CN105606811A - Diagnostic kit for nasopharynx cancer - Google Patents
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- CN105606811A CN105606811A CN201610072104.2A CN201610072104A CN105606811A CN 105606811 A CN105606811 A CN 105606811A CN 201610072104 A CN201610072104 A CN 201610072104A CN 105606811 A CN105606811 A CN 105606811A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
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Abstract
The invention discloses a diagnostic kit for nasopharynx cancer. The diagnostic kit is an EBV-VCA-IgA and CypA combined detection ELISA kit which comprises an EBV-VCA-IgA detection reagent and a CypA detection reagent, wherein the EBV-VCA-IgA detection reagent comprises an EBV-VCA-IgA positive control, an EBV-VCA-IgA negative control, an ELISA working solution, a substrate solution A, a substrate solution B and a terminating solution; the CypA detection reagent comprises a biotin labeled anti-human CypA antibody, horse radish peroxidase labeled avidin, a TMB solution and a terminating solution. According to the diagnostic kit for the nasopharynx cancer, serum CypA detection and EBV-VCA-IgA detection are combined in use, so that the senility is high and can reach 93.6%; an enzyme-linked immunosorbent assay is economic, convenient and rapid, and the detection of protein is not limited by immunosuppression conditions; the diagnostic kit can be used as a clinic diagnostic measure for the nasopharynx cancer, and particularly, the CypA detection rate to EBV-VCA-IgA negative patients with the nasopharynx cancer is 72.22%.
Description
Technical field
The present invention relates to tumor diagnosis kit, relate in particular to nasopharyngeal carcinoma diagnosis kit.
Background technology
Nasopharyngeal carcinoma (nasopharyngealcarcinoma, NPC) is multiple country in Southeast Asia and the south China be born inA kind of epithelium source property tumour. Early stage nasopharyngeal carcinoma is taking radiotherapy as main, but hidden because of this disease primary lesion, clinical symptoms are not obvious,Easy to cause missed diagnosis, mistaken diagnosis, therefore be difficult for early detection. This disease grade malignancy is higher, and 75%-90% patient has belonged to late period when medical, andThere is metastases in local lymph node or the transfer of organ at a distance. Therefore, early detection and diagnosis of nasopharyngeal carcinoma, improve diagnostic accuracy,For clinical raising result for the treatment of and prognosis, thereby raising survival rate and patients ' life quality have very important meaning.
Epidemiology finds, nasopharyngeal carcinoma is not only with heredity, environment dietary factor is relevant, also with Epstein-Barr virus (Epstein-BarrVirus, is called for short EBV) infect closely relatedly, wherein dye the relevant antibody such as EA, VCA to nose for virus multiplication sexualityPharynx cancer has higher specificity. However,, because the reasons such as immunosupress easily occur tumour patient, some patients are to EB diseaseIt is low that poison infects generation antibody response, only detects EBV antibody horizontal and can not reflect tumorigenic actual conditions completely, at noseIt is high that pharynx cancer patient detects EBV-VCA-IgA specificity, but positive rate only has 70% left and right, and sensitiveness is not high enough. Therefore, to nosePharynx cancer patient carries out early diagnosis, so that early treatment significantly improves patient's prognosis and survival rate, is the research heat of rhinopharyngeal neoplasmPoint.
Past has some to use other antibody as the method for the detection associating VCA-IgA of EBV-EA antibody, to improve detectionSensitiveness, but still can not avoid the low problem of immunosupress patient antibody response. Epstein-Barr virus and the close phase of nasopharyngeal carcinomaClose, still, because immunosupress easily occurs tumour patient, some patients produce the hyporeactive of antibody to ebv infection,Only detect antibody horizontal sensitiveness not high enough, in Patients With Npc, detect conventionally only have an appointment 70% sun of EBVVCA-IgAProperty rate.
Cyclophilin A (CyclophilinsA, CypA) is a molecular chaperone protein, and tumour cell can be secreted CypA to born of the same parentsOutward. Cyclophilin A (CypA) gene is positioned at chromosome 7p13 site, and long 2276bp includes 5 extrons, and molecular weight of albumen approximately18kDa, forms containing 165 amino acid whose polypeptide strands by one. CypA is mainly made up of beta sheet, has peptidyl prolylIsomerase activity (PPIase) and molecular chaperones effect, the transformation between the proline cis-trans-isomer on energy catalytic polypeptide, thereforeCypA plays an important role in regulatory transcription, immune response, secretory protein and injury of mitochondria. CypA is a kind of outer secreting property eggIn vain, tumour cell can be secreted into extracellular by CypA, in some diseases patients serum, can detect. CypA can promote that tumour is thinBorn of the same parents' propagation, infiltration and transfer, participate in increase Angiogenesis, the process of antitumor cell apoptosis. It is reported, CypA is at pancreasExpression in the Several Kinds of Malignancies such as cancer, lung cancer, liver cancer, the cancer of the esophagus, breast cancer, melanoma is increased, and CypA positive patient is pre-Bad afterwards.
Summary of the invention
The object of the invention is to solve EBV-VCA-IgA antibody assay kit quick for diagnosis and the examination of nasopharyngeal carcinomaThe problem that perception is not high, provides a kind of sensitivity up to more than 90% nasopharyngeal carcinoma diagnosis kit.
Nasopharyngeal carcinoma diagnosis kit of the present invention is EBV-VCA-IgA and CypA joint-detection ELISA kit, shouldELISA kit comprises that EBV-VCA-IgA detects reagent and CypA detects reagent.
Wherein, EBV-VCA-IgA detection reagent comprises EB-VCA-IgA positive control, EB-VCA-IgA negative control, enzymeMark working solution, substrate solution A and substrate solution B, stop buffer; CypA detects reagent and comprises biotin labeled anti-human CypA antibody, pepperyRoot peroxidase labelling Avidin, TMB solution and stop buffer.
Inventor finds: the expression rising of the CypA in tumor tissues occurs to show in early days nasopharyngeal carcinoma, in addition, and inventorThe CypA that is further unequivocally established in patients with nasopharyngeal carcinoma of team expresses and rises.
On above-mentioned Research foundation, inventor's parasyndesis detects VCA-IgA and CypA in patients with nasopharyngeal carcinoma, sensitiveDegree can bring up to 93.6%, detects apparently higher than VCA-IgA or the CypA of individual event; Especially when serum VCA-IgA detects feminine gender,Can, by detecting the diagnosis of the auxiliary nasopharyngeal carcinoma of CypA, reduce rate of missed diagnosis, thereby improve diagnosis efficiency. Therefore, with ELISA method connectionClose detection EBV antibody and CypA antibody for serodiagnosis and the examination of nasopharyngeal carcinoma, can improve the sensitiveness of diagnosis, improvedTo the value of nasopharyngeal carcinoma serological diagnostic and examination.
Utilize the Epstein-Barr virus VCA-IgA antibody in enzyme linked immunosorbent assay (ELISA) qualitative detection human serum, by serum sampleIn this, EBV-IgA antibody coated Epstein-Barr virus VCA antigen in micropore is combined, and washes away unconjugated other compositions, then adds pepperyThe mouse-anti people IgA antibody of root peroxidase (HRP) mark, finally, with tmb substrate colour developing, detects absorbance by ELIASAValue, qualitatively judges IgA antibody and whether exists; It is also to utilize enzyme linked immunosorbent assay that described CypA detects reagent detection principle(ELISA) the coated anti-human CypA antibody in micropore of the CypA in serum sample is combined, adds biotin labeled anti-humanAfter CypA antibody, then add the Avidin of HRP mark, after thorough washing with substrate TMB colour developing. TMB is at peroxidaseUnder catalysis, change into blueness, and be converted into final yellow under sour effect, the CypA in the depth and the sample of color is justRelevant, under 450nm wavelength, measure absorbance (OD value) with ELIASA, calculate concentration of specimens.
In the present invention, the serology of two kinds of objects (CypA and EBV-VCA-IgA antibody) detects and all uses ELISA. ApplicationELISA joint-detection serum cyclophilin A and Epstein-Barr virus VCA-IgA antibody, convenient sources, detects fast, economical, and assay is moreTruly, accurately, contribute to nasopharyngeal carcinoma Precise Diagnosis and examination, thereby be that early treatment, raising Nasopharyngeal Carcinoma Patients survival rate haveImportant meaning.
Brief description of the drawings
The ROC curve map that Fig. 1: EBV-VCA-IgA detects;
The parameter of the ROC curve map that Fig. 2: EBV-VCA-IgA detects;
The ROC curve map that Fig. 3: CypA detects;
The parameter of the ROC curve map that Fig. 4: CypA detects;
The ROC curve map that when Fig. 5: EBV-VCA-IgA is negative, CypA detects;
The parameter of the ROC curve map that when Fig. 6: EBV-VCA-IgA is negative, CypA detects;
Detailed description of the invention
Embodiment 1: the preparation of diagnostic kit of the present invention
Diagnostic kit of the present invention is EBV-VCA-IgA and CypA joint-detection ELISA kit, and this ELISA kit comprisesEBV-VCA-IgA detects reagent and CypA detects reagent. Wherein, EBV-VCA-IgA detection reagent comprises the EB-VCA-IgA positiveContrast, EB-VCA-IgA negative control, enzyme mark working solution, substrate solution A and substrate solution B, stop buffer; CypA detects reagent and comprises lifeThe anti-human CypA antibody of thing element mark, Horseradish peroxidase-conjugated avidin, TMB solution and stop buffer.
Wherein, CypA detection reagent preparation process is:
1. with the coated microwell plate of anti-human CypA antibody, every hole adds 100ul, rocks gently evenly, is covered with film, and 4 DEG C are spent the night. 2.The unnecessary liquid of sucking-off, every hole adds 100ul5% hyclone sealing, 37 DEG C of incubations 2 hours. Discard liquid, dry, wash plate 3Inferior. Invade bubble 5 minutes, 350ul/ hole, dries at every turn.
Embodiment 2: the use of diagnostic kit of the present invention
(1) first sample is carried out to EBV-VCA-IgA detection
1. take out kit and put equilibrium at room temperature 30 minutes. 20 × cleaning solution is done to 20 times of dilutions with distilled water. Add sample: take outCoated plate, performs mark, stays a hole blank, and lath is fixed on grillage, and remaining lath seals and puts with adhesive stickerEnter in sealing bag and preserve. Negative control 3 holes, positive control 1 hole, each 100ul control serum. Blank 1 hole is vacant. All the other are everyHole adds 100ul sample diluting liquid, and every hole adds sample 10ul to be checked, and reaction plate light shaking is made to sample blending. Incubation:With after shrouding film shrouding, put in 37 DEG C of incubators and react 30 minutes. Wash plate: after incubation, shrouding film is taken off, blotted liquid in hole,Wash plate 5 times with washing plate liquid, invade bubble 30-60 second at every turn. Enzyme-added mark working solution: every hole adds 100ul enzyme mark working solution, blank wellExcept. Incubation: with after shrouding mould shrouding, put in 37 DEG C of incubators and react 30 minutes. Wash plate: after incubation, shrouding film is connect, blotLiquid in hole, washes plate 5 times with washing plate liquid, invades bubble 30-60 second at every turn. Colour developing: every hole adds substrate A, the each 50ul of B liquid, gentlyConcussion mixes, and with after shrouding film shrouding, puts in 37 DEG C of incubators and reacts 15 minutes. Stop: every hole adds stop buffer 50ul, gently shakeSwing and mix. Measure: setting ELIASA wavelength is 450nm, measures each hole optical density (OD value). Read in 10 minutes in cessation reactionNumber.
Data processing: calculate critical value: the average OD value of cutoff=0.10+ negative control is (when the average OD value of negative controlBe less than at 0.05 o'clock, calculate by 0.05; In the time that the average OD value of negative control is greater than 0.05 by calculated with actual values). Result is judged: feminine genderResult: sample OD value < cutoff value is negative. Positive findings: sample OD value > cutoff value is positive.
(2) again sample is carried out the use of CypA detection
1. various reagent is moved to equilibrium at room temperature at least 30 minutes. Application of sample: establish respectively standard items hole, sample to be tested hole, every hole is dividedDo not add standard items or sample to be tested 100ul, rock and mix gently, be covered with plate and paste, 37 DEG C of incubations 2 hours. Discard liquid, dry,Need not wash. Every hole adds biotin labeling antibody working solution 100ul, be covered with new plate and paste, 37 DEG C 1 hour. Discard liquid in hole,Dry, wash plate 3 times. Invade bubble 2 minutes, 200ul/ hole, dries at every turn. Every hole adds Horseradish peroxidase-conjugated avidin working solution100ul, is covered with new plate and pastes, 37 DEG C of incubations 1 hour. Discard liquid in hole, dry, wash plate 5 times. Invade bubble 2 minutes at every turn,200ul/ hole, dries. Every hole adds substrate solution 90ul successively, 37 DEG C of lucifuge colour developing 15-30 minute. Every hole adds stop bath successively50ul, cessation reaction. After reaction terminating, in 5 minutes, sequentially measure the optical density (OD in each hole at 450nm wavelength with ELIASAValue).
2. data processing
In professional curve plotting software curveexpert1.4, the OD value of each standard items deducts after the OD value of blank wellDo
Figure. Taking OD value as abscissa, the concentration of standard items is ordinate, draws calibration curve, obtains the fit equation of curveFormula. By the fit equation formula of the OD value substitution calibration curve of sample, calculate sample concentration.
Embodiment 3: the clinical practice of kit of the present invention
Inventor, by detecting 117 routine Nasopharyngeal Carcinoma Patients and 54 routine normal persons' serum specimen, has obtained respectively each serum markOD value and the CypA concentration value of this EBV-VCA-IgA. Because EBV-VCA-IgA detection kit can qualitatively judge detected bloodFinal proof basis, therefore inventor show that the positive rate of EBV-VCA-IgA in 117 routine Nasopharyngeal Carcinoma Patients is 69.2%(81/117), feminine genderRate is 30.8%(36/117). CypA detection kit quantitatively detects CypA concentration in serum, need to by ROC tracing analysis itsTo the size of nasopharyngeal carcinoma diagnosis usefulness.
ROC curve (ReceiverOperatingCharacteristic, experimenter's working curve), for two classificationDifferentiate the A+E of effect, general principle is by the movement of critical value (cutoff value), obtain multipair sensitivity withFalse Rate (1-specificity), taking sensitivity as the longitudinal axis, taking False Rate as transverse axis, connects each point curve plotting, then calculated curveUnder area ROC.
The value of ROC TG-AUC reflection diagnostic test, (0.50,0.70], represent that diagnostic value is lower;(0.70,0.90], represent that diagnostic value is medium; More than 0.90 represent that diagnostic value is higher. Be that TG-AUC is larger, diagnosis effectCan be higher. From curve, can obtain the excellent diagnostics standard of certain diagnostic test, and the spirit of this diagnostic test under this standardSensitivity and specificity. In the drawings, the corresponding diagnostic criteria of point that approaches the upper left corner (0,100) is most excellent diagnostics standard.MedCalc software is a medical computing device designing for medical worker specially, aspect processing ROC curve, and operation letterSingle.
Inventor obtains by the ROC curve functional analysis of MedCalc software: EBV-VCA-IgA(refers to Fig. 1 and Fig. 2)Cutoff value: 0.144; Sensitivity: 77.78%; Specificity: 96.30%; ROC area under a curve (AUC): 0.833.CypA(refers to Fig. 3 and Fig. 4) cutoff value: 9.312; Sensitivity: 75.21%; Specificity: 55.56%; Under ROC curveArea (AUC): 0.663. Obviously, both diagnostic values are medium. Now, taking the critical value of 9.312 as CypA etiologic diagnosis, can obtainPositive rate to CypA in Nasopharyngeal Carcinoma Patients is 75.21%(88/117), the positive rate in normal person is 46.3%(25/117)(referring to table 1).
Table 1: the positive rate of indices in Nasopharyngeal Carcinoma Patients
Joint-detection serum cyclophilin A and Epstein-Barr virus VCA-IgA antibody, and obtain the associating spirit after both associatings by following formulaSensitivity and associating specificity:
Associating susceptibility=susceptibility A+(1-susceptibility A) × susceptibility B=74.07%+(1-74.07%) × 75.21%=93.6%
Associating specificity=specificity A × specificity B=96.30% × 55.56%=53.5%
After both associatings, sensitivity improves, and refers to table 2 up to 93.6%(), show joint-detection serum cyclophilin A and Epstein-Barr virusThis method of VCA-IgA antibody can be applicable to nasopharyngeal carcinoma diagnosis and examination, makes assay sensitive, accurate, quick, thereby improvesThe recall rate of nasopharyngeal carcinoma, improves clinical diagnosis efficiency.
Table 2: indices is the sensitivity in ROC curve and specificity alone or in combination
Show that by above-mentioned dying relevant VCA-IgA antibody to virus multiplication sexuality has higher specificity (can reach to nasopharyngeal carcinoma96.30%), but sensitivity only 77.78%, also has part patient to fail to pinpoint a disease in diagnosis or mistaken diagnosis. The present invention is by joint-detection serum parent ringElement A and Epstein-Barr virus VCA-IgA antibody, made up this deficiency preferably.
Inventor finds, uses in the positive case of Epstein-Barr virus VCA-IgA antibody kit testing result, wherein in 82 examplesNasopharyngeal Carcinoma Patients 81 examples, normal person's 1 example, serum cyclophilin A can auxiliary diagnosis. Because Epstein-Barr virus VCA-IgA antibody has betterSpecificity, now can examination go out most of nasopharyngeal cancer patient. But for patient EBV-VCA-IgA of mistaken diagnosis easy to cause missed diagnosis,89 examples are used (wherein Nasopharyngeal Carcinoma Patients 36 examples, normal person in the negative case of Epstein-Barr virus VCA-IgA antibody kit testing result53 examples), joint-detection serum cyclophilin A (referring to table 3), positive rate is 72.22%(26/36), prompting Nasopharyngeal Carcinoma Patients orWhen people at highest risk cannot detect with the high Epstein-Barr virus VCA-IgA antibody assay kit of specificity, can take associating inspectionSurvey the CypA in serum, thereby help to improve clinical diagnosis efficiency. When Fig. 5 and Fig. 6 are EBV-VCA-IgA feminine gender, CypA detectsROC curve map and parameter thereof, the cutoff value of CypA when EBV-VCA-IgA is negative: 8.334; Sensitivity: 77.8%; SpecialDegree: 50.9%; ROC area under a curve (AUC): 0.637.
CypA testing result in the Nasopharyngeal Carcinoma Patients of table 3 serum Epstein-Barr virus VCA-IgA feminine gender
| CypA （+） | CypA（-） | Positive rate | |
| EBV-VCA-IgA（-） | 26 | 10 | 72.22%（26/36） |
Claims (2)
1. a nasopharyngeal carcinoma diagnosis kit, is characterized in that this kit is EBV-VCA-IgA and CypA joint-detection ELISAKit, this ELISA kit comprises that EBV-VCA-IgA detects reagent and CypA detects reagent.
2. nasopharyngeal carcinoma diagnosis kit as claimed in claim 1, is characterized in that: EBV-VCA-IgA detects reagent by EB-VCA-IgA positive control, EB-VCA-IgA negative control, enzyme mark working solution, substrate solution A and substrate solution B, stop buffer composition;CypA detects reagent by biotin labeled anti-human CypA antibody, Horseradish peroxidase-conjugated avidin, TMB solution and terminationLiquid composition.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109633170A (en) * | 2018-12-29 | 2019-04-16 | 郑州安图生物工程股份有限公司 | A kind of Epstein-Barr virus shell antigen I gA antibody assay kit |
| US20200319186A1 (en) * | 2018-02-13 | 2020-10-08 | Sun Yat-Sen University Cancer Center (Sysucc) | Markers for diagnosis and prognostic prediction of npc and application thereof |
| CN116694768A (en) * | 2023-06-27 | 2023-09-05 | 清远市人民医院 | Application of human leukocyte antigen HLA-B5801 gene in nasopharyngeal carcinoma susceptibility screening |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20200319186A1 (en) * | 2018-02-13 | 2020-10-08 | Sun Yat-Sen University Cancer Center (Sysucc) | Markers for diagnosis and prognostic prediction of npc and application thereof |
| CN109633170A (en) * | 2018-12-29 | 2019-04-16 | 郑州安图生物工程股份有限公司 | A kind of Epstein-Barr virus shell antigen I gA antibody assay kit |
| CN116694768A (en) * | 2023-06-27 | 2023-09-05 | 清远市人民医院 | Application of human leukocyte antigen HLA-B5801 gene in nasopharyngeal carcinoma susceptibility screening |
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