CN1624478A - Detecting reagent box of nagopharynx tissue specificity gene coding protein, its preparation and application - Google Patents

Abstract

The invention relates to a immunology test for secreted protein. Firstly, design a sect of polypeptide to prepare for polypeptide antibody of rabbit to human in accordance with amino acid sequence of nasopharynx tissue idiosyncratic gene NASG, meanwhile, use confluent protein of NASG to prepare confluent protein antibody of rabbit to human; make the purified NASG recombinant protein as the standard, prepare for ELISA kit for NASG protein by double antibody sandwich method; choose secretion from nasopharynx to do ELISA test, test protein expressing quality of NASG in secretion of nasopharynx from different groups and do premature and rapid diagnosis without injure for nasopharyngeal carcinoma subject.

Description

Detection kit, preparation and the application thereof of nasopharyngeal tissue specific gene encoding proteins

Technical field:

The present invention relates to the immunology detection of biological substance, be specifically related to a kind of detection of secreted protein.

Background technology:

Nasopharyngeal carcinoma (NPC) is China's south common malignancy, and Guangdong is the district occurred frequently.Nasopharyngeal carcinoma is meant the cancerous swelling that the mucous membrane of nasopharynx epithelium takes place, mostly be low differentiated squamous-cell carcinomas greatly, its grade of malignancy height, site of pathological change is hidden, particularly pharyngeal recess and nasopharynx top person, early symptom is not obvious, thereby being difficult to early detection, the sing misdiagnosis and mistreatment rate is higher, can reach 12.2%, thereby in the nasopharyngeal carcinoma of making a definite diagnosis, its 5 years survival rates are paced up and down about 50%～60% for a long time.Nasopharyngeal carcinoma is carried out early diagnosis so that early treatment, and the survival rate that improves the patient is one of important topic of NPC clinical research always.At present have multiple to the early stage detection method of nasopharyngeal carcinoma, include: the antibody test of Epstein-Barr virus serology label such as viral capsid antigen one immunoglobulin A (EBVCA 2IgA), Epstein-Barr virus latent membrane protein, the early stage complex antigen of Epstein-Barr virus (EBV 2-EA) IgG, the detection of nasopharyngeal carcinoma related neoplasms label such as interleukins, TNF, iuntercellular adhesion molecule, CYFRA2121 and Telomerase etc.Though the independent or use in conjunction of these indexs provides Useful Information for the diagnosis of nasopharyngeal carcinoma, yet they all lack enough sensitivity and specificity.We know, Epstein-Barr virus is not only relevant with the generation of nasopharyngeal carcinoma, and it is closely related with infection of Burkitt lymthoma, the upper respiratory tract etc., recently also be reported in the various t cell lymphomas and detect epstein barr virus dna, thereby nasopharyngeal carcinoma patient Epstein-Barr virus antibody and pathology are made a definite diagnosis and compared positive coincidence rate only for being 72.6%～77.3%.And be not that the generation of all nasopharyngeal carcinoma is all relevant with Epstein-Barr virus, 90% above crowd infected EBV before growing up, detect EBV and can not prove that also EBV is directly relevant with NPC in the NPC patients serum.Therefore, though the generation of Epstein-Barr virus associated antibodies titre and nasopharyngeal carcinoma, development have very high correlativity, its detection method also has superiority, but still can only be as a kind of index of correlation and play suggesting effect, can not be used as nasopharyngeal carcinoma examining finger veins mark really.If lack pathological diagnosis,, also be difficult to as the foundation of implementing radiation and chemotherapy even Epstein-Barr virus associated antibodies titre is high again.Except pathologic diagnosis, still there is not other early stage, non-invasive inspection method at present to advanced nasopharyngeal carcinoma (NPC); Even pathologic diagnosis also is difficult to the disposable pathological tissues of getting, can't avoid that minimal disease repeatedly had a sampling of wound property.Though the case of nasopharyngeal carcinoma atypia focus type or mucous membrane mo(u)ld bottom half is through repeatedly biopsy, Chang Yinwu pathology section positive findings and delay treatment; For the case after the radiotherapy, when its nasopharyngeal tissue performance is not true to type, judging that its pathology is on recurrence or the radiotherapy side effect, also in a dilemma, especially the patient who occurs cranium brain symptom after some radiotherapy, it is that RE or cancer kitchen range develop to encephalic that every inspection (comprising pathologic finding) all is difficult to accurately determine the end, greatly influence treatment decision-making.In sum, set up a kind of non-invasive detection method that can carry out examination, diagnosis to the nasopharyngeal carcinoma patient, significant to early screening, diagnosis, prevention and the treatment etc. of nasopharyngeal carcinoma.

By inhibition subtractive hybridization associating cDNA microarray technology, we have cloned specific expressed nasopharyngeal carcinoma tumor susceptibility gene candidate---the NASG of the upper respiratory tract, (number of patent application: 02139605.1, gene order accession number: AF439448).We analyze by structure and function to the NASG gene (claiming the SPLUNC gene again) of new clone, find that NASG albumen is a kind of secreted protein, expressed in abundance in normal person and pharynx nasalis chronic inflammation patient's nasopharyngeal tissue, and in nasopharyngeal carcinoma patient's nasopharyngeal tissue, express significantly downward modulation.Thereby how the content by detecting NASG albumen and do not obtain with how having wound NASG albumen then be set up early stage, no wound detect need solution problem.

Summary of the invention:

The present invention is intended to detect the no wound quick diagnosis of the content realization of NASG albumen to early stage nasopharyngeal carcinoma patient by preparing a kind of NASG protein detection kit thereby obtain secretion from pharynx nasalis.

The technical scheme that realizes the foregoing invention purpose is:

The detection kit of a kind of nasopharyngeal tissue specific gene encoding proteins NASG, comprise that bag is anti-by good ELISA Plate, HRP mark two, common reagent Tween-20 and chromogenic substrate tetramethyl benzidine TMB, be that other is equipped with the anti-NASG polypeptide antibody of the rabbit of horseradish peroxidase HRP mark through the NASG fusion antibody that coating buffer is handled and retardance liquid is handled in the ELISA Plate hole of this kit; The concentration of the NASG fusion antibody in the kit ELISA Plate hole is good with 1mg/ml.The preparation method of the detection kit of nasopharyngeal tissue specific gene encoding proteins NASG comprises the preparation of the anti-human polypeptides antibody of rabbit, and the preparation of the anti-people NASG of rabbit fusion antibody is when (1), the anti-human polypeptides antibody of preparation rabbit, according to the amino acid sequence of NASG, designing and synthesizing 21 amino acid whose peptide sequences is: Cys, Lys, Val, The, Asp, Pro, Gln, Leu, Leu, Glu, Leu, Gly, Leu, Val, Gln, Ser, Pro, Asp, Gly, His, Arg; (2), with the anti-people NASG of rabbit fusion antibody, be cushioned liquid with bag and be diluted to 1ug/ml and join in the ELISA Plate hole, 4 ℃ of bags are spent the night; (3), remove not wrap the liquid of quilt, add the phosphate buffer PBST that contains 0.5% bovine serum albumin with containing 0.1%Tween-20 phosphate buffer PBST flushing back, hatched 1 hour for 37 ℃, the PBST wash-out, it is standby to put 4 ℃ of preservations.

Nasopharyngeal tissue specific gene encoding proteins test kit is applied to as the detection method of NASG protein content in the human body nasopharyngeal secretions of nasopharyngeal carcinoma early diagnosis index:

(1), obtaining of nasopharyngeal secretions: normal saline flushing nasal cavity, under the nose visor, be inserted into pharynx nasalis with thin cotton swab, placed 5～8 minutes, take out cotton swab, with cotton balls put into the bottom hollow, be with the little centrifuge tube of centrifugal collection tube outward, at 4 ℃ of centrifugal nasopharyngeal secretionses that obtain of following 7500xg;

(2), with antigen-nasopharyngeal secretions of obtaining by usefulness physiological saline dilution in 1: 10 after, join in the ELISA Plate hole of several kits, the NASG gene recombinant protein that adds purifying simultaneously in several do not add the ELISA Plate hole of determined antigen is done standard sample; Under 37 ℃ of conditions, in 100 rev/mins shaking table, hatched 3 hours,, blot residual liquid with the unconjugated nasopharyngeal secretions of PBST flush away;

(3) the anti-people NASG of the rabbit of HRP mark polypeptide antibody is joined in the ELISA Plate that adds antigen, hatch not binding antibody of back flush away, add chromogenic substrate TMB, room temperature was placed 20 minutes, join the sulfuric acid cessation reaction of 2M, in wavelength readings, the metering of microplate reader 420nm;

(4), judge the height of protein content in patient's nasopharyngeal secretions with NASG concentration contrast in the concentration that records and the normal population nasopharyngeal secretions.

Description of drawings

Be described in further detail the present invention below in conjunction with accompanying drawing:

The NASG-GST fusion of Fig. 1 for from Escherichia coli, inducing;

The ELISA detection kit that Fig. 2 prepares after the ELISA Plate for antibody sandwich;

Fig. 3 detects the expression of NASG in normal nasopharyngeal secretion and nasopharyngeal carcinoma secretion for Western blot;

1,3,4: nasopharyngeal carcinoma patient's nasopharyngeal secretions; 2,5-8; Normal person and chronic inflammation patient nasopharyngeal secretions

Fig. 4 is the expression of NASG in normal nasopharyngeal tissue and nasopharyngeal carcinoma biopsy specimen

A:Northern blot, I nasopharyngeal carcinoma tissue, the II normal structure,

B:RT-PCR; .1,2. normal adult nasopharynx 3. people's embryo nasopharynxs

4.HNE1 5～8.NPC biopsy specimen

One, the preparation of the ELISA kit of NASG (SPLUNC):

1, the preparation of NASG (SPLUNC) antibody:

1), the anti-human polypeptide antibody of preparation rabbit:

1., according to the amino acid sequence of NASG, design and 21 amino acid whose NASG polypeptide of chemical synthesis, the sequence of this polypeptide is: Cys, Lys, Val, The, Asp, Pro, Gln, Leu, Leu, Glu, Leu, Gly, Leu, Val, Gln, Ser, Pro, Asp, Gly, His, Arg, polypeptide and keyhole limpet hemocyanin (keyhole limpet hemocyanin, KLH) is crosslinked to increase immunogenicity.

2., polypeptide is dissolved in phosphate (PBS) buffer solution, be injected to new zealand rabbit, initial dose is 300 μ g/kg, and the dosage of booster immunization is about about 1/4 of initial dose. Per 2～3 all booster immunizations once, immunity is 4 times altogether, and the antiserum of acquisition is purified into the IgG (NASG antibody 1) of anti-NASG (PLUNC) with the affinitive layer purification kit (concrete steps are strictly undertaken by the specification of Pierce company) of Pierce company;

2), the anti-human NASG fusion antibody of preparation rabbit

1., the total length of pcr amplification NASG, be cloned into the prokaryotic expression carrier PGEX with GST target will, transform e. coli jm109, lower induce the NASG protein expression with isopropyl-β-D-thiogalactoside (IPTG) in 30 ℃, be purified into NASG-GST fusion (Fig. 1) (concrete operations are strictly undertaken by the specification of company) with the GST protein purification kit of Pharmacia company.

2., with this fusion protein immunization NZw, the antibody (NASG antibody 2) that the preparation rabbit is anti-human and usefulness affinitive layer purification (method is with aforementioned polypeptide antibody).

3), polypeptide amalgamation protein antibody tires and specific detection

1., synthetic polypeptide be cushioned liquid (take by weighing 8.4 gram sodium bicarbonates, 3.56 gram sodium carbonate are dissolved in 1 liter of deionized water, accent pH value to 9.50) with bag be dissolved as 10ug/ml, be added to 96 hole ELISA Plate by the 100ul/ hole, 4 ℃ are spent the night.

2., take out ELISA Plate, outwell bag and be cushioned liquid, contain PBS (PBST) flushing 3 times of 0.1%Tween-20, add 200ul retardance liquid (PBST that contains 1% bovine serum albumin(BSA)) and stop nonspecific binding site, hatched 1 hour PBST wash-out 2 times for 37 ℃.

3., by 1: 500; 1: 1000; 1: 2000; 1: 5000: 1: 10000; 1: 100000; 1: 1000000 totally 7 dilutabilitys (dilution: dissolving 80 gram sodium chloride in one liter of deionized water, 11.6 gram sodium hydrogen phosphates, 2 gram potassium dihydrogen phosphates, 2 gram potassium chloride are transferred pH value to 7.2) add the two kinds of NASG antiserums in front.Hatched 1 hour for 37 ℃, PBST washes 3 times.

4., add the two anti-of horseradish peroxidase (HRP) mark, hatched 45 minutes for 37 ℃, PBST washes 5 times.

5., mix 6ml tetramethyl benzidine (TMB) reagent A and 6ml TMB reagent B, the 100ul/ hole joins 96 orifice plates, incubated at room 15 minutes, the hole of the visible antibody test positive is blueness, we prepare tiring of antibody can reach 1: 1000000.

6., get nasopharyngeal secretions and human nasopharyngeal epithelioma 1 lysate (50mM Tris, 150mM NaCl, the 1mM disodium ethylene diamine tetraacetate, 1%Triton X-100, PH:8.0), carry out the SDS-PAGE electrophoresis, be transferred to nitrocellulose filter, program is carried out Western blot analysis routinely, and ECL is luminous, radioautograph, routine is developed a film.The band that as seen one clauses and subclauses are only arranged in nasopharyngeal secretions and the human nasopharyngeal epithelioma 1 lysate illustrates that we have the specificity of height by the antibody of made.

2, the preparation of the ELISA detection kit of NASG

1), the selection of coated antibody and enzyme labelled antibody working concentration

According to the BCA determination of protein concentration kit instructions operation of Pierce company, measure the concentration of antibody and antigen.Adopt the chessboard assay method of standard then, be cushioned liquid (prescription the same) with bag and NASG fusion antibody (antibody 2) be diluted to concentration is 10,1,0.1ug/ml, wrap quilt respectively on elisa plate, each concentration comprises three stringers, 4 ℃ are spent the night, PBST washing 3 times.Therein one walk crosswise each bag added strong positive antigen liquid (the NASG-GST fusion of 10ug/ml) in the hole, add weak positive antigen liquid (the NASG-GST fusion of 0.001ug/ml) during another is walked crosswise, the third line adds negative control.37 ℃ are incubated 2 hours, PBST washing 4 times.The NASG polypeptide antibody (enzyme labelled antibody 1) that adds the HRP mark, 37 ℃ are incubated 1 hour, PBST washing 3 times.Add tmb substrate, room temperature was placed 15 minutes, added stop buffer (2M H ₂SO ₄), reading on the microplate reader is 1ug/ml thereby select the concentration of coated antibody.

2), the prepared in batches of kit

1., the anti-people's of rabbit NASG fusion antibody be cushioned liquid with bag be diluted to 1ug/ml, it is joined 96 hole ELISA Plate, 4 ℃ of bags are spent the night.

2., remove not wrap the liquid of quilt, contain PBS (PBST) flushing 3 times of 0.1%Tween-20, add 200ul retardance liquid and stop nonspecific binding site, hatched 1 hour PBST wash-out 2 times for 37 ℃.Put 4 ℃ of batches and preserve standby (Fig. 2).

3., other reagent of kit such as the anti-NASG antibody of rabbit of HRP mark presses the consumption packing, common reagent such as Tween-20, TMB purchases and packing from Pierce company.

3), the detection of sensitivity, specificity, stability

1., NASG albumen is diluted to 200ug/ml with PBS, 100ug/ml, 50ug/ml, 25ug/ml, 10ug/ml, 2ug/ml, 0.5ug/ml, 0.05ug/ml, 0.01ug/ml 0ug/ml, every hole 100ul join above-mentioned bag by in the good ELISA Plate, hatch 2 hours for 37 ℃.The PBS (PBST) that contains 0.1%Tween-20 washes 3 times.

2., by 1: 1000 with the HRP-NASG antibody dilution, every hole adds 100ul, hatches 1 hour for 37 ℃, PBST washes 4 times.

3., adding 100ul tmb substrate room temperature placed the sulfuric acid of adding 2M, reading under the microplate reader of 420nm wavelength 15 minutes.

4., detect the amount of minimum NASG antigen, our result shows that this reagent can detect the concentration of 0.01ug/ml NASG albumen, illustrates that our kit has higher sensitivity.

Two, the detection step of the EL1SA kit of NASG (SPLUNC):

1, the method for drawing material of nasopharyngeal secretions: behind the normal saline flushing nasal cavity, a thin cotton swab is inserted into pharynx nasalis (tumor surface) under the nose visor, place after 5-8 minute, take out cotton swab fast, cotton balls is put into the hollow little centrifuge tube in bottom, centrifuge tube puts centrifugal collection tube outward again, and 7500 * g is centrifugal 1 minute under 4 ℃, just can obtain nasopharyngeal secretions.This method is non-invasive drawing materials, and can generally be accepted by the people.

2, the NASG gene recombinant protein of purifying is as standard items, with antigen (nasopharyngeal secretions) by 1: 10 the dilution after, join the front and wrapped by in the good ELISA Plate, 37 ℃ of shaking tables were hatched 3 hours with 100 rev/mins speed, with washing the unconjugated antigen of plate liquid flush away.

3, PBST washes plate 3-4 time, blot residual liquid, the NASG polypeptide antibody that adds the anti-people of rabbit of HRP mark is hatched, the unconjugated antibody of flush away adds chromogenic substrate TMB, and room temperature was placed 20 minutes, as seen each hole shows the blueness (accompanying drawing 2) of different depth, the sulfuric acid cessation reaction that adds 2M, microplate reader are calculated the content of NASG albumen in the sample in the 420nm wavelength readings.The concentration that can obtain NASG albumen in the nasopharyngeal secretions of normal population is 30-80ug/ml, and the concentration of NASG albumen is less than 5ug/ml in the nasopharyngeal carcinoma patient nasopharyngeal secretions.

4, we have detected the content of the NASG albumen in 80 routine nasopharyngeal carcinoma patient's nasopharyngeal secretionses by this method, only in the nasopharyngeal carcinoma patient's nasopharyngeal secretions after the two routine radiotherapies NASG protein content greater than 5ug/ml, thereby more than the rate of accuracy reached to 90% of diagnosis nasopharyngeal carcinoma, utilize NASG protein content in the nasopharyngeal secretions come to nasopharyngeal carcinoma make in early days, non-invasive diagnosis fast, have good specificity.

5, get same normal person and nasopharyngeal carcinoma patient's nasopharyngeal secretions, utilizing this method to carry out ELISA measures, measure once every day, repeat altogether 10 times, by formula (S is a standard deviation to the coefficient of variation (CV)=S/X * 100%, X is a mean value) calculate batch between and the variation within batch coefficient, be respectively 4.63% and 4.98% with interassay coefficient of variation in can criticizing.This detection method good stability is described.

We have not only cloned the specific expressed nasopharyngeal carcinoma CDKN2 of nasopharyngeal tissue through years of researches, and have proved that it for a kind of secreted protein, can detect in nasopharyngeal secretions.Therefore, we have prepared the antibody of anti-NASG (SPLUN1), the ELISA detection kit that further prepares NASG albumen through double antibody sandwich method, get secretion from pharynx nasalis and carry out the ELISA detection, NASG expressing quantity in each crowd's nasopharyngeal secretions of detection by quantitative, thereby the prediction nasopharyngeal carcinoma ill risk, the examination susceptible person, and to the nasopharyngeal carcinoma patient make in early days, non-invasive diagnosis fast.Primary Study result shows, this kit is to outpatient service first visit patient's accuracy rate of diagnosis consistent with pathological examination (Fig. 3), this detection fast, the patient is not made damage, patient and normally can accepting per capita; Simultaneously we find that the NASG protein expression in the nasopharyngeal carcinoma patient normal side pharynx nasalis secretion also reduces, in conjunction with our bioinformatic analysis to the NASG gene, illustrate that the NASG down-regulated expression is the early stage incident (Fig. 4) that nasopharyngeal carcinoma takes place, thereby the research not only can be used for diagnosis of nasopharyngeal carcinoma, and be applicable to the early stage large-scale crowd examination and the ill risk profile of nasopharyngeal carcinoma, for diagnosis of nasopharyngeal carcinoma and control provide powerful technical support.On the one hand, we can carry out early stage prevention to the crowd of NASG protein expression downward modulation in the nasopharyngeal secretions, thereby reduce the incidence of disease; On the other hand,, thereby reduce case fatality rate, improve patient's quality of life because the cause of death major part of nasopharyngeal carcinoma causes that because of shifting the late period of tumour utilize this detection method, we can diagnose nasopharyngeal carcinoma in early days, accomplish early diagnosis, early treatment.Thereby this research has great economy and society and is worth, and is worth applying widely.

Specific implementation method:

1, a certain patient's pharynx nasalis normal saline flushing is got patient right side nasopharyngeal secretions 20ul by preceding method, simultaneously to patient's row pathological biopsy.The nasopharyngeal secretions of obtaining is diluted to 200ul with PBS, begins to utilize this kit that the NASG albumen in the secretion is carried out detection by quantitative;

2, refrigerator takes out and has wrapped by good ELISA Plate, placed 20 minutes under the room temperature, simultaneously standard items are pressed following concentration dilution: 25ug/ml, 12.5ug/ml, 6.25ug/ml, 3.12ug/ml, 1.56ug/ml, 0.78ug/ml, 0.39ug/ml, 0ug/ml (blank), the order (96 lattice in the following table are 96 holes in the ELISA Plate) that dilution is respectively got in the 100ul according to the form below joins ELISA Plate;

3,10 times of dilutions of nasopharyngeal secretions (quantitative, as to contain NASG albumen 6ug/ml) of taking from the normal person are got 100ul adding ELISA Plate (gauge orifice), add patient's sample to be tested again, and various sample standard deviations add diplopore:

4, after 37 ℃ of shaking tables are hatched 3 hours with 100 rev/mins speed,, blot residual liquid with washing the unconjugated antigen of plate liquid flush away;

	?1	?2	?3	?4	?5	?6	?7	?8	?9	?10	?11	?12
													A	?25	?25	Standard	Standard
B	?12.5	?12.5	Patient	Patient
													C	?6.25	?6.25
D	?3.12	?3.12
													E	?1.56	?1.56
F	?0.78	?0.78
													G	?0.39	?0.39
H	?0	?0

5, every hole added 100ul, hatched 1 hour for 37 ℃ with the HRP-NASG antibody dilution in 1: 1000, and PBST washes 4 times;

6, add 100ul tmb substrate room temperature and placed 15 minutes, add 2M sulfuric acid (stop buffer), reading under the microplate reader of 420nm wavelength;

7, take advantage of extension rate according to the NASG protein concentration that records, the NASG protein content that draws in this patient's nasopharyngeal secretions is 4.27ug/ml.Show that this patient suffers from nasopharyngeal carcinoma, two days later, pathological examination shows that this patient is the low differentiated squamous-cell carcinomas of nasopharynx.

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Claims (4)

1, the detection kit of a kind of nasopharyngeal tissue specific gene encoding proteins NASG, comprise the ELISA Plate, ELIAS secondary antibody, common reagent Tween-20 and the chromogenic substrate tetramethyl benzidine TMB that are coated with antibody, it is characterized in that being the NASG fusion antibody of handling through coating buffer and retardance liquid is handled in the ELISA Plate hole of this kit, other is equipped with the anti-NASG polypeptide antibody of the rabbit of horseradish peroxidase HRP mark.

2, by the detection kit of the described a kind of nasopharyngeal tissue specific gene encoding proteins NASG of claim 1, it is characterized in that the concentration of the NASG fusion antibody in the kit ELISA Plate hole is good with 1ug/ml.

3, the preparation method of the detection kit of a kind of nasopharyngeal tissue specific gene encoding proteins NASG as claimed in claim 1 comprises the preparation of the anti-human polypeptides antibody of rabbit, and the preparation of the anti-people NASG of rabbit fusion antibody is characterized in that:

(1), during the anti-human polypeptides antibody of preparation rabbit, according to the amino acid sequence of NASG, 21 amino acid polypeptide sequences that design and synthesize are: Cys, Lys, Val, The, Asp, Pro, Gln, Leu, Leu, Glu, Leu, Gly, Leu, Val, Gln, Ser, Pro, Asp, Gly, His, Arg;

(2), with the anti-people NASG of rabbit fusion antibody, be cushioned liquid with bag and be diluted to 1ug/ml and join in the ELISA Plate hole, 4 ℃ of bags are spent the night;

(3), remove not wrap the liquid of quilt, add the phosphate buffer that contains 0.5% bovine serum albumin with the phosphate buffer that contains 0.1%Tween-20-PBST flushing back, hatched 1 hour for 37 ℃, the PBST wash-out, it is standby to put 4 ℃ of preservations.

4, a kind of application of nasopharyngeal tissue specific gene encoding proteins detection kit as claimed in claim 1 is characterized in that this kit is used for as the detection method of the human body nasopharyngeal secretions NASG protein content of nasopharyngeal carcinoma early diagnosis index being:

(1), obtaining of nasopharyngeal secretions: normal saline flushing nasal cavity, under the nose visor, be inserted into pharynx nasalis with thin cotton swab, placed 5～8 minutes, take out cotton swab, with cotton balls put into the bottom hollow, be with the little centrifuge tube of centrifugal collection tube outward, at 4 ℃ of centrifugal nasopharyngeal secretionses that obtain of following 7500xg;

(2), with antigen-nasopharyngeal secretions of obtaining by usefulness physiological saline dilution in 1: 10 after, join in the ELISA Plate hole of several kits, the NASG gene recombinant protein that adds purifying simultaneously in several do not add enzymes mark apical pore of determined antigen is done standard sample; Under 37 ℃ of conditions, in 100 rev/mins shaking table, hatched 3 hours,, blot residual liquid with the unconjugated nasopharyngeal secretions of PBST flush away;

(3) the anti-people NASG of the rabbit of HRP mark polypeptide antibody is joined in the ELISA Plate that adds antigen, hatch not binding antibody of back flush away, add chromogenic substrate TMB, room temperature was placed 20 minutes, added 2M sulfuric acid cessation reaction, in wavelength readings, the metering of microplate reader 420nm;

(4), judge the height of protein content in patient's nasopharyngeal secretions with NASG concentration contrast in the concentration that records and the normal population nasopharyngeal secretions.

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