CN104777304A - Dry fluorescence immunoassay kit and detection method used for quantitative determination of human epidermal growth factor receptor 2 - Google Patents

Abstract

The invention discloses a dry fluorescence immunoassay kit and a detection method used for quantitative determination of human epidermal growth factor receptor 2. The dry fluorescence immunoassay kit comprises a HER-2 reagent card, a HER-2 buffer solution, and a HER-2 standard card; the HER-2 reagent card is a membrane carrier used for dry immunochromatography experiments, and comprises a lining plate with an adhesive; a sample pad, an antibody carrying membrane, and a water absorbent pad overlapped with each other are arranged on the lining plate; the antibody carrying membrane is coated with rabbit anti-mouse secondary antibody as a quality control line, and monoclonal antibody of mouse anti-human HER-2 as a detection line; the HER-2 buffer solution contains HER-2 monoclonal antibody, tris(hydroxymethyl)aminomethane (TRIS), MAK33, and methylcellulose. The dry fluorescence immunoassay kit and the detection method used for quantitative determination of human epidermal growth factor receptor 2 are high in specificity and sensitivity; time used for obtaining detection results is short; operation mode is simple; and detection results are accurate and reliable.

Description

The dry type fluorescence immunoassay kit of quantitative detection human epidermal growth factor receptor 2 and detection method

Technical field

The present invention relates to and a kind ofly measure whole blood, serum, the dry type fluorescence immunoassay kit of blood plasma and method of testing thereof, especially relate to dry type fluorescence immunoassay kit and the detection method of a kind of quantitative detection human epidermal growth factor receptor 2.

Background technology

EGF-R ELISA (epidermal growth factor receptor, abbreviation EGFR) is one of ErbB family member, and having tyrosine kinase activity, is a kind of important transmembrane receptor.EGFR is by after ligand activation, start intracellular signal transduction, the cascade reaction of adaptin, enzyme in tenuigenin, regulate transcribing of transcription factor activation gene, instruct cell migration, stick, breed, break up, apoptosis, and with the formation of tumour with worsen closely related.

Current breast cancer has become the modal malignant tumour of women, is also one of common female tumor cause of death.The patient with breast cancer HER-2/neu high expressed of nearly 20% ~ 30%, the poor prognosis of HER-2/neu high expressed and patient is closely related.HER-2/neu gene is the 2nd member of the long factor family of people's epidermis dirt.Be positioned chromosome 17q21, its a kind of molecular weight of encoding is the transmembrane glycoprotein of 185ku, has tyrosine kinase activity, structure is divided into the active territory of tyrosine kinase in the outer ligand binding domain of film, cross-film district and film.The outer part of born of the same parents of HER-2/neu albumen can drop to blood from the surface of cell, forms the soluble protein HER-ZECD that molecular weight is approximately 105ku.The cracking of HER-ZECD starts the receptor phosphorylation on cell membrane, thus have activated her-2 in nucleus and cause genetic transcription, cell proliferation; Or after ECD cracking, the intracellular tyrosine kinase holding signal transmissibility of brachymemma, corresponding experimental result thinks that the cracking of ECD can make the p95 on cell membrane send out dirt phosphorylation, thus causes the propagation of tumour cell.

HER-2 albumen is only expressed at fetal period usually, only low expression level in only a few tissue of growing up later.But there are some researches show in the human tumor of more than 30% amplification/the overexpression (as breast cancer, oophoroma, carcinoma of endometrium, carcinoma of fallopian tube, cancer of the stomach and prostate cancer etc.) that there is HER-2 gene; Wherein the primary infiltrative breast carcinoma of 20%-30% has the amplification/overexpression of HER2 gene.Research confirms: the overexpression of HER-2 and sending out dirt and attacking relevant of tumour (especially breast cancer), can improve the danger of transfer; The cell of transfection and animal model confirm, it can change the susceptibility of tumour to hormone and chemotherapeutics.

China works out in October, 2006 and has issued " breast cancer HER2 guide detection ".U.S. clinical tumourassociation (ASCO) and pathology association of the U.S. (CAP) combine and have issued " the ASCO/CAP guide that breast cancer HER2 detects is known together " on Dec 11st, 2006 and again have updated this guide in 2013, highlight the link, inside and the external mass that easily occur error in detection to control and guarantee program, be intended to make the running program of detection and the standardization to result interpretation, improve repeatability and the accuracy of HER2 detection.Therefore, quantitatively detect people's epidermis dirt growth factor receptor body 2 (HER-2) to have very important significance.The present invention relates to dry type fluorescence immunoassay detect HER-2 kit its detect improve accuracy prerequisite under, for more favourable human epidermal growth factor receptor 2 (HER-2) practical operation of ease for use aspect detect reduce personal error.

Summary of the invention

The object of this invention is to provide a kind of high specificity, highly sensitive, the time obtaining testing result is short, and mode of operation is easy, and testing result quantitatively detects dry type fluorescence immunoassay kit and the detection method of people's epidermis dirt growth factor receptor body 2 (HER-2) accurately and reliably.

The dry type fluorescence immunoassay kit of quantitative detection human epidermal growth factor receptor 2 of the present invention, the dry type fluorescence immunoassay kit of quantitative detection people epidermis dirt growth factor receptor body 2, is characterized in that: this kit comprises HER-2 reagent card, HER-2 damping fluid and HER-2 standard card;

Described HER-2 reagent card is a kind of membrane carrier for dry type immunity-chromatography test, HER-2 reagent card comprises the liner plate with bonding agent, liner plate is provided with the sample pad, antibody carrier film and the adsorptive pads that mutually overlap, and wherein, described antibody carrier film is nitrocellulose filter; Described antibody carrier film scribbles the monoclonal antibody of the mouse anti human HER-2 as detection line of the rabbit anti-mouse 2 as nature controlling line of wire or band shape anti-and wire or band shape; Described HER-2 damping fluid contains anti-HER-2 monoclonal antibody and trishydroxymethylaminomethane (TRIS), MAK33 and methylcellulose; HER-2 typical curve is stored in described HER-2 standard card.

The dry type fluorescence immunoassay kit of quantitative detection people epidermis dirt growth factor receptor body 2 of the present invention, wherein said HER-2 reagent card adopts following steps to make:

1,7g trishydroxymethylaminomethane (TRIS), 1g NaN3 and 6g methylcellulose is taken in container, take 4g polysorbas20 (TWEEN-20), add after suitable quantity of water makes polysorbas20 (TWEEN-20) dissolve completely, pour in said vesse;

2, Proclin-300 measured after 0.2ml dissolves completely in the beaker of 10ml purified water with pipettor, pour in said vesse, then add 800ml purified water in a reservoir, fully stir;

3, add the pH of solution in hydrochloric acid or sodium hydroxide solution adjustment container, control pH is between 7.95-8.05;

4, take bovine serum albumin(BSA) (BSA) 3g, tetracycline 0.04g, neomycinsulphate 1g, all pour in said vesse, fully mix;

5, by purified water, the solution in container is settled to 1000ml, with 0.2u μm of frit, obtains immune damping fluid, for subsequent use;

6,1.0mg disuccinimidyl suberate is dissolved in 50ul dimethyl sulfoxide (DMSO) (DMSO), for subsequent use; Get 2mg rabbit anti-mouse antibody be dissolved in the 0.1mol/L PB damping fluid of PH9.5 to cumulative volume be 1ml, obtain primary antibody solution, the for subsequent use disuccinimidyl suberate be dissolved in 50ul dimethyl sulfoxide (DMSO) (DMSO) is drawn with liquid-transfering gun, join in described primary antibody solution, put room temperature 90min, obtain secondary antibody solution;

7, join in concentration tube by the secondary antibody solution that step 6 obtains, then put in high speed freezing centrifuge, under 3000g, centrifugal 30min, is concentrated into 0.5ml, obtains three grades of antibody-solutions;

8,0.5ml fluorescent grain is got, add in 5ml reaction cup, after staticly settling 2 minutes, draw supernatant, then in reaction cup, add 1.5ml PH9.50.1mol/L PB, mix 30 seconds, then add in three grades of antibody-solutions of step 7 acquisition in reaction cup, keep mixing state, room temperature reaction 4 hours, obtain the conserving liquid of the fluorescent grain marked;

9, in the reaction cup described in step 8, add the TRIS solution room temperature reaction 30 minutes of 0.3ml1mol/L, in reaction cup, then add the conserving liquid that 1.5ml PH7.20.1mol/L PB cleans the fluorescent grain marked, mix 30 seconds;

10, vial is proceeded to the fluorescent grain marked that step 9 obtains by 10ml PH7.20.1mol/L PB;

11,1.5mg mouse anti human HER-2 antibody is dissolved in the conserving liquid of the fluorescent grain marked that 5ml step 9 obtains, the conserving liquid of the fluorescent grain this marked again joins in the vial in step 10, mixing reaction 2 hours, in vial, add 5ml PH7.20.1mol/LPB again clean the Immunofluorescent particles marked, and mix 30 seconds;

12, with 50ml fluorescent grain conserving liquid, Immunofluorescent particles is proceeded to vial, prepare Immunofluorescent particles concentration be 0.05% normal concentration fluorescent grain use liquid;

13, the immune damping fluid that solution step 12 obtained and step 5 obtain mixes according to the volume ratio of 1:1, obtains HER-2 fluorescent grain reagent (its working concentration is 0.025%);

14, by the HER-2 fluorescent grain reagent of three of step 7 gained grades of antibody-solutions and step 13 gained respectively wire or banded connection on antibody carrier film to form nature controlling line and detection line.And mutually bonding sample pad, antibody carrier film, absorbing membrane with on the liner plate of bonding agent, obtain described HER-2 reagent card overlap joint;

Described HER-2 damping fluid adopts following steps to make

1, get Tris6.06g, NaCl13.0g, Zncl20.05g, Proclin-3000.2ml and MgCl20.05g in flask, then in flask, add 800ml purified water, fully stir, solid is wherein dissolved completely;

2, add pH value of solution in hydrochloric acid or sodium hydroxide solution tune flask, control pH is within the scope of 7.35-7.45;

3, taking bovine serum albumin(BSA) (BSA) 3g pours in above-mentioned beaker;

4, be settled to 1000ml by purified water, with 0.2u μm of frit, obtain reagent 1 dilution, for subsequent use;

5, get the anti-HER-2 monoclonal antibody of 10mg, add in 5ml physiological saline, join in concentration tube, centrifugal 20 minutes of 3000RPM, is concentrated into 1 milliliter, obtains concentrate;

6, obtain in concentrate the 0.1M NaIO4 solution adding 0.2ml to step 5, under room temperature, lucifuge stirs 20 minutes, and then load in bag filter, dialyse with the sodium-acetate buffer of 1mM pH4.4,4 DEG C are spent the night, and collects and retains liquid;

7, obtain to retain in liquid to step 6 and add 0.2M PH9.5 carbonate buffer solution, make pH be elevated to 9.0, then add the anti-HER-2 monoclonal antibody of 2.5mg immediately, stirs 2 hours, then add the 4mg/ml NaBH4 solution of 0.1ml, mix, at putting 4 DEG C 2 hours;

8, step 7 is obtained solution to load in bag filter, dialyse with 0.15M PH7.4PBS, 4 DEG C are spent the night, and collect and retain liquid;

9, obtain to retain in liquid to step 8 and dropwise add equal-volume saturated ammonium sulfate, put 4 DEG C 1 hour, then 3000rpm centrifugal half an hour, abandon supernatant, sediment semi-saturation ammonium sulfate washes secondary, and last sediment is dissolved in the PBS of 0.15M pH7.4 of 20ml, obtains solution;

10, solution step 9 obtained loads in bag filter, dialyse 5 hours with the PB damping fluid of 0.15M PH7.4, remove ammonium ion, collect and to retain after liquid 10, 000rpm removes precipitation in centrifugal 30 minutes, collect supernatant, supernatant according to volume ratio is 1 volume: the ratio of the MgCl2 of 100 volumes, add 1M MgCl2 solution, obtain the conjugate of anti-HER-2 monoclonal antibody, by the conjugate of anti-HER-2 monoclonal antibody collected, with reagent 1 dilution that above-mentioned steps 4 obtains, conjugate with the anti-HER-2 monoclonal antibody of 1 volume: the volume ratio of reagent 1 dilution of 1000 volumes mixes, obtain HER-2 damping fluid,

Described HER-2 standard card adopts following steps to make:

1, HER-2 antigen concentration is respectively 0,15,45,90,80, the titer of 350ng/ml to add in 6 HER-2 damping fluids after mixing respectively, then solution after mixing is got in the sample pad on 100ul instillation HER-2 reagent card, wait for after 15 minutes, dry type fluorescence immunity analyzer is used to detect, fluorescent value detection obtained is coordinate axis Y-axis, above-mentioned concentration of standard solution is that X-axis carries out four parameter fittings, then 4 reference records the Fitting Calculation obtained, in standard card, both obtained HER-2 standard card.

The detection method of the dry type fluorescence immunoassay kit of quantitative detection people epidermis dirt growth factor receptor body 2 of the present invention, comprises the steps:

1), by sample to be detected get 10ul to join in HER-2 damping fluid, then slightly mix, do not produce dirt bubble;

2), taking out 100ul in HER-2 damping fluid upon mixing joins in the sample pad of HER-2 reagent card;

3), after 15 minutes, HER-2 reagent card and HER-2 standard card are inserted in dry type fluorescence immunity analyzer respectively and detects, carry out the Fitting Calculation according to four parameters in detected fluorescent value and described HER-2 standard card, the HER-2 concentration of sample to be detected can be obtained.

The dry type fluorescence immunoassay kit of quantitative detection people epidermis dirt growth factor receptor body 2 of the present invention and detection method, comprise HER-2 reagent card, HER-2 damping fluid and HER-2 standard card; Described HER-2 reagent card is a kind of membrane carrier for dry type immunity-chromatography test, HER-2 reagent card comprises the liner plate with bonding agent, liner plate is provided with the sample pad, antibody carrier film and the adsorptive pads that mutually overlap, and wherein, described antibody carrier film is nitrocellulose filter; Described antibody carrier film scribbles the monoclonal antibody of the mouse anti human HER-2 as detection line of the rabbit anti-mouse 2 as nature controlling line of wire or band shape anti-and wire or band shape; Described HER-2 damping fluid contains anti-HER-2 monoclonal antibody and trishydroxymethylaminomethane (TRIS), MAK33 and methylcellulose; HER-2 typical curve is stored in described HER-2 standard card.Shown by the great many of experiments of thousands of example, the dry type fluorescence immunoassay kit of quantitative detection people epidermis dirt growth factor receptor body 2 of the present invention and detection method, there is high specificity, highly sensitive, the time obtaining testing result is short, and mode of operation is easy, and testing result is feature accurately and reliably, accuracy rate in experiment, up to 100%, has outstanding substantive distinguishing features and significant progress.

Do to describe in detail into-step to the quantitative detection dry type fluorescence immunoassay kit of human epidermal growth factor receptor 2 of the present invention and detection method below.

Embodiment

The dry type fluorescence immunoassay kit of quantitative detection people epidermis dirt growth factor receptor body 2 of the present invention, this kit comprises HER-2 reagent card, HER-2 damping fluid and HER-2 standard card;

Described HER-2 reagent card is a kind of membrane carrier for dry type immunity-chromatography test, HER-2 reagent card comprises the liner plate with bonding agent, liner plate is provided with the sample pad, antibody carrier film and the adsorptive pads that mutually overlap, and wherein, described antibody carrier film is nitrocellulose filter; Described antibody carrier film scribbles the monoclonal antibody of the mouse anti human HER-2 as detection line of the rabbit anti-mouse 2 as nature controlling line of wire or band shape anti-and wire or band shape; Described HER-2 damping fluid contains anti-HER-2 monoclonal antibody and trishydroxymethylaminomethane (TRIS), MAK33 and methylcellulose; HER-2 typical curve is stored in described HER-2 standard card.

Above-mentioned HER-2 reagent card adopts following steps to make:

1,7g trishydroxymethylaminomethane (TRIS), 1g NaN3 and 6g methylcellulose is taken in container, take 4g leaf temperature 20 (TWEEN-20), add after suitable quantity of water makes polysorbas20 (TWEEN-20) dissolve completely, pour in said vesse;

2, Proclin-300 measured after 0.2ml dissolves completely in the beaker of 10ml purified water with pipettor, pour in said vesse, then add 800ml purified water in a reservoir, fully stir;

3, add the pH of solution in hydrochloric acid or sodium hydroxide solution adjustment container, control pH is between 7.95-8.05;

4, take bovine serum albumin(BSA) (BSA) 3g, tetracycline 0.04g, neomycinsulphate 1g, all pour in said vesse, fully mix;

5, by purified water, the solution in container is settled to 1000ml, with 0.2 μ μm frit, obtains immune damping fluid, for subsequent use;

6,1.0mg disuccinimidyl suberate is dissolved in 50ul dimethyl sulfoxide (DMSO) (DMSO), for subsequent use; Get 2mg rabbit anti-mouse antibody be dissolved in the 0.1mol/LPB damping fluid of PH9.5 to cumulative volume be 1ml, obtain primary antibody solution, the for subsequent use disuccinimidyl suberate be dissolved in 50ul dimethyl sulfoxide (DMSO) (DMSO) is drawn with liquid-transfering gun, join in described level antibody-solutions, put room temperature 90min, obtain secondary antibody solution;

7, join in concentration tube by the secondary antibody solution that step 6 obtains, then put in high speed freezing centrifuge, under 3000g, centrifugal 30min, is concentrated into 0.5ml, obtains three grades of antibody-solutions;

8,0.5ml fluorescent grain is got, add in 5ml reaction cup, after staticly settling 2 minutes, draw supernatant, then in reaction cup, add 1.5m1PH9.50.1mol/L PB, mix 30 seconds, then add in three grades of antibody-solutions of step 7 acquisition in reaction cup, keep mixing state, room temperature reaction 4 hours, obtain the conserving liquid of the fluorescent grain marked;

9, in the reaction cup described in step 8, add the TRIS solution room temperature reaction 30 minutes of 0.3ml 1mol/L, in reaction cup, then add the conserving liquid that 1.5ml PH7.20.1mol/L PB cleans the fluorescent grain marked, mix 30 seconds;

10, vial is proceeded to the fluorescent grain marked that step 9 obtains by 10ml PH7.20.1mo1/L PB;

11,1.5mg mouse anti human HER-2 antibody is dissolved in the conserving liquid of the fluorescent grain marked that 5ml step 9 obtains, the conserving liquid of the fluorescent grain this marked again joins in the vial in step 10, mixing reaction 2 hours, in vial, add 5ml PH7.20.1mol/L PB again clean the Immunofluorescent particles marked, and mix 30 seconds;

12, with 50ml fluorescent grain conserving liquid, Immunofluorescent particles is proceeded to vial, prepare Immunofluorescent particles concentration be 0.05% normal concentration fluorescent grain use liquid;

13, the immune damping fluid that solution step 12 obtained and step 5 obtain mixes according to the volume ratio of 1:1, obtains HER-2 fluorescent grain reagent (its working concentration is 0.025%);

14, by the HER-2 fluorescent grain reagent of three of step 7 gained grades of antibody-solutions and step 13 gained respectively wire or banded connection on antibody carrier film to form nature controlling line and detection line.And mutually bonding sample pad, antibody carrier film, absorbing membrane with on the liner plate of bonding agent, obtain described HER-2 reagent card overlap joint;

Described HER-2 damping fluid adopts following steps to make:

1, get Tris6.06g, NaCl13.0g, Zncl20.05g, Proclin-3000.2ml and MgCl20.05g in flask, then in flask, add 800ml purified water, fully stir, solid is wherein dissolved completely;

2, add pH value of solution in hydrochloric acid or sodium hydroxide solution tune flask, control pH is within the scope of 7.35-7.45;

3, taking bovine serum albumin(BSA) (BSA) 3g pours in above-mentioned beaker;

4, be settled to 1000ml by purified water, with 0.2 μ μm frit, obtain reagent 1 dilution, for subsequent use;

5, get the anti-HER-2 monoclonal antibody of 10mg, add in 5ml physiological saline, join in concentration tube, centrifugal 20 minutes of 3000RPM, is concentrated into 1 milliliter, obtains concentrate;

6, obtain in concentrate the 0.1M NaIO4 solution adding 0.2ml to step 5, under room temperature, lucifuge stirs 20 minutes, and then load in bag filter, dialyse with the sodium-acetate buffer of 1mM pH4.4,4 DEG C are spent the night, and collects and retains liquid;

7, obtain to retain in liquid to step 6 and add 0.2M PH9.5 carbonate buffer solution, make pH be elevated to 9.0, then add the anti-HER-2 monoclonal antibody of 2.5mg immediately, stirs 2 hours, then add the 4mg/ml NaBH4 solution of 0.1ml, mix, at putting 4 DEG C 2 hours;

8, step 7 is obtained solution to load in bag filter, dialyse with 0.15M PH7.4PBS, 4 DEG C are spent the night, and collect and retain liquid;

9, obtain to retain in liquid to step 8 and dropwise add equal-volume saturated ammonium sulfate, put 4 DEG C 1 hour, then 3000rpm centrifugal half an hour, abandon supernatant, sediment semi-saturation ammonium sulfate washes secondary, and last sediment is dissolved in the PBS of 0.15M pH7.4 of 20ml, obtains solution;

10, solution step 9 obtained loads in bag filter, use 0.15M PH7, the PB damping fluid of 4 is dialysed 5 hours, remove ammonium ion, collect and to retain after liquid 10, 000rpm removes precipitation in centrifugal 30 minutes, collect supernatant, supernatant according to volume ratio is 1 volume: the ratio of the MgCl2 of 100 volumes, add 1M MgCl2 solution, obtain the conjugate of anti-HER-2 monoclonal antibody, by the conjugate of anti-HER-2 monoclonal antibody collected, with reagent 1 dilution that above-mentioned steps 4 obtains, conjugate with the anti-HER-2 monoclonal antibody of 1 volume: the volume ratio of reagent 1 dilution of 1000 volumes mixes, obtain HER-2 damping fluid,

Described HER-2 standard card adopts following steps to make:

1, HER-2 antigen concentration is respectively 0,15,45,90,80, the titer of 350ng/ml to add in 6 HER-2 damping fluids after mixing respectively, then solution after mixing is got in the sample pad on 100ul instillation HER-2 reagent card, wait for after 15 minutes, dry type fluorescence immunity analyzer is used to detect, fluorescent value detection obtained is coordinate axis Y-axis, above-mentioned concentration of standard solution is that X-axis carries out four parameter fittings, then 4 reference records the Fitting Calculation obtained, in standard card, both obtained HER-2 standard card.

The detection method of the dry type fluorescence immunoassay kit of quantitative detection human epidermal growth factor receptor 2 of the present invention, comprises the steps:

1), by sample to be detected get 10ul to join in HER-2 damping fluid, then slightly mix, do not produce dirt bubble;

2), taking out 100ul in HER-2 damping fluid upon mixing joins in the sample pad of HER-2 reagent card;

3), after 15 minutes, HER-2 reagent card and HER-2 standard card are inserted in dry type fluorescence immunity analyzer respectively and detects, carry out the Fitting Calculation according to four parameters in detected fluorescent value and described HER-2 standard card, the HER-2 concentration of sample to be detected can be obtained.

Claims (3)

1. quantitatively detect the dry type fluorescence immunoassay kit of human epidermal growth factor receptor 2, it is characterized in that: this kit comprises HER-2 reagent card, HER-2 damping fluid and HER-2 standard card;

Described HER-2 reagent card is a kind of membrane carrier for dry type immunity-chromatography test, HER-2 reagent card comprises the liner plate with bonding agent, liner plate is provided with the sample pad, antibody carrier film and the adsorptive pads that mutually overlap, and wherein, described antibody carrier film is nitrocellulose filter; Described antibody carrier film scribbles the monoclonal antibody of the mouse anti human HER-2 as detection line of the rabbit anti-mouse 2 as nature controlling line of wire or band shape anti-and wire or band shape; Described HER-2 damping fluid contains anti-HER-2 monoclonal antibody and trishydroxymethylaminomethane (TRIS), MAK33 and methylcellulose; HER-2 typical curve is stored in described HER-2 standard card.

2., according to the dry type fluorescence immunoassay kit of quantitative detection people epidermis dirt growth factor receptor body 2 according to claim 1, it is characterized in that: described HER-2 reagent card adopts following steps to make:

1,7g trishydroxymethylaminomethane (TRIS), 1g NaN is taken ₃with 6g methylcellulose in container, take 4g polysorbas20 (TWEEN-20), add after suitable quantity of water makes polysorbas20 (TWEEN-20) dissolve completely, pour in said vesse;

2, Proclin-300 measured after 0.2ml dissolves completely in the beaker of 10ml purified water with pipettor, pour in said vesse, then add 800ml purified water in a reservoir, fully stir;

3, add the pH of solution in hydrochloric acid or sodium hydroxide solution adjustment container, control pH is between 7.95-8.05;

4, take bovine serum albumin(BSA) (BSA) 3g, tetracycline 0.04g, neomycinsulphate 1g, all pour in said vesse, fully mix;

5, by purified water, the solution in container is settled to 1000ml, with 0.2 μ μm frit, obtains immune damping fluid, for subsequent use;

6,1.0mg disuccinimidyl suberate is dissolved in 50ul dimethyl sulfoxide (DMSO) (DMSO), for subsequent use; Get 2mg rabbit anti-mouse antibody be dissolved in the 0.1mol/L PB damping fluid of PH9.5 to cumulative volume be 1ml, obtain primary antibody solution, the for subsequent use disuccinimidyl suberate be dissolved in 50ul dimethyl sulfoxide (DMSO) (DMSO) is drawn with liquid-transfering gun, join in described primary antibody solution, put room temperature 90min, obtain secondary antibody solution;

7, join in concentration tube by the secondary antibody solution that step 6 obtains, then put in high speed freezing centrifuge, under 3000g, centrifugal 30min, is concentrated into 0.5ml, obtains three grades of antibody-solutions;

8,0.5ml fluorescent grain is got, add in 5ml reaction cup, after staticly settling 2 minutes, draw supernatant, then in reaction cup, add 1.5ml PH9.50.1mol/L PB, mix 30 seconds, then add in three grades of antibody-solutions of step 7 acquisition in reaction cup, keep mixing state, room temperature reaction 4 hours, obtain the conserving liquid of the fluorescent grain marked;

9, in the reaction cup described in step 8, add the TRIS solution room temperature reaction 30 minutes of 0.3ml 1mol/L, in reaction cup, then add the conserving liquid that 1.5ml PH7.20.1mol/L PB cleans the fluorescent grain marked, mix 30 seconds;

10, vial is proceeded to the fluorescent grain marked that step 9 obtains by 10ml PH7.20.1mol/L PB;

11,1.5mg mouse anti human HER-2 antibody is dissolved in the conserving liquid of the fluorescent grain marked that 5ml step 9 obtains, the conserving liquid of the fluorescent grain this marked again joins in the vial in step 10, mixing reaction 2 hours, in vial, add 5mlPH7.20.1mol/LPB again clean the Immunofluorescent particles marked, and mix 30 seconds;

12, with 50ml fluorescent grain conserving liquid, Immunofluorescent particles is proceeded to vial, prepare Immunofluorescent particles concentration be 0.05% normal concentration fluorescent grain use liquid;

13, the immune damping fluid that solution step 12 obtained and step 5 obtain mixes according to the volume ratio of 1:1, obtains HER-2 fluorescent grain reagent (its working concentration is 0.025%);

14, by the HER-2 fluorescent grain reagent of three of step 7 gained grades of antibody-solutions and step 13 gained respectively wire or banded connection on antibody carrier film to form nature controlling line and detection line.And mutually bonding sample pad, antibody carrier film, absorbing membrane with on the liner plate of bonding agent, obtain described HER-2 reagent card overlap joint;

Described HER-2 damping fluid adopts following steps to make:

1, Tris6.06g, NaCl13.0g, Zncl is got ₂0.05g, Proclin-3000.2ml and MgCl ₂0.05g, in flask, then adds 800ml purified water, fully stirs in flask, and solid is wherein dissolved completely;

2, add pH value of solution in hydrochloric acid or sodium hydroxide solution tune flask, control pH is within the scope of 7.35-7.45;

3, taking bovine serum albumin(BSA) (BSA) 3g pours in above-mentioned beaker;

4, be settled to 1000ml by purified water, with 0.2 μ μm frit, obtain reagent 1 dilution, for subsequent use;

5, get the anti-HER-2 monoclonal antibody of 10mg, add in 5ml dirt reason salt solution, join in concentration tube, centrifugal 20 minutes of 3000RPM, is concentrated into 1 milliliter, obtains concentrate;

6, the 0.1M NaIO adding 0.2ml is obtained in concentrate to step 5 ₄solution, under room temperature, lucifuge stirs 20 minutes, and then load in bag filter, dialyse with the sodium-acetate buffer of 1mM pH4.4,4 DEG C are spent the night, and collects and retains liquid;

7, in step 6 acquisition reservation liquid, add 0.2M PH9.5 carbonate buffer solution, make pH be elevated to 9.0, then add the anti-HER-2 monoclonal antibody of 2.5mg immediately, stir 2 hours, then add the 4mg/ml NaBH of 0.1ml ₄solution, mixing, at putting 4 DEG C 2 hours;

8, step 7 is obtained solution to load in bag filter, dialyse with 0.15M PH7.4PBS, 4 DEG C are spent the night, and collect and retain liquid;

9, obtain to retain in liquid to step 8 and dropwise add equal-volume saturated ammonium sulfate, put 4 DEG C 1 hour, then 3000rpm centrifugal half an hour, abandon supernatant, sediment semi-saturation ammonium sulfate washes secondary, and last sediment is dissolved in the PBS of the 0.15MpH7.4 of 20ml, obtains solution;

10, solution step 9 obtained loads in bag filter, dialyses 5 hours with the PB damping fluid of 0.15M PH7.4, removes ammonium ion, collect and to retain after liquid 10,000rpm removes precipitation in centrifugal 30 minutes, and collecting supernatant, is the supernatant of 1 volume according to volume ratio: the MgCl of 100 volumes ₂ratio, add 1M MgCl ₂solution, obtain the conjugate of anti-HER-2 monoclonal antibody, by the conjugate of anti-HER-2 monoclonal antibody collected, with reagent 1 dilution that above-mentioned steps 4 obtains, conjugate with the anti-HER-2 monoclonal antibody of 1 volume: the volume ratio of reagent 1 dilution of 1000 volumes mixes, and obtains HER-2 damping fluid;

Described HER-2 standard card adopts following steps to make:

1, HER-2 antigen concentration is respectively 0,15,45,90,80, the titer of 350ng/ml to add in 6 HER-2 damping fluids after mixing respectively, then solution after mixing is got in the sample pad on 100ul instillation HER-2 reagent card, wait for after 15 minutes, dry type fluorescence immunity analyzer is used to detect, fluorescent value detection obtained is coordinate axis Y-axis, above-mentioned concentration of standard solution is that X-axis carries out four parameter fittings, then 4 reference records the Fitting Calculation obtained, in standard card, both obtained HER-2 standard card.

3. the detection method quantitatively detecting the dry type fluorescence immunoassay kit of people's epidermis dirt growth factor receptor body 2 as claimed in claim 1 or 2, is characterized in that: comprise the steps:

1), by sample to be detected get 10ul to join in HER-2 damping fluid, then slightly mix, do not produce bubble;

2), taking out 100ul in HER-2 damping fluid upon mixing joins in the sample pad of HER-2 reagent card;

3), after 15 minutes, HER-2 reagent card and HER-2 standard card are inserted in dry type fluorescence immunity analyzer respectively and detects, carry out the Fitting Calculation according to four parameters in detected fluorescent value and described HER-2 standard card, the HER-2 concentration of sample to be detected can be obtained.