CN101988925A - ELISA (Enzyme Linked Immunosorbent Assay) reagent kit for detecting Hum j3 specific IgE antibodies - Google Patents

Abstract

The invention provides an ELISA (Enzyme Linked Immunosorbent Assay) reagent kit for detecting Hum j3 specific-sIgE (Hum j3 specific IgE) levels in blood of scandent hop pouen allergy patients. Capture antibodies of the reagent kit are Hum j3 monoclonal antibodies which do not generate the competitive inhibition with Hum j3 specific IgE antibodies. The reagent kit has good sensitivity, specificity, linear detection range and color development stability, and provides a fire-new and easily-standardized path for the monitoring of the Hum j3 specific IgE level in blood samples of the scandent hop pouen major sensitization protein Hum j3 allergy patients and the subsequent molecular immunization therapy.

Description

Be used to detect the ELISA kit of Humj3 specific IgE antibody

Technical field

The present invention relates to a kind of ELISA kit that is used for detecting pollen humuli scandentis autopath blood Hum j3specific-sIgE (Hum j3 specific IgE) level based on mouse hybridoma monoclonal antibody and the main allergic protein Hum of natural pollen humuli scandentis purifying j3, the molecular immune treatment of Hum j3 allergy provides brand-new, has been easy to standardized approach for future.

Background technology

The annual voluble herb plant of Rosidae Cannabaceae humulus grass (Hum μ lus japonicus or Hum μ lus scandens) originates in China, is distributed widely in the Far East Area, is a kind of important pollen sensibiligen summer and autumn.All there is distribution China each provinces and regions except that Xinjiang, Qinghai, and various countries, East Asia (Korea, Japan, Russia, Vietnam) and Europe, U.S.West Coast is regional that distribution also arranged.Last century, the eighties was passed in the sensitization pollen survey by the national gas first time that BJ Union Hospital takes the lead, in 79 pollen bleeding points, there are 53 points can collect pollen humuli scandentis, be only second to artemisia pollen, its distribution range covers the whole nation basically, sends out the peak and be eight, September ^[1]BJ Union Hospital finds that first pollen humuli scandentis is the important sensibiligen that China is only second to artemisia pollen summer and autumn, be the important cause of disease that causes occurring clinically pollinosis symptoms such as allergic rhinitis, allergic conjunctivitis, pollen dermatitis, seasonal asthma, influenced patient's quality of life to a great extent ^[2]

Present stage is for the diagnosis and the treatment of pollinosis, depend on specific allergen's vaccine (Specific Allergenic Vaccines), and the special immunization therapy of allergen (Allergen-Specific Immunotherapy SIT) is the ripe at present method at the pollinosis etiological treatment.Allergen vaccine is meant the biopreparate that comprises allergenic extract and other composition, can be effective to diagnosis, prevention and the treatment of human anaphylactia ^[3]Immunization therapy (the Allergen-Specific Immunotherapy that allergen is special, SIT) function and the quantity by regulating antigen presenting cell, bone-marrow-derived lymphocyte and the lymphocytic immune response of T and regulating mediation allergic reaction effector cell, thus patient's symptom alleviated ^[4]WHO issues policy paper ^[5]Point out that SIT uniquely can influence the anaphylactia natural history, effectively stops allergic rhinitis progress to be the treatment of asthma method.

Although SIT is the ripe at present method at the pollinosis etiological treatment, but present stage, allergic disease immunodiagnosis and the treatment pattern based on allergenic extract be not to be optimal pattern, and, then represented a kind of new direction based on the diagnosis of allergen component (allergen component-resolved diagnosis andtreatment) and the trial of treatment pattern ^[6,7]Even because for a kind of pollen hypersensitivity, different patients' serum sensitized albumino reaction spectrum is also different, for example be all humulus grass autopath, because pollen humuli scandentis allergen kind is a lot, may contain specific IgE (sIgE) in its Different Individual serum at different sensitization composition reactions in the pollen humuli scandentis allergenic extract.Therefore, as skin test and external IgE detectable, only can draw information with pollen extract, but can't confirm which kind of allergic protein allergy to this pollen hypersensitivity.Recently, because molecular biological progress, a large amount of allergens is purified, evaluation and recombinant expressed, based on the diagnosis of allergen component (allergen component-resolved diagnosis andtreatment) and the trial of treatment pattern, more the reaction of science allergic protein serum sIgE response spectrum in autopath's the body, so diagnose more special, the corresponding purpose that also just can reach the individuation immunization therapy.Simultaneously owing to got rid of to insignificant allergic protein of individual patient and non-allergic protein, make that also this individuation immunization therapy is safer, make the special immunization therapy of allergen in medical practice, can reach the quality definition of WHO, i.e. " the professional diagnosis standard of height ", " effectively utilizing resource ", " patient accepts the risk minimum ", " patient is highly satisfied ", " and continuity of patient treatment " about health care ^[8-16]

Recently, have and morely deliver based on the diagnosis of allergen component and the article of treatment ^[8-15]As B.K.Ballmer-weber etc. ^[13]With the reorganization the original in-vitro diagnosis autopath of carrot allergic effect actually for which kind of carrot allergen allergy.The author finds that this diagnostic mode has sensitivity and specificity preferably, can be as a kind of diagnostic mode of standard.And G.Pittner ^[7]Coming that with the natural of purifying and reorganization mite allergen the dermatophagoides pteronyssinus autopath is carried out in-vitro diagnosis research also finds, dust mite allergy patient greater than 95% can unite diagnosis by nDer p1 and rDer p2, this method can be used to screen be applicable to immunization therapy the autopath and the immune state monitoring of accepting the patient of immunization therapy.A.Reuter etc. ^[17]Utilize the rPru av 1 of reorganization, 3,4 three kinds of molecules are respectively to have carried out the evaluation that serodiagnosis is tested with the mode of mixing to the cherry autopath separately, and its susceptibility of the diagnosis of recombinant molecule mixed mode is 95%, and the diagnosis susceptibility of cherry allergenic extract only is 65%.And aspect immunization therapy, Adriano Mari etc. ^[18]The pattern of specific active immunotherapy has been done new discussion, and thinking has some monotropic patients that answer original molecule sensitization, can carry out immunization therapy with the allergen molecule of purifying or reorganization; And, then can carry out immunization therapy by traditional allergenic extract to a kind of multiple allergen of pollen or cause the allergen of the different pollen of cross reaction; For the patient of multiple pollen sensitization, then can carry out going again after the mixing of allergen molecule immunization therapy according to the pattern of " cocktail ".

Carry out the serodiagnosis of anaphylactogen with the pattern of Chimeric ELISA, ultimate principle is, with the special mouse monoclonal antibody of allergen is the capture antibody coated elisa plate, the allergen that adds allergenic extract or purifying then, add standard serum, test serum and negative serum then, add mouse anti human IgE monoclonal antibody at last, colour developing is after the microplate reader reading, by contrasting, calculate in the test serum sample at this allergenic sIgE with typical curve ^[19], mode chart is seen accompanying drawing 1.Martin etc. ^[19]Think that this Chimeric ELISA pattern and CAP system detect the special sIgE of allergen correlativity is preferably arranged, in clinical and epidemic research, of great use, because it is simple and practical, can operate again at basic hospital.Trombone AP ^[20]Deng having researched and analysed in the dust mite allergy patient blood IgE antibody with the method for Chimeric ELISA for the response situation of dust mite allergen Derp1 and Der p2, and detect with the CAP system and to contrast for the situation of mite body allergenic extract Dpt, find that the former the former with the latter is obviously relevant.Among all dust mite allergy patients, 70% patient is for the sIgE test positive of Dpt, and 76.5% patient is to Der p1 seropositivity, and 79.2% patient is to Der p2 seropositivity, and 83.1% is positive for Der p1 and Derp2 joint-detection.The author thinks the Cut-off value of working as Der p1 or Der p2 in 2IU/ml, detects the positive desired value of Dpt greater than 95% for the CAP system.

Because pollen humuli scandentis is researcher's attention extremely both at home and abroad because geographic distribution is wide, epidemiological significance is great.Lee is eastern at home ^[21]Deng to the carrying out of Wuhan Area pollen humuli scandentis allergenic proteins component Two-dimensional Electrophoresis Analysis, Sun Xiuzhen ^[22]Study Deng to pollen humuli scandentis allergen sensitization component; Liu Yun ^[23]Deng made up humulus grass and Preliminary Identification pollen cDNA expression library, Tao Ailin etc. ^[24]Created the mosaic gene of humulus grass and artemisiifolia anaphylactogen, and its antigenicity has been made an appraisal; Yin Jia ^[25]Detect clinical value in diagnosis pollen humuli scandentis disease etc. Deng having studied intracutaneous test and serological specificity IgE.But all the time, never have enough understanding for the main allergen of humulus grass.As of late, take the lead in by purifying, clone, pollen humuli scandentis allergic proteins of evaluation such as the Yin Jia of allergic reaction section of BJ Union Hospital, its autopath's serum sIgE combination rate is about 74%, is defined as the main allergic protein of humulus grass, drafts Hum j3 by name.

The present application people utilizes the main allergic protein of natural humulus grass of purifying, the immunity BALB/c mouse, obtained the hybridoma monoclonal antibody, by screening to monoclonal antibody, made up the ELISA method of the Hum j3 specific-sIgE level in the pollen humuli scandentis autopath blood of measuring with the ChimericELISA method first, this method has good susceptibility, specificity, linear detection range, color stability, and that the patient who carries out the molecular immune treatment for screening and monitoring Hum j3 provides is brand-new, be easy to standardized means.

Summary of the invention

The object of the present invention is to provide a kind of ELISA kit that is used to detect Hum j3 specific IgE antibody.

For achieving the above object, the present invention at first utilizes by the main allergic protein Hum of purifying j3 immunity BALB/c mouse, obtain mouse hybridoma monoclonal antibody through screening, this monoclonal antibody requires to catch Hum j3 molecule, but does not suppress with Hum j3-specific-sIgE antibody competition.The present invention obtains the good hybridoma cell strain (Hum j3-#87-mAb) of the comprehensive proterties of a strain by screening, this hybridoma on July 28th, 2009 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, classification called after mouse hybridoma cell, preserving number is CGMCC No.3208.

Based on said monoclonal antibody, the invention provides a kind of ELISA kit of the Hum of detection j3 specific IgE antibody, the described ELISA Plate that comprises the antibody that is hunted down, described capture antibody are Hum j3 monoclonal antibody, and this antibody not with the inhibition of competing of Hum j3 specific IgE antibody.

Kit of the present invention also can comprise one or more in the following reagent: 1) cleansing solution; 2) substrate colour developing liquid; 3) reaction terminating liquid; 4) Hum j3 standard items; 5) detect antibody.

Described detection antibody is enzyme labeling anti human IgE antibody, as enzyme labeling mouse-anti human IgE monoclonal antibody.The marker enzyme of described detection antibody is horseradish peroxidase, alkaline phosphatase, glucose oxidase thing enzyme or beta galactosidase.

When using concrete marker enzyme, substrate colour developing liquid, stop buffer should adapt.When for example using horseradish peroxidase to serve as a mark enzyme, developer can be made up of colour developing liquid A liquid and colour developing liquid B liquid, colour developing liquid A liquid is hydrogen peroxide or urea peroxide, and described colour developing liquid B liquid is o-phenylenediamine or tetramethyl benzidine, and stop buffer is sulfuric acid or the hydrochloride buffer of 1～2mol/L; When alkaline phosphatase served as a mark enzyme, colour developing liquid was for being 1～2mol/L sodium hydroxide solution to nitro phosphate buffer, stop buffer.

Described cleansing solution can be PBST, or its concentrate.

The be hunted down bag of ELISA Plate of antibody of described bag can be added the capture antibody of 100 μ l, 5 μ g/L by every hole, spends the night (12h) in 4 ℃ of coated elisa plates, after the PBST washing, with 1%BSA-PBST confining liquid sealing 1.5h.

Be to be understood that monoclonal antibody of the present invention is not limited only to prepare above-mentioned detection kit, it can be used to prepare the ELISA reagent of any detection Humj 3 specific IgE antibodies.

The present invention is setting up the ELISA method that detects in the humulus grass autopath blood at Hum j3 molecule sIgE level first, and relevant detection kit is provided; Inquired into the utilization of this method aspect the irritated patient's clinical diagnosis of Hum j3 first, for monitoring main allergic protein of humulus pollen Hum j3 autopath's Hum j3 specific IgE level and later molecular immune treatment provide brand-new, have been easy to standardized approach.

Description of drawings:

Fig. 1 .Chimeric ELISA method is measured the ELISA method synoptic diagram of Hum j3specific-sIgE level in the pollen humuli scandentis autopath blood;

W22sIgE level that Fig. 2 .UniCAP system detects and Chimeric ELISA method are measured the relation between the Hum j3 specific-sIgE level;

Fig. 3 .Chimeric ELISA method is measured the ROC curve of Hum j3specific-sIgE level in the pollen humuli scandentis autopath blood;

Fig. 4 .Chimeric ELISA method is measured the typical curve of Hum j3specific-sIgE level in the pollen humuli scandentis autopath blood.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are used to illustrate the present invention, limit the scope of the invention and be not used in.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition (as the antibody technique experiment guide, Ed Harlow, David Lane etc. work, Shen Guanxin, Gong Feili etc. translate, Science Press, and the requirement advised of relevant manufacturer, first published in 2000).

The percentage sign that relates among the present invention " % " if do not specify, is meant mass percent; But the number percent of solution except as otherwise herein provided, is meant and contains the some grams of solute among the solution 100ml; Number percent between the liquid is meant the ratio of capacity in the time of 20 ℃.

The foundation of embodiment 1Chimeric ELISA method

1, the acquisition of main allergic protein of humulus pollen Hum j3

Obtain the main allergic protein Hum of natural pollen humuli scandentis purifying j3 by molecular gel chromatography, ion-exchange chromatography, affinity chromatography.Specifically work referring to patents such as Yin Jia---main allergic protein of humulus pollen application number: 200710122176.4.

2, at the acquisition of the mouse hybridoma monoclonal antibody of Hum j3

After obtaining the main allergic protein Hum j3 of natural purifying humulus grass, (chapter 11 P351-364) is finished the preparation and the primary dcreening operation of mouse hybridoma monoclonal antibody for Ahmedabad year chief editor, " Immunization Update learns a skill and uses " with reference to " hybridoma technology and Monoclonal Antibody ".Specifically work referring to patents such as Yin Jia---monoclonal antibody of main allergic protein of humulus pollen application number: 200710122175.X.

Obtain the monoclonal antibody of the anti-Hum j3 of many strains stably excreting by screening, suppress inhibition ELISA that serum combines with Hum j3 by monoclonal antibody and can know monoclonal antibody by inference and combine with the non-sIgE binding site of Hum j3 molecule.Experimental implementation program and condition are as follows: the Hum j3 of 100 μ l, 1 μ g/ml purifying in 4 ℃ of bags by 12h; The 1%BSA-PBST sealing is after 1.5 hours; The monoclonal antibody that adds 50 μ l 0.1mg/ml, pooled serum pond (20 the humulus grass autopath serum mixing that adds 50 μ l, 1/4 dilution again, it is 4 grades that former serum pond w22sIgE detects, 48.5kUA/ml, CAP System), mixing monoclonal antibody and serum, 37 ℃ of incubation 3h (contrast only adds the pooled serum pond of 50ul 1/4 dilution, adds 50 μ l 1%BSA-PBST confining liquids then); Adding HRP mark: the mouse anti human IgE monoclonal antibody of dilution in 1: 2000,37 ℃ of incubation 2h; Colour developing at last is in OD492nm place reading.Calculate inhibiting rate.Obtain the good hybridoma cell strain of the comprehensive proterties of a strain by this method, its inhibiting rate only is 4.74%, and this strain has been carried out preservation on July 28th, 2009 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation.

So after can catching Hum j3 with this monoclonal antibody, in conjunction with the Hum j3-specific-sIgE antibody among the patients serum, close as two resistive connections with the anti-IgE mouse of HRP enzyme target monoclonal antibody then, Xian Se mode makes up the method that detects Hum j3-specific-sIgE level at last.Because #87 monoclonal antibody energy specific recognition Hum j3 molecule, so the Hum j3 molecule that the pollen humuli scandentis allergenic extract that this Chimeric ELISA also can directly demarcate with process substitutes purifying has been save the complicated processes that needs purifying Hum j3 molecule as standard items.

3, the collection and the diagnostic criteria of humulus grass autopath standard pooled serum pond, negative control sera pond, test serum

(1) the clinical comprehensive diagnos standard of pollen humuli scandentis autopath is referring to document ^[25]: 1) typical case's pollen hypersensitivity medical history summer and autumn: seasonal rhinitis, eye conjunctivitis and or bronchial astehma, summer and autumn (July～October) morbidity, pollen humuli scandentis immersion liquid intracutaneous test 〉=+, pollen humuli scandentis RAST sIgE 〉=I level (0.35kUa/l) simultaneously; Or 2) medical history and symptom typical case, simple pollen humuli scandentis immersion liquid intracutaneous test 〉=++ the person; Or 3) medical history and symptom typical case, simple pollen humuli scandentis RASTsIgE 〉=II level (0.70kUa/l).

(2) not irritated but to other pollen hypersensitivity patient's diagnostic criteria (for example trees pollen, other weeds pollen, herbage pollen): 1) typical pollen hypersensitivity medical history to pollen humuli scandentis: seasonal rhinitis, eye conjunctivitis and or bronchial astehma; 2) sensitization pollen extract intracutaneous test 〉=++; 3) sensitization pollen RAST sIgE 〉=II level (0.70kU/l); 4) pollen humuli scandentis RAST sIgE detects negative.

(3) normal healthy controls experimenter standard: 1) no pollen hypersensitivity medical history; 2) pollen humuli scandentis immersion liquid intracutaneous test feminine gender; 3) pollen humuli scandentis RAST test is negative.

According to above-mentioned diagnostic criteria, collect experimenter's serum.Wherein, pollen humuli scandentis autopath 30 examples, other pollen hypersensitivity but pollen humuli scandentis be allergy sufferers 15 examples not, healthy patients 10 examples.

4, detect the structure of the Chimeric ELISA method of Hum j3-specific-sIgE level

Optimize the condition that the ELISA method detects Humj3-specific-sIgE level in the main allergic protein of humulus pollen autopath blood: the dilute concentration of the anti-concentration of Sheet, humulus grass allergenic extract or purifying main allergic protein of humulus pollen, the gradient dilution scope in standard serum pond, the dilute concentration of enzyme mark mouse anti human IgE monoclonal antibody, developing time;

Related experiment condition and operation are determined as follows:

(1) spends the night with 4 ℃ of coated elisa plate 12h of 100 μ l#87-mAb (0.5 μ g/ hole);

(2) 1 * PBST washing lotion cleansing solutions are washed plate 5 times, 1%BSA-PBST confining liquid 1.5h;

(3) 1 * PBST washing lotion cleansing solutions are washed plate 5 times, and the initial concentration that adds continuous 11 2 * gradient dilutions is the main allergic protein Hum j3 standard items 100 μ l of natural purifying humulus grass (or Hum j3 content humulus grass allergenic extract standard items through demarcating) of 0.05 μ g/ml;

(4) 1 * PBST washing lotion cleansing solutions are washed plate 5 times, add by 20 humulus grass autopath pooled serum pond dilutabilitys did since 1: 2.5 10 2 * doubling dilution until 1: 1280; Add 30 parts of humulus grass autopath test serums, 15 parts of other pollen hypersensitivities then but pollen humuli scandentis not allergy sufferers serum, 10 parts of healthy blanks of non-allergy.The test serum dilutability is 1: 10 during detection.

(5) 1 * PBST washing lotion cleansing solutions are washed plate 5 times, add the horseradish peroxidase-labeled mouse anti human IgE monoclonal antibody 100 μ l of dilution in 1: 2000.

(6) add OPD-H ₂O ₂Colour developing liquid damping fluid, color development stopping behind the 5min, OD492 reading.

(7) dose-effect curve with the main allergic protein Hum j3 standard items of humulus grass is a typical curve, calculation sample Hum j3 content.

5, determine that ELISA detects the whole bag of tricks index of Hum j3 specific-sIgE in the pollen humuli scandentis autopath blood: susceptibility, specificity, the range of linearity.

1) will be selected in the experimenter according to clinical comprehensive diagnos standard is divided into ill and not ill, dilutability (or corresponding OD colour developing value) with the standard serum pond is different separation, match ROC curve determines that this Chimeric ELISA diagnostic test has the cut-off value of optimum sensitivity and specificity.

Result (Fig. 3), the positive threshold value of best serum dilution fixes on 1: 320 dilutability place, and this moment, the OD492 absorbance was 0.18.Be lower than this value, then be judged as Hum j3specific-sIgE testing result feminine gender.When this cut-off value, diagnosing the susceptibility of pollen humuli scandentis allergy as molecular labeling and Chimeric ELISA method as detection method with Hum j3 is 93.1%, and specificity is 90.9%, and positive desired value is 93.5%, and its negative desired value is 90.5%.And when OD492＞0.63, its specificity is 95%; When OD＞1.5,100% specificity is arranged.Illustrate that Hum j3 can be used as a molecular labeling of pollen humuli scandentis allergy; The ROC curve display of match, this diagnostic test ROC area under curve A is 0.901, illustrates that its diagnostic value is higher, Chimeric ELISA can be used as the diagnostic test of pollen humuli scandentis allergy.

2) when best cut-off value, the result who detects with Chimeric ELISA testing result and UniCAPSystem makes consistency analysis, and whether judge when diagnosing pollen humuli scandentis irritated as molecular labeling and Chimeric ELISA method as detection method with Hum j3 suitable.

The absorbance of correspondence is 0.18 for boundary when the positive threshold value of best serum dilution is 1: 320 dilutability, result that Chimeric ELISA detects and the testing result (〉=1 grade of positive criterion) of UniCAP System, the consistance of two diagnostic tests is κ=0.840, and good consistance (Fig. 2) is arranged.

3) in Chimeric ELISA, the typical curve of standard serum pond institute match is analyzed, judge the best range of linearity, so that the Hum j3 specific-sIgE of test serum is carried out classification.

Table 1 humulus grass autopath's serum sample essential information and testing result

Remarks: [1]. humulus grass autopath's catalogue number(Cat.No.) represents with P.[2]. in the sex, F represents the woman, and M represents the man.[3]. diagnosis abbreviation: P, pollinosis; BA, bronchial astehma; AA, allergic asthma; AR, allergic rhinitis; DA, drug allergy.[4]. allergen sIgE testing result (progression/detected value) allergen is abridged referring to the explanation of the Sweden ImmunoCAP of Phadia company anaphylactogen detection system.[5]. for ease of statistical study, serum sample (OD value≤blank OD value) assignment that detects less than Humj 3specific-sIgE level with chimeric ELISA is OD492=0.02.[6]. the haemolysis sample.[7]. the numerical value disappearance.

The essential information and the testing result of other weeds pollen hypersensitivity patients serum's samples of table 2 and non-irritated normal healthy controls serum sample

Remarks: [1]. the irritated but catalogue number(Cat.No.) non-humulus grass autopath of other weeds represents with " NHAP "; The catalogue number(Cat.No.) of normal healthy controls represents with " HC ".[2]. in the sex, F represents the woman, and M represents the man.[3]. diagnosis abbreviation: P, pollinosis; BA, bronchial astehma; AA, allergic asthma; AR, allergic rhinitis; DA, drug allergy.[4]. allergen sIgE testing result (progression/detected value) allergen is abridged referring to the explanation of the Sweden ImmunoCAP of Phadia company anaphylactogen detection system.[5]. for ease of statistical study, serum sample (OD value≤blank OD value) assignment that detects less than Hum j 3specific-sIgE level with chimeric ELISA is OD492=0.02.[6]. in the sample of normal healthy controls, "-" expression " nothing " or " need not detect ".

Be positioned at 1 at the standard serum dilutability: 2.5-1: during seven 2 * doubling dilution scopes of 160, then between Log Dilution and the OD492 best linear fit relation (Fig. 4), y=1.5691x+3.5872, R arranged ²=0.9901.Can be the grade scale of Hum j3 specific-sIgE with the dosage-linear relationship of typical curve, finally determine a Hum j3specific-sIgE level that quantizes relatively.

The establishment of embodiment 2 ChimericELISA kits

Based on above-mentioned experiment, this example provides a kind of kit that pollen humuli scandentis autopath blood Humj3 specific IgE detects that is used for, and it is composed as follows:

1) ELISA Plate of Sheet clonal antibody; (#87 monoclonal antibody, package amount are 0.5 μ g/ hole)

2) horseradish peroxidase-labeled two is anti-; (horseradish peroxidase-labeled mouse-anti human IgE monoclonal antibody)

3) cleansing solution; (PBST)

4) substrate colour developing liquid:

Substrate buffer solution (pH 5.0):

A liquid: 0.1mol/L citric acid (2.1g C ₆H ₈O ₇H ₂O/100ml), 4 ℃ of preservations;

B liquid: 0.2mol/L phosphoric acid hydrogen two is received (7.163gNa ₂HPO ₄12H ₂O/100ml), room temperature preservation;

Get A liquid 24.3ml, B liquid 25.7ml mixes, again adding distil water 50ml, 100ml altogether.4 ℃ of preservations.

OPD-H ₂O ₂Colour developing liquid damping fluid: substrate buffer solution 10ml, OPD 4mg, 30%H ₂O ₂15 μ l.The lucifuge preparation.

5) reaction terminating liquid (2mol/L H ₂SO ₄);

6) Hum j3 standard items.

Description of test:

1, experiment material

(1) 20 humulus grass autopath serum pond: specificity sIgE testing result 48.5KUA/L.Wherein this 20 humulus grass autopaths get clinical comprehensive diagnos standard by Yin Jia etc. ^[25]Established standards is made a definite diagnosis

(2) natural purifying Hum j3 standard items are pressed patented claim: 200710122176.4 preparations, purity 98%.

(3) sodium carbonate (Na ₂CO ₃), sodium bicarbonate (NaHCO ₃), sodium chloride (NaCl), potassium chloride (KCl), potassium dihydrogen phosphate (KH ₂PO ₄), phosphoric acid hydrogen two receives (Na ₂HPO ₄12H ₂O), citric acid (C ₆H ₈O ₇H ₂O), sulfuric acid (H ₂SO ₄) to be homemade analysis pure.

(4) bovine serum albumin(BSA) (BSA) is available from Beijing Heng Shengma of unit biotechnology research institute.

(5) Tween-20 is available from U.S. Fluka.

(6) horseradish peroxidase mouse anti human IgE monoclonal antibody: preclinical medicine institute of the Chinese Academy of Medical Sciences.

(7) o-phenylenediamine (OPD) is a U.S. Amresco product, the packing of river, Beijing biotechnology in morning development company.

(8) 30% hydrogen peroxide (H ₂O ₂): Beijing northization fine chemicals company limited.

(9) U.S. Bio-rad Laboratories, Inc. determining the protein quantity coloring agent Protein Assay DyeReagent Concentrate (classification number: 500-0006).

The removable polystyrene ELISA Plate in (10) instrument and equipment: BIOFIL 96 holes (Canadian JET BiochemicalsInt ' l., Inc); Anthos 2010 microplate reader (Austrian anthos labtec instrumentsGes.m.b.h); The Auslab Australia Si Leibo enzyme mark laboratory room managing 2.2.0.0 of system (Beijing Ao Sibang medical data company limited).

(11) other chemical reagent: acrylamide, methylene diacrylamide: U.S. SIGMA; Lauryl sodium sulfate: Sweden Pharmacia biotech AB; Tris (MW:121.14): B.M., Beijing side moisten biological center; Hydrochloric acid: Beijing Yili Fine Chemicals Co., Ltd.; Glycocoll: B.M., Beijing side moisten biological center; Beta-mercaptoethanol: Hong Kong Farco chemical supplies; Bromophenol blue: BioMark, Beijing side moisten biological center; Glycerine (glycerine): Beijing Century Red Star chemical industry Ltd; TEMED: U.S. Fluka, Beijing side moisten biological center; Ammonium Persulfate 98.5: U.S. Amresco; Agar: U.S. SIGMA, Beijing side moisten biological center; Coomassie brilliant blue R-250: U.S. SIGMA, Beijing side moisten biological center; Methyl alcohol: Beijing Yili Fine Chemicals Co., Ltd.; Acetate (glacial acetic acid): Beijing chemical reagents corporation; Absolute ethyl alcohol: Beijing Century Red Star chemical industry Ltd; 50% glutaraldehyde: Beijing Yili Fine Chemicals Co., Ltd.; 37% formaldehyde: Beijing chemical reagents corporation; Sodium thiosulfate: Beijing Century Red Star chemical industry Ltd; Silver nitrate, natrium carbonicum calcinatum: Beijing Yili Fine Chemicals Co., Ltd.; Low molecular weight protein molecular weight standard: molecular weight ranges 97～14.4kDa (table 1.3), U.S. Amersham.

(12) instrument: UniCAP100, Sweden pharmacia company.W22 is the pollen humuli scandentis allergenic extract.

2, solution preparation

(1) bag is cushioned liquid (pH 9.6): Na ₂CO ₃0.16g, NaHCO ₃0.293g dissolved in distilled water is to 100ml.4 ℃ of preservations.

(2) cleansing solution (PBS/0.05%Tween-20, pH 7.4): NaCl 8.0g, KCl 0.2g, KH ₂PO ₄0.2g, Na ₂HPO ₄12H ₂O 2.9g, Tween-20 0.5ml, adding distil water adds to 1000ml.4 ℃ of preservations.

(3) confining liquid double one anti-, two anti-dilutions (1%BSA/PBST): matching while using.

(4) substrate buffer solution (pH 5.0):

(5) A liquid: 0.1mol/L citric acid (2.1g C ₆H ₈O ₇H ₂O/100ml), 4 ℃ of preservations.

(6) B liquid: 0.2mol/L phosphoric acid hydrogen two is received (7.163gNa ₂HPO ₄12H ₂O/100ml), room temperature preservation.

(7) get A liquid 24.3ml, B liquid 25.7ml mixes, again adding distil water 50ml, 100ml altogether.4 ℃ of preservations.

(8) colour developing liquid: substrate buffer solution 10ml, OPD 4mg, 30%H ₂O ₂15 μ l.Face time spent lucifuge preparation.

Stop buffer (2mol/L H ₂SO ₄): 50ml dropwise adds among the distilled water 400ml with 98% concentrated sulphuric acid (18.4mol/l), and continuous stirring and dissolving.

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Claims (10)

1. ELISA kit that is used to detect Hum j3 specific IgE antibody, it comprises the ELISA Plate of the antibody that is hunted down, described capture antibody is a Hum j3 monoclonal antibody, and this antibody not with the inhibition of competing of Hum j3 specific IgE antibody.

2. kit as claimed in claim 1 is characterized in that, described capture antibody is that hybridoma Hum j3-#87-mAb CGMCC No.3208 secretion obtains.

3. kit as claimed in claim 1 or 2 is characterized in that, it also comprises in the following reagent one or more:

1) cleansing solution;

2) substrate colour developing liquid;

3) reaction terminating liquid;

4) Hum j3 standard items;

5) detect antibody.

4. kit as claimed in claim 3 is characterized in that, described detection antibody is enzyme labeling mouse-anti human IgE monoclonal antibody.

5. kit as claimed in claim 4 is characterized in that, the marker enzyme of described detection antibody is horseradish peroxidase, alkaline phosphatase, glucose oxidase thing enzyme or beta galactosidase.

6. kit as claimed in claim 4 is characterized in that, described cleansing solution is PBST.

7. kit as claimed in claim 4 is characterized in that, described substrate colour developing liquid is OPD-H ₂O ₂Colour developing liquid, stop buffer are 2mol/L H ₂SO ₄, the marker enzyme of described detection antibody is a horseradish peroxidase.

8. kit as claimed in claim 1 or 2, it is characterized in that, the be hunted down method for coating of ELISA Plate of antibody of described bag is that 4 ℃ of coated elisa plate 12h of capture antibody that 100 μ l, 5 μ g/L are added in every hole spend the night, after the PBST washing, with 1%BSA-PBST confining liquid sealing 1.5h.

9. secrete the monoclonal antibody that produces by hybridoma Hum j3-#87-mAb CGMCC No.3208.

10. the application of the described monoclonal antibody of claim 9 in the ELISA reagent of preparation detection Hum j3 specificity sIgE antibody.

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