 40 μL, 30 mM carboxyl activation reagent EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 40 μL, 30 mM protein cross-linking agent Sulfo -NHS (N-hydroxyl sulfosuccinimide), then vortexed to mix;

(g) Ultrasonic treatment in an ice bath (ultrasound conditions: 20kHz, 320W, each ultrasonic 15s, stop 15s, a total of 60 times);

(h) centrifuge at 13000rpm for 10min, discard the supernatant;

(i) Add 1.2mL DMSO-MES buffer solution (33% (V/V) DMSO, 10mM MES aqueous solution) and mix slightly, then perform ultrasonic treatment (ultrasonic conditions: 20kHz, 320W, each ultrasonic 15s, stop 15s , a total of 3 times);

(j) centrifuge at 13000rpm for 10min, discard the supernatant;

(k) repeat steps (i) and (j) 3 times;

(l) Add 1mL DMSO-MES buffer solution (containing 33% (vol/vol) DMSO, 10mM MES aqueous solution) and mix slightly, then perform ultrasonic treatment (ultrasound conditions: 20kHz, 320W, each ultrasonic 15s, stop 15s, 3 times in total);

(m) Add 0.5 mL, 1 mg/mL anti-HNL Ab1 solution to the above UCP suspension, invert and mix well and incubate at room temperature for 3 h, and ensure that UCP does not precipitate during the incubation process;

(n) After adding 150 μL of 2M glycine buffer solution with pH=11, perform ultrasonic treatment and mix well (ultrasound conditions: 20 kHz, 320 W, each ultrasonic 15 s, stop 15 s, a total of 3 times);

(o) Centrifuge at 13000rpm for 10min, discard the supernatant;

(p) Add 5mL of UCP-labeled antibody preservation solution (25mmol/L glycerol, 0.02% (vol/vol) Triton and 0.1% (wt/vol) NaN ₃ aqueous solution, carry out ultrasonic treatment and mix (ultrasonic conditions: 20kHz, 320W, each ultrasound for 15s, stop for 15s, a total of 3 times);

(q) Repeat steps (o) and (p) once overnight;

(r) Centrifuge at 13000rpm for 10min, discard the supernatant;

(s) Add an aqueous solution containing 25 mmol/L glycerol, 0.02% (vol/vol) Triton-100 and 0.1% (wt/vol) NaN ₃ to the obtained precipitate, and mix to obtain a UCP-labeled antibody solution.

2) Preparation of UPT immunochromatographic test strips

a) Blood filtration membrane preparation

The blood filtration membrane material is glass cellulose membrane (Whatman, LF1), which is composed of two upper and lower layers staggered by 3/4; the front layer and the subsequent layer of glass cellulose membrane are soaked in 14U/mL heparin sodium solution and then dried for later use; The size of each layer of blood filtration membrane is 10mm long and 5mm wide.

b) Sample Flow Pad Preparation

The material of the sample flow pad is a highly hydrophilic glass cellulose membrane (Milipore, Hi-FlowTM Plusmembrane, HF 075), which is used to connect the blood filtration membrane and the UCP binding pad; the size is 10mm long and 5mm wide; the front end of the sample flow pad overlaps by 2mm On the blood filtration membrane, the back end of the flow pad 2mm rests on the UCP binding pad.

c) UCP binding pad

The UCP binding pad is a glass cellulose membrane (Milipore, Pad Materials, G028), according to the amount of 0.5mL/ ^cm2 , the concentration of 1mg/mL UCP-labeled anti-HNL Ab1 antibody solution was evenly added to the UCP binding pad, and dried at 30°C; then cut into 8mm long and 5mm wide; Its front end is 2 mm long and rests on the sample flow pad, and its rear end 2 mm long rests on the analysis membrane.

c) Analytical membrane T/C line preparation

The analysis membrane material is nitrocellulose membrane (Millipore, AE99, 120-160s/4cm); with the help of a micropipette, the anti-matrix metalloproteinase-9 ( MMP-9) antibody (T1) solution, anti-monomeric HNL antibody (T2) solution, anti-homodimeric HNL antibody (T3) solution and anti-mouse IgG antibody (C) solution, the antibody concentration used was 1 mg/mL, and finally The spray volume is 0.05mL/mm, which is equivalent to spraying 50ng antibody per millimeter of T/C line; T1 area is 10mm away from the front end of the analysis membrane, T2 area is 15mm away from the front end of the analysis membrane, T3 area is 20mm away from the front end of the analysis membrane, and C area is away from the front end of the analysis membrane 25mm; the analysis membrane is cut according to the size of Figure 2 in the accompanying drawings; the front end of the analysis membrane is 2mm long and placed under the UCP binding pad, and the rear end of the analysis membrane is 2mm long and placed under the absorbent pad.

d) Absorbent pads

The material of the absorbent pad is absorbent filter paper (Whatman, CF4); the size is 25 mm in length and 5 mm in width); the front end of the absorbent pad is 2 mm long and rests on the analytical membrane.

e) Liner

The material of the liner is a self-adhesive polyvinyl chloride rubber sheet or a material with similar functions.

f) Test strip assembly

Assemble the liner, two-layer blood filtration membrane filter, UCP binding pad, sample flow pad, analysis membrane and absorption pad according to the sequence shown in Figure 1 of the accompanying drawings, and further package it with a plastic shell.

Embodiment 3: A kind of standard curve of simultaneous quantitative detection different molecular form human neutrophil lipocalin (HNL) immunochromatography device

1) Solution preparation

a) Preparation of sample diluent

The sample diluent is obtained by diluting the sample diluent lyophilized powder.

Sample diluent lyophilized powder is 20mL, pH 7.2 containing 100mmol/L HEPES solution of 200mmol/L NaCl, 0.5% (vol/vol) Tween-20, 1% (wt/vol) BSA. μmol/L filter to filter and freeze-dry. The sample diluent is obtained by adding 20 mL of deionized water to the lyophilized powder of the above sample diluent and mixing.

b) Preparation of HNL standard concentration gradient solution

The mother solution of HNL standard concentration gradient solution is obtained from standard freeze-dried powder plus deionized water. The standard freeze-dried powder contains 400 μg/mL monomer, homodimer, or heterodimer HNL, 10 μL each of 10 mmol/L PBS buffer solution at pH = 7.2, obtained by lyophilization.

The specific preparation steps of HNL standard concentration gradient solution are as follows:

(a) Add 10 μL of deionized water to the lyophilized powder of the above-mentioned standard products and mix well to obtain the standard product mother solution, add 990 μL sample diluent to the standard product mother solution and mix well, and mark it as S _single , S _same and S _different .

(b) Take 7 centrifuge tubes and mark them as S1, S2, S3, S4, S5, S6 and S7 respectively.

(c) Add 0.5 mL each of S _single , S _homo and S _different solutions to tube S1, then add 0.5 mL of sample diluent, and vortex to mix.

(c) Take 1mL of the solution from the S1 tube to the S2 tube, then add 1mL of the sample diluent to the S2, and vortex to mix.

(d) Transfer 1mL of the solution from tube S2 to tube S3, then add 1mL of sample diluent to tube S3 and vortex to mix.

(e) Transfer 1mL of solution from tube S3 to tube S4, then add 1mL of sample diluent to tube S4, and vortex to mix.

(f) Transfer 1mL of solution from tube S4 to tube S5, then add 1mL of sample diluent to tube S5 and vortex to mix.

(g) Transfer 1mL of solution from tube S5 to tube S6, then add 1mL of sample diluent to tube S6, and vortex to mix.

(h) Add 1 mL of sample diluent to tube S7.

2) Determination of HNL concentration by UPT immunochromatography

(a) Place 7 HNL UPT immunochromatographic test strips prepared in Example 2 on the horizontal tabletop and mark U7, U6, U5, U4, U3, U2 and U1;

(b) Add 100 μL of the solutions in tubes S7, S6, S5, S4, S3, S2 and S1 to the corresponding wells of U7, U6, U5, U4, U3, U2 and U1 immunoassay strips.

3) After waiting for 15 minutes, UPT readings were performed on T1, T2 and T3 of the analysis membranes on U7, U6, U5, U4, U3, U2 and U1 in sequence, and the peak area (AUC) was collected, which was recorded as T1 (1 ^st ), T2(1 ^st ) and T3(1 ^st ).

4) Repeat steps 2) and 3) to collect peak areas (AUC), which are recorded as T1(2 ^nd ), T2(2 ^nd ) and T3(2 ^nd ), respectively.

5) Draw a standard curve.

Table 1 is the peak area (AUC) of the HNL standard concentration gradient solution and the corresponding UPT readings in this embodiment. Among them, T1 refers to the AUC value of heterodimer HNL, T2 refers to the AUC value of monomeric HNL, and T3 refers to the AUC value of homodimer; 1 ^st and 2 ^nd refer to the results of two experiments.

Table 1: UCP readings for standard samples

According to the average value of the peak area (AUC) of the two (1 ^st and 2 ^nd ) UPT readings in Table 1 and the corresponding standard concentration, draw the standard curve of the three molecular forms of HNL corresponding to the UCP peak area at different concentrations, as shown in Figure 3.

Example 4: An immunochromatographic device for simultaneous quantitative detection of different molecular forms of human neutrophil lipocalin (HNL) is used for the concentration of three molecular forms of HNL in urine samples of acute bacterial infection, viral infection and normal control group Quantitative

In this example, the UPT immunochromatographic test strip prepared in Example 2 and the operation steps in Example 3 were used to dilute the clinical urine sample and use the method of the present invention to quantify the three molecular forms of HNL.

1) The urine sample dilution method is as follows:

(a) Take several 1.5mL centrifuge tubes and mark them.

(b) Take 360 μL of sample diluent to each 1.5 mL centrifuge tube, add 40 μL of urine sample, and vortex to mix.

2) Results

Table 2: Area under UPT peak (AUC) readings for different molecular forms of HNL in urine samples

Fig. 4 is the standard curve of the UPT HNL immune test paper strip that the present embodiment adopts embodiment 3 method to establish, can calculate healthy control group, suffer from influenza group and bacterium by the trend line formula on this standard curve and table 2 data Concentrations of different molecular forms of HNL in urine samples from a group of patients with chronic inflammation.

Claims (8)

1. the immunochromatography of synchronization combining detection by quantitative different molecular form human neutrophil lipocalin protein (HNL) Device, it is characterised in that: it is made up of sample diluting liquid lyophilized powder, standard substance lyophilized powder and immuno-chromatographic test paper strip；Described immunity Chromatograph test strip is made up of liner plate, two layers of blood separating films, sample flow pad, UCP pad, analyzing film and absorption pad；Two layers of blood separatings Film is divided into preposition hemofiltration film and rearmounted hemofiltration film, two layers of blood separating films, sample flow pad, UCP pad, analyzing film and absorption pad from A-P sequentially overlaps and is pasted on liner plate, and conventional method assembles or is packaged with plastic housing；Described hemofiltration film through concentration is The fMLP solution of 0.0～10 μm ol/L and the heparin sodium aqua of 7.0～28.5U/mL soak to dry after moistening and obtain；Described UCP Pad is coated and can identify monomer, homodimer and three kinds of molecular forms human neutrophil lipocalin eggs of heterodimer simultaneously Anti-three kinds of molecular forms HNL antibody of the UCP labelling of white HNL；Described analyzing film be provided with parallel three detection zone T1, T2, T3 and quality control region C, described three detection zones be coated successively anti-Matrix Metalloproteinase-9 antibody, anti-monomer HNL antibody and Anti-homodimer HNL antibody, described antibody for antigen behaviour source HNL；It is dynamic that quality control region C is coated anti-UCP traget antibody source The antibody of thing immunoglobulin；Described liner plate is for supporting two layers of blood separating films, sample flow pad, UCP pad, analyzing film and suction Receive the assembling of pad.

2. a kind of synchronization combining detection by quantitative different molecular form human neutrophil lipocalin egg as claimed in claim 1 The device for immunochromatography of (HNL) in vain, it is characterised in that: described sample diluting liquid lyophilized powder is by 20mL, pH=7.2～7.5, Containing 150～250mmol/L NaCl, 0.25～0.5% (vol/vol) Tween-20,0.5%～the 50 of 2% (wt/vol) BSA ～100mM HEPES buffer is after the stirring fully of mechanically or magnetically power, uses 0.22 μm or 0.45 μm filter to carry out filtration sterilization Rear lyophilizing obtains.

3. a kind of synchronization combining detection by quantitative different molecular form human neutrophil lipocalin egg as claimed in claim 1 The device for immunochromatography of (HNL) in vain, it is characterised in that: described standard substance lyophilized powder be the monomer of 400 μ g/mL, homodimer or The pH=7.2～7.4 of three kinds of molecular forms HNL of heterodimer, the PBS buffer solution 10 μ L lyophilizing of 10mmol/L obtain.

4. a kind of synchronization combining detection by quantitative different molecular form human neutrophil lipocalin egg as claimed in claim 1 The device for immunochromatography of (HNL) in vain, it is characterised in that: above forward luminescent material UCP be mean diameter be 400～600nm through two Silicon oxide is coated the crystalline material being made up of thulium with surface-functionalized modification.

5. a kind of synchronization combining detection by quantitative different molecular form human neutrophil lipocalin egg as claimed in claim 1 The device for immunochromatography of (HNL) in vain, it is characterised in that: first standard substance lyophilized powder is added after 10 μ L deionized waters fully dissolve Prepare standard substance mother solution, then with sample diluting liquid, standard substance mother solution is carried out 2 times of method gradient dilutions and obtain the mark of variable concentrations Quasi-product gradient concentration solution, takes 50～150 μ L standard substance gradient concentration solution and adds the adding of device for immunochromatography of horizontal positioned Sample hole, well is preposition hemofiltration film or the position being positioned on preposition hemofiltration film corresponding plastic housing future insufficiency；15～20min Inside utilize UPT readout instrument that corresponding T1, T2 and T3 band is scanned reading, draw standard according to standard substance peak area reading bent Line；Then, by serum to be measured, blood plasma, urine sample or whole blood sample after sample diluting liquid dilutes 5～200 times, 50～150 μ L are taken Add the well of the device for immunochromatography of horizontal positioned；Utilize UPT readout instrument to corresponding T1, T2 in 15～20min and T3 band is scanned reading, then carries out the quantitative of different molecular form NGAL in different sample according to the standard curve drawn； Sample diluting liquid lyophilized powder adds acquisition sample diluting liquid after 20mL deionized water fully dissolves before using.

6. a kind of synchronization combining detection by quantitative different molecular form human neutrophil lipocalin protein described in claim 1 (HNL) preparation method of device for immunochromatography, its step is as follows:

A) preparation of sample diluting liquid lyophilized powder

By 20mL, pH=7.2～7.5, containing 150～250mmol/L NaCl, 0.25～0.5% (vol/vol) Tween-20, 50～the 100mM HEPES buffer of 0.5%～2% (wt/vol) BSA after the stirring fully of mechanically or magnetically power, sample diluting liquid 0.22 μm or 0.45 μm filter is used to carry out filtration sterilization, room temperature preservation after lyophilizing；

B) preparation of standard substance lyophilized powder

The monomer of 400 μ g/mL, homodimer or the pH=7.2～7.4 of three kinds of molecular forms HNL of heterodimer, 10mmol/L PBS buffer solution 10 μ L lyophilizing obtains；

C) preparation of hemofiltration film

Hemofiltration membrane material is that glass fibre element film or the membrane material with similarity, preposition hemofiltration film and rearmounted hemofiltration film are upper and lower Two-layer mutually staggers overlap joint, is 0.0～10 μm ol/L fMLP solution and 7.0～28.5U/mL heparin sodium aquas soak through concentration；

D) preparation of sample flow pad

Sample flow cushion material is the glass fibre of high-hydrophilic or has the membrane material of similarity, be used for being connected hemofiltration film and UCP pad；

E) preparation of UCP pad

UCP pad is nitrocellulose filter or the membrane material with similarity, by 0.25～0.5mL/cm²Amount by concentration Antibody-solutions for the UCP labelling of 1mg/mL is uniformly added on UCP pad, and 30 DEG C～35 DEG C are dried；

F) preparation in analyzing film T/C district

Analyzing film material is nitrocellulose filter or the membrane material with similarity；Utilize manual method or special point sample instrument From hemofiltration film toward absorption pad direction successively even application anti-Matrix Metalloproteinase-9 antibody-solutions, anti-monomer on analyzing film HNL antibody-solutions, anti-homodimer HNL antibody-solutions and the antibody-solutions of anti-UCP traget antibody source animal immunoglobulin, Forming parallel three detection zone T1, T2, T3 and quality control region C respectively, antibody concentration used is 1mg/mL, final quantity for spray It is 0.025～0.05mL/mm, is equivalent to every millimeter of T/C wire spraying 25ng～50ng antibody；Spacing between each line is according to UCP Readout instrument detection window sets, and dries；

G) preparation of absorption pad

Absorption pad is absorbent filter or the material with similar functions；

H) preparation of liner plate

The material of liner plate is to have Self-adhesive character polyvinyl chloride plastic sheet or have the material of similar functions；

I) test strips assembles

Liner plate, two layers of blood separating membrane filtrations, UCP pad, sample flow pad, analyzing film and absorption pad are assembled according to a conventional method or use Plastic housing is packaged, thus obtains a kind of synchronization combining detection by quantitative different molecular form human neutrophil lipocalin egg The device for immunochromatography of white HNL.

7. a kind of synchronization combining detection by quantitative different molecular form human neutrophil lipocalin egg as claimed in claim 1 The preparation method of the device for immunochromatography of (HNL) in vain, it is characterised in that: antibody-solutions is that antibody is dissolved in system after antibodies buffer For obtaining；In antibody-solutions, antibody concentration is 1mg/mL, and antibodies buffer is containing 1% (vol/vol) methanol, pH=8.0 100mM Tris-HCL solution, or the 10mmol/L PBS of pH=7.2～7.4.

8. a kind of synchronization combining detection by quantitative different molecular form human neutrophil lipocalin egg as claimed in claim 1 The preparation method of the device for immunochromatography of white HNL, it is characterised in that: the preparation process of the antibody-solutions of UCP labelling is as follows,

Surface silanization and the UCP suspension of carboxylated modification that a () is averaged a diameter of 400～600nm, concentration is 5mg/mL 1mL adds 13000rpm in centrifuge tube and is centrifuged 10min, abandons supernatant；

B () adds 1mL DMSO-MES buffer in gained precipitates, water-soluble for containing 33% (V/V) DMSO and 10mM MES Liquid, carries out supersound process after slight mixing；Ultrasound condition is 20kHz, 320W, each ultrasonic 15s, stops 15s, 3 times altogether；

C () is centrifuged 10min then at 13000rpm, abandon supernatant；

(d) repetition step (b) and (c) twice；

E () addition 0.8mL DMSO-MES buffer in gained precipitates carries out supersound process after slightly mixing；

Ultrasound condition is 20kHz, 320W, each ultrasonic 15s, stops 15s, 3 times altogether；

F () adds the protein-crosslinking agent Sulfo-of the carboxyl group activating reagents EDC and 40 μ L, 30mM of 40 μ L, 30mM of new preparation NHS, then vortex concussion mixing；

G () carries out supersound process in ice bath, ultrasound condition is 20kHz, 320W, and each ultrasonic 15s stops 15s, 60 times altogether；

H () 13000rpm is centrifuged 10min, abandon supernatant；

I () adds 1.2mL DMSO-MES buffer, for the aqueous solution containing 33% (V/V) DMSO and 10mM MES, slightly mix After carry out supersound process, ultrasound condition is 20kHz, 320W, each ultrasonic 15s, stops 15s, 3 times altogether；

J () 13000rpm is centrifuged 10min, abandon supernatant；

(k) repetition step (i) and (j) 3 times；

L () adds 1mL DMSO-MES buffer, for the aqueous solution containing 33% (V/V) DMSO, 10mM MES, slight mixing is laggard Row supersound process, ultrasound condition is 20kHz, 320W, and each ultrasonic 15s stops 15s, 3 times altogether；

M () adds the anti-HNL antibody-solutions of 0.5mL, 1mg/mL, reverse mixing incubated at room 3h toward above-mentioned UCP suspension liquid, incubate Guarantee during educating that UCP does not precipitates；

N () carries out supersound process mixing after adding the 2M glycine buffer of 150 μ L, pH=11, ultrasound condition be 20kHz, 320W, each ultrasonic 15s, stop 15s, 3 times altogether；

O () 13000rpm is centrifuged 10min, abandon supernatant；

P () adds 5mL UCP traget antibody and preserves liquid, for 25mmol/L glycerol, 0.02% (vol/vol) Triton and 0.1% (wt/vol)NaN₃Aqueous solution, carry out supersound process mixing, ultrasound condition is 20kHz, 320W, each ultrasonic 15s, stops 15s, 3 times altogether；

Q () repeats step (o) with (p) once the most afterwards；

R () 13000rpm is centrifuged 10min, abandon supernatant；

S () adds containing 25mmol/L glycerol, 0.02% (vol/vol) Triton-100 and 0.1% (wt/ in gained precipitates vol)NaN₃Aqueous solution, mixing obtain UCP labelling antibody-solutions.