CN103823066A - Kit used for fast diagnosis of early stage and middle stage lung cancer and detection method of kit - Google Patents
Kit used for fast diagnosis of early stage and middle stage lung cancer and detection method of kit Download PDFInfo
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- CN103823066A CN103823066A CN201410093875.0A CN201410093875A CN103823066A CN 103823066 A CN103823066 A CN 103823066A CN 201410093875 A CN201410093875 A CN 201410093875A CN 103823066 A CN103823066 A CN 103823066A
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57423—Specifically defined cancers of lung
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a kit used for fast diagnosis of early stage and middle stage lung cancer. Capturing antibodies comprise monoclonal antibodies manufactured by galactose agglutinin, cell cutin segment antigen 21-1, and human aspartyl-beta-hydroxylase respectively, the kit further comprises detection antibodies; the detection antibodies comprises HRP marked polyclonal antibodies which are manufactured by galactose agglutinin, cell cutin segment antigen 21-1, and human aspartyl-beta-hydroxylase respectively. The kit provided by the invention has the following benefits: high sensitivity and good accuracy both higher than 90%, and capability of effectively lowering detection cost, and reducing operating steps and detection errors.
Description
Technical field:
The present invention relates to the kit field that cancer diagnosis detects use, be specially a kind of for lung cancer early metaphase quick diagnosis reagent kit and detection method thereof.
Background technology:
Lung cancer is the highest cancer of mortality ratio in global range.This treatment of cancer difficulty, especially refractory of the lung cancer of transfer.Nearly 75% patients with lung cancer has been metastatic or late period in the time making a definite diagnosis, and its 5 years survival rates are only 6%.Recent decades in past, although the treatment measures such as chemotherapy, radiotherapy and operation have had development fast, 5 years survival rates of lung cancer are still lower.Be mainly because also do not have a kind of safe method to screen it, early stage in disease---the best opportunity for the treatment of, just find in time that patient suffers from the possibility of lung cancer very little.
At present in the urgent need to the effective screening strategy of one.Unclear when CT scan, blood testing can help doctor to make better judgement.Adopt CT scan to screen to asymptomatic patient, not only costliness but also danger.This screening has very high false positive, and some of them patient will accept unnecessary lung's biopsy.
Tumor markers (tumor markers, TM) refers in tumour generation and breeding,, release synthetic by tumour cell itself, or mark tumour existence tumour cell reaction being produced by body and a class material of growth.These materials do not exist in normal adult or the level that occurs in cancer patient is significantly higher than normal person.At present tumor markers detection technique is considered to the unique channel of the asymptomatic micro-kitchen range tumour of early detection, and this detection technique can be found tumour prior to physical examination such as X-ray, ultrasonic, CT, MRI or PET-CT.Can be used for the examination of people at highest risk's malignant tumour, diagnosing tumor and antidiastole, the effect of assessment treatment, prediction or supervision tumor recurrence or transfer.At present, the pulmonary cancer diagnosis kit that hospital occurs is all to detect some common tumor markers, and sensitivity and accuracy are all on the low side.Because being mainly that selected tumor marker individual event detection often has significant limitation, be difficult to meet the requirement of Rapid&Early diagnosis.
At present, on market, also do not come out for the rapidly and efficiently diagnostic kit of lung cancer, badly influence lung cancer early detection and treatment.
Summary of the invention:
The object of the invention is for the deficiency existing with above-mentioned existing pulmonary cancer diagnosis, provide a kind of highly sensitive, accuracy is strong and easy to use efficiently for lung cancer early metaphase quick diagnosis reagent kit.
Another object of the present invention is to provide a kind of detection method for lung cancer early metaphase quick diagnosis reagent kit.
In order to realize the object of the invention, the technical scheme that the present invention takes is: for lung cancer early metaphase quick diagnosis reagent kit, comprise capture antibody, described capture antibody comprises galactose agglutinin and cytokeratin fragment antigen 21-1(CYFRA21-1) monoclonal antibody that makes respectively.
Described capture antibody also comprises the monoclonal antibody that mankind's asparagus fern amine acyl group B-hydroxylase (HAAH) makes.
Described kit also comprises detection antibody, and described detection antibody comprises the polyclonal antibody after the HRP mark that galactose agglutinin, cytokeratin fragment antigen 21-1 (CYFRA21-1) and mankind's asparagus fern amine acyl group B-hydroxylase (HAAH) make respectively.
Described capture antibody is for to be cloned into carrier for expression of eukaryon by galactose agglutinin, cytokeratin fragment antigen 21-1 (CYFRA21-1) and mankind's asparagus fern amine acyl group B-hydroxylase (HAAH); and in mammalian cell, realize the expression of albumen; after purifying, obtain corresponding antigen, the corresponding monoclonal antibody that described antigen immune mammal is obtained.
The step that the preparation method of described capture antibody comprises is as follows:
(1) preparation of antigen: the Gene cloning of galactose agglutinin, cytokeratin fragment antigen 21-1 and mankind's asparagus fern amine acyl group B-hydroxylase to carrier for expression of eukaryon, and in mammalian cell, realize the expression of albumen, after purifying, obtain antigen, this antigen also can be used as standard items and uses;
(2) preparation of capture antibody: by above-mentioned antigen immune mammal, obtaining corresponding monoclonal antibody is capture antibody.
Described detection antibody is for to be cloned into carrier for expression of eukaryon by galactose agglutinin, cytokeratin fragment antigen 21-1 (CYFRA21-1) and mankind's asparagus fern amine acyl group B-hydroxylase (HAAH); and in mammalian cell, realize the expression of albumen; after purifying, obtain corresponding antigen; the corresponding polyclonal antibody that described antigen immune mammal is obtained, carries out HRP mark to described polyclonal antibody.
The step that the preparation method of described detection antibody comprises is as follows:
(1) preparation of antigen: the Gene cloning of galactose agglutinin, cytokeratin fragment antigen 21-1 and mankind's asparagus fern amine acyl group B-hydroxylase to carrier for expression of eukaryon, and in mammalian cell, realize the expression of albumen, after purifying, obtain antigen, this antigen also can be used as standard items and uses;
(2) preparation of detection antibody: by above-mentioned antigen immune mammal, obtain corresponding polyclonal antibody for detecting antibody;
(3) HRP mark: described polyclonal antibody is carried out to HRP mark.
Described galactose agglutinin preferably galactose galectin-3 and Galectin-8.
Described capture antibody is coated in the hole of microtiter plate in advance, can simplify step, improves detection efficiency.
For the preparation method of lung cancer early metaphase quick diagnosis reagent kit, it comprises the following steps:
(1) preparation of antigen: by the Gene cloning of galactose agglutinin, cytokeratin fragment antigen 21-1 and mankind's asparagus fern amine acyl group B-hydroxylase to carrier for expression of eukaryon; and in mammalian cell, realize the expression of albumen; after purifying, obtain required antigen; wherein, described antigen also can be used as standard items use.
(2) preparation of capture antibody: above-mentioned antigen immune mammal is obtained to corresponding monoclonal antibody, and described monoclonal antibody is as the capture antibody of this kit.
(3) preparation of detection antibody: above-mentioned antigen immune mammal is obtained to corresponding polyclonal antibody, described polyclonal antibody is carried out to HRP mark.The present invention detects antibody and has mark function, and the operation steps that can save labelled antibody and add labelled antibody is effectively cost-saving, reduces operation steps, reduces and detects error.
(4) capture antibody is coated:
1.. be the hole of the coated microtiter plate of capture antibody of 1 μ g/ml by concentration with carbonate/bicarbonate damping fluid (pH9.6); 2.. capping microtiter plate overnight incubation at 4 ℃; 3.. discard coating buffer (capture antibody of carbonate/bicarbonate damping fluid dilution), and with twice of cleansing solution washing microtiter plate, in micropore, add 200 μ l PBST(phosphate tween damping fluids) at every turn, whipping microtiter plate gently above tank, remove cleansing solution, on paper handkerchief, pat microtiter plate, remove remaining drop, dry be put in 4 ℃ of environment for subsequent use.
(5) sealing: 200 μ l sealing damping fluids (1.2%BSA/PBS) are added in every hole, remaining protein binding site in the coated hole of sealing.
For the detection method of lung cancer early metaphase quick diagnosis reagent kit, it comprises the following steps:
(1) preparation of antigen: by Galectin-3, HAAH and CYFRA21-1 Gene cloning be to carrier for expression of eukaryon, and in mammalian cell, realize the expression of albumen, after purifying, obtain required antigen;
(2) preparation of capture antibody: above-mentioned antigen immune mammal is obtained to corresponding monoclonal antibody, and described monoclonal antibody is as the capture antibody of this kit;
(3) preparation of detection antibody: above-mentioned antigen immune mammal is obtained to corresponding polyclonal antibody, described polyclonal antibody is carried out to HRP mark;
(4) capture antibody is coated:
1.. be the hole of the coated microtiter plate of capture antibody of 1 μ g/ml by concentration with carbonate/bicarbonate damping fluid (pH9.6);
2.. capping microtiter plate overnight incubation at 4 ℃;
3.. discard coating buffer (capture antibody of carbonate/bicarbonate damping fluid dilution), and with twice of cleansing solution washing microtiter plate, in micropore, add 200 μ l PBST(phosphate tween damping fluids) at every turn, whipping microtiter plate gently above tank, remove cleansing solution, on paper handkerchief, pat microtiter plate, remove remaining drop, dry be put in 4 ℃ of environment for subsequent use.
(5) sealing
1.. add 200 μ l sealing damping fluids (1.2%BSA/PBS) in each hole of microtiter plate, remaining protein binding site in the coated hole of sealing;
2.. capping microtiter plate is also hatched 1 hour at 37 ℃;
(6) application of sample
1.. the sample of 100 μ l is added to each hole of microtiter plate, at 37 ℃, hatch 60 minutes; Obtain quantitative result accurately, way is the signal of comparison unknown sample and typical curve conventionally.The necessary bioassay standard product (double mensuration or triplicate) of each ELISA Plate and blank sample, to guarantee accuracy;
2.. discard sample, and wash microtiter plate three times, in micropore, add 200 μ l PBST(phosphate tween damping fluids at every turn);
3.. the detection antibody that is 0.5 μ g/ml by 100 μ l concentration adds each hole to;
4.. capping microtiter plate is also hatched 1 hour at 37 ℃;
5.. with PBST washing microtiter plate four times;
(7) detect
1.. by TMB(3,3', 5,5'-tetramethyl benzidine) solution adds each hole to, hatches 15-30 minute, adds isopyknic stop buffer, then reads optical density at 450nm place.
2.. the data drawing standard curve being obtained by serial dilutions, it is upper that concentration is marked on X-axis (logarithmically calibrated scale), and absorbance is marked in Y-axis (linear scale).On this typical curve, draw sample concentration by interpolation method.
The present invention is from numerous tumor markers; by various permutation and combination; filter out 3 tumor marker (CBP-35s; mankind's asparagus fern amine acyl group B-hydroxylase (HAAH), cytokeratin fragment antigen 21-1 (CYFRA21-1) composition lung cancer quick diagnosis reagent kit.Wherein, mankind's asparagus fern amine acyl group B-hydroxylase (Human Aspartyl/Asparaginyl β-hydroxylase; HAAH) be to be present in intracellular a kind of enzyme embryonic period, embryonic phase; belong to and rely on α-ketoglutaric acid dioxygenase family, the beta carbon hydroxylation on can aspartic acid or the asparagine residue in catalysis specific protein Concentrations of Epidermal Growth Factor (EGF) acceptor spline structure territory.Research shows the high expressed of HAAH at tumor cell surface, is a kind of molecular marked compound of novel malignant tumour high specific.Nearest research also confirms that HAAH is the ideal mark thing of lung cancer.CBP-35 (Galectin-3) is a kind of galactose-binding protein, is unique member with embedded structure in Glectin family.CBP-35 (Galectin-3) is distributed widely in tumor tissues, and the expression of hL-31 is with the invasion and attack of its tumour and shift closely related.Galectin-3 can be combined and impel integrin at cell surface clustering with the glycosylated CD98 of cell surface, indirectly strengthens the affinity of integrin and its part; Also can be directly in conjunction with integrin alpha 1 β 1 and CD11b/18, positivity or negativity regulate the activity of integrin, affect the combination of integrin and extracellular ligand.Galectin-3 can also increase the expression of integrin at cell surface, and promotes the secretion of collagen at cell compartment.Because CBP-35 has affinity to polysaccharide, it also can be directly in conjunction with glycosylated cells epimatrix composition, the sticking of mediated cell and matrix.Studies show that in Serum of Patients with Lung Cancer, Galectin-3 level obviously exceedes healthy population.Cytokeratin fragment antigen 21-1 (CYFRA21-1) is the soluble fragments of Cyfra21-1 (CYK-19), and CYK-19 is extensively distributed in normal structure surface, in stratiform or scaly epithelium.In Malignant Epithelium cell, the proteinase of activation has accelerated the degraded of cell, makes a large amount of cytokeratin fragment CYFRA21-1 be released into blood, and in malign lung cancerous tissue, CYFRA21-1 rich content especially has high expressed in lung squamous cancer.
Compared with prior art, beneficial effect of the present invention is as follows: have the advantages that highly sensitive accuracy is strong, its sensitivity and accuracy all reach more than 90%, can effectively reduce in addition testing cost, reduces operation steps and reduce and detect error.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of the present invention for lung cancer early metaphase quick diagnosis reagent kit;
Fig. 2 is the comparative selection figure of the present invention for the detection antibody best effort concentration of lung cancer early metaphase quick diagnosis reagent kit;
Fig. 3 is the present invention detects Galectin-3 variation comparison diagram for lung cancer early metaphase quick diagnosis reagent kit blood before and after lung cancer therapy;
Fig. 4 is the present invention detects CYFRA21-1 variation comparison diagram for lung cancer early metaphase quick diagnosis reagent kit blood before and after lung cancer therapy;
Fig. 5 is the present invention detects HAAH variation comparison diagram for lung cancer early metaphase quick diagnosis reagent kit blood before and after lung cancer therapy.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail to explanation for lung cancer early metaphase quick diagnosis reagent kit.
For lung cancer early metaphase quick diagnosis reagent kit, comprise capture antibody, capture antibody sealing damping fluid, standard items, the detection antibody of mark HRP, cleansing solution and nitrite ion etc.Described capture antibody comprises the monoclonal antibody that galactose agglutinin and cytokeratin fragment antigen 21-1 (CYFRA21-1) make respectively; By the common fast detecting to galactose agglutinin and cytokeratin fragment antigen 21-1 (CYFRA21-1), can effectively improve Detection accuracy and sensitivity, effectively overcome the deficiency causing only according to detecting CYFRA21-1.
Described capture antibody also comprises the monoclonal antibody that mankind's asparagus fern amine acyl group B-hydroxylase (HAAH) makes; by detecting galactose agglutinin, cytokeratin fragment antigen 21-1 (CYFRA21-1) and mankind's asparagus fern amine acyl group B-hydroxylase (HAAH), its sensitivity and accuracy are all reached more than 90%.
Described capture antibody is for to be cloned into carrier for expression of eukaryon by galactose agglutinin, cytokeratin fragment antigen 21-1 (CYFRA21-1) and mankind's asparagus fern amine acyl group B-hydroxylase (HAAH); and in mammalian cell, realize the expression of albumen; after purifying, obtain corresponding antigen, the corresponding monoclonal antibody that described antigen immune mammal is obtained.
The step that the preparation method of described capture antibody comprises is as follows:
(1) preparation of antigen: the method by molecular cloning is Galectin-3, HAAH and CYFRA21-1 Gene cloning be to carrier for expression of eukaryon, and in mammalian cell, realize the expression of albumen, obtains antigen after purifying, and this antigen also can be used as standard items and uses; The preparation of described antigen also can be used existing conventional method preparation, no longer burdensome at this.
(2) preparation of capture antibody: by above-mentioned antigen immune mammal, obtaining corresponding monoclonal antibody is capture antibody.Described mammal is mouse and rabbit, preferably mouse.The preparation of described capture antibody also can be used existing conventional method preparation, no longer burdensome at this.
Described detection antibody comprises the polyclonal antibody after the HRP mark that galactose agglutinin, cytokeratin fragment antigen 21-1 (CYFRA21-1) and mankind's asparagus fern amine acyl group B-hydroxylase (HAAH) make respectively.
Described detection antibody is for to be cloned into carrier for expression of eukaryon by galactose agglutinin, cytokeratin fragment antigen 21-1 (CYFRA21-1) and mankind's asparagus fern amine acyl group B-hydroxylase (HAAH); and in mammalian cell, realize the expression of albumen; after purifying, obtain corresponding antigen, what described antigen immune mammal was obtained carries out the polyclonal antibody after HRP mark.The step that the preparation method of described detection antibody comprises is as follows:
(1) preparation of antigen: the method by molecular cloning the Gene cloning of galactose agglutinin, cytokeratin fragment antigen 21-1 and mankind's asparagus fern amine acyl group B-hydroxylase to carrier for expression of eukaryon, and in mammalian cell, realize the expression of albumen, after purifying, obtain antigen, this antigen also can be used as standard items and uses;
(2) preparation of detection antibody: by above-mentioned antigen immune mammal, obtain corresponding polyclonal antibody;
(3) HRP mark: described polyclonal antibody is carried out to HRP mark.
Described mammal is mouse and rabbit, preferably rabbit.Described polyclonal antibody is carried out HRP mark and can be used the existing method of current routine.
Wherein, described galactose agglutinin preferably galactose galectin-3 or Galectin-8; The preferred serum cytokeratin of described cytokeratin fragment antigen 21-1 fragment antigen 21-1.
Wherein, described capture antibody can be coated in the hole of microtiter plate of PVC material in advance, can simplify step, improves detection efficiency.
For the preparation method of lung cancer early metaphase quick diagnosis reagent kit, it comprises the following steps:
(1) preparation of antigen: the method by molecular cloning is by Galectin-3, HAAH and CYFRA21-1 Gene cloning be to carrier for expression of eukaryon, and in mammalian cell, realize the expression of albumen, obtains required antigen after purifying, wherein, described antigen also can be used as standard items use;
(2) preparation of capture antibody: above-mentioned antigen immune mammal is obtained to corresponding monoclonal antibody, and described monoclonal antibody is as the capture antibody of this kit;
(3) preparation of detection antibody: above-mentioned antigen immune mammal is obtained to corresponding polyclonal antibody, described polyclonal antibody is carried out to HRP mark; Described detection antibody has mark function, and the operation steps that can save labelled antibody and add labelled antibody is effectively cost-saving, reduces operation steps, reduces and detects error.
(4) capture antibody is coated:
1.. be coated in the hole of microtiter plate with the capture antibody that carbonate/bicarbonate damping fluid (pH9.6) is 1 μ g/ml by concentration;
2.. use bond plastic goods capping microtiter plate and overnight incubation at 4 ℃;
3.. discard coating buffer (capture antibody of carbonate/bicarbonate damping fluid dilution), and with twice of cleansing solution washing microtiter plate, in micropore, add 200 μ l PBST(phosphate tween damping fluids) at every turn, whipping microtiter plate gently above tank, remove cleansing solution, on paper handkerchief, pat microtiter plate, remove remaining drop, dry be put in 4 ℃ of environment for subsequent use; Described cleansing solution is PBS(phosphate buffer) in add a certain amount of Tween20, the mass percent concentration of described Tween20 is 0.05%;
(5) sealing
1.. add the PBS(phosphate buffer of 200 μ l sealing damping fluids (for containing 1.2%BSA(bovine serum albumin(BSA)) in every hole of microtiter plate)), for sealing the remaining protein binding site in coated hole;
2.. capping microtiter plate is also hatched 1 hour at 37 ℃.
For the detection method of lung cancer early metaphase quick diagnosis reagent kit, it comprises the following steps:
(1) preparation of antigen: the method by molecular cloning is by Galectin-3, HAAH and CYFRA21-1 Gene cloning be to carrier for expression of eukaryon, and in mammalian cell, realize the expression of albumen, obtains required antigen after purifying, wherein, described antigen also can be used as standard items use;
(2) preparation of capture antibody: above-mentioned antigen immune mammal is obtained to corresponding monoclonal antibody, and described monoclonal antibody is as the capture antibody of this kit;
(3) preparation of detection antibody: above-mentioned antigen immune mammal is obtained to corresponding polyclonal antibody, described polyclonal antibody is carried out to HRP mark;
(4) capture antibody is coated:
1.. be the hole of the coated microtiter plate of capture antibody of 1 μ g/ml by concentration with carbonate/bicarbonate damping fluid (pH9.6);
2.. use bond plastic goods capping microtiter plate and overnight incubation at 4 ℃;
3.. discard coating buffer (capture antibody of carbonate/bicarbonate damping fluid dilution), and with twice of cleansing solution washing microtiter plate, in micropore, add 200 μ l PBST(phosphate tween damping fluids at every turn, can be the phosphate buffer containing the pH7.4 of 0.05% Tween-20), whipping microtiter plate gently above tank, removes cleansing solution, pats microtiter plate on paper handkerchief, remove remaining drop, dry be put in 4 ℃ of environment for subsequent use; Described cleansing solution is PBS(phosphate buffer) in add a certain amount of Tween20, the mass percent concentration of described Tween20 is 0.05%;
(5) sealing
1.. add 200 μ l sealing damping fluids (1.2%BSA/PBS) in each hole of microtiter plate, remaining protein binding site in the coated hole of sealing;
2.. hatch 1 hour with bond plastic goods capping microtiter plate and at 37 ℃;
(6) application of sample
1.. 100 μ l are suitably diluted to each hole that the sample of (diluting 20 times) adds microtiter plate to, at 37 ℃, hatch 60 minutes; Obtain quantitative result accurately, way is the signal of comparison unknown sample and typical curve conventionally.The necessary bioassay standard product (double mensuration or triplicate) of each ELISA Plate and blank sample, to guarantee accuracy;
2.. discard sample, and wash microtiter plate three times, in micropore, add 200 μ l PBST(phosphate tween damping fluids at every turn);
3.. the detection antibody that is 0.5 μ g/ml by 100 μ l concentration adds each hole to;
4.. with bond plastic goods capping microtiter plate and hatch at 37 ℃ 1 hour;
5.. with PBST washing microtiter plate four times;
(7) detect
1.. by TMB(3,3 ', 5,5 '-tetramethyl benzidine) solution adds each hole to, hatches 15-30 minute, adds isopyknic stop buffer (2M H
2sO
4), then read optical density with enzyme-linked immunosorbent assay instrument at 450nm place.
2.. the data drawing standard curve being obtained by serial dilutions, it is upper that concentration is marked on X-axis (logarithmically calibrated scale), and absorbance is marked in Y-axis (linear scale).On this typical curve, draw sample concentration by interpolation method.
The present invention comprises CBP-35 (Galectin-3) for lung cancer early metaphase quick diagnosis reagent kit, mankind's asparagus fern amine acyl group B-hydroxylase (HAAH), two in tri-indexs of CYFRA21-1 raise as the standard of diagnosis early metaphase breast cancer simultaneously, detect principle as shown in Figure 1; CBP-35 (Galectin-3), mankind's asparagus fern amine acyl group B-hydroxylase (HAAH), two in tri-indexs of CYFRA21-1 decline as the standard of lung cancer therapy recruitment evaluation simultaneously.Can be for the result for the treatment of of lung cancer being carried out to dynamic evaluation by the height that detects 3 tumor marker levels in blood clinically; Can also be used in addition the application of the relapse and metastasis to lung cancer and prognosis judgement clinically.
Test effect explanation
The selection of double-antibody sandwich elisa optimum experimental condition
Coated mouse-anti people Galectin-3, the selection of HAAH and CYFRA21-1 monoclonal antibody best effort concentration: while determining that according to square formation method coated concentration is 1ug/mL, the OD value of monoclonal antibody is 1.03, so its best coated concentration is 1ug/mL.As shown in Figure 2, the anti-human Galectin-3 of rabbit, the selection of the how anti-best effort concentration of HAAH and CYFRA21-1: along with detecting the increase of antibody dilution multiple, lung cancer case serum to be measured and normal human serum OD value have the trend of successively decreasing, in the time that antibody concentration is 1:200, positive control (positive control OD value deducts blank OD value) is higher with the ratio (being called for short P/N value) of normal control (normal control OD value deducts blank OD value) A450nm, is 1:200 therefore select the anti-human antibody best effort of rabbit concentration.The best effort concentration that serum is groped is 1:25.It is 1.2%BSA that confining liquid is groped best effort solubility.
Clinical serum specimen detects
400 parts of serum specimens are detected altogether, with hospital through being diagnosed as the positive control group of lung cancer I-II phase patients serum (110 example), other benigns patient and the negative control group of normal population serum (290 examples (normal 170 examples, benign 120 examples)), PBST is blank, is undertaken qualitative and is quantitatively detected clinical serum specimen by above-mentioned double antibodies sandwich ELISA method.As shown in the table:
Take P/N value >2 as double antibodies sandwich ELISA Positive judgement standards, take pathological diagnosis as standard, clinical serum specimen is detected, lung cancer result detection sensitivity (SN) is 95.45%(105/110), accuracy rate (SP) reaches 95.25%(381/400).
Clinical the result for the treatment of of lung cancer is carried out to dynamic evaluation
For determining that can this kit be used for the result for the treatment of of breast cancer to carry out dynamic evaluation, we have collected the serum before and after 102 parts for the treatment of in patients with lung cancers.102 patients' testing result is referring to Fig. 3-5, and result shows Galectin-3 in the serum before and after treatment in patients with lung cancer, and there were significant differences for the level of HAAH and CYFRA21-1 (P<0.05).Effectively Galectin-3 in the rear serum for the treatment of, the level of HAAH and CYFRA21-1 can decline greatly, points out this kit can be used for the result for the treatment of of lung cancer to carry out dynamic evaluation.
More than describe preferred embodiment of the present invention in detail, should be appreciated that the ordinary skill of this area just can design according to the present invention be made many modifications and variations without creative work.Therefore, all technician in the art according to the present invention design on prior art basis by logic analysis, reasoning or according to the available technical scheme of limited experiment, all should be among by the determined protection domain of these claims.
Claims (9)
1. for lung cancer early metaphase quick diagnosis reagent kit, comprise capture antibody, it is characterized in that, described capture antibody comprises the monoclonal antibody that galactose agglutinin and cytokeratin fragment antigen 21-1 make respectively.
2. according to claim 1ly it is characterized in that for lung cancer early metaphase quick diagnosis reagent kit, described capture antibody also comprises the monoclonal antibody that mankind's asparagus fern amine acyl group B-hydroxylase makes.
3. according to claim 2 for lung cancer early metaphase quick diagnosis reagent kit; it is characterized in that; described kit also comprises detection antibody, and described detection antibody comprises the polyclonal antibody after the HRP mark that galactose agglutinin, cytokeratin fragment antigen 21-1 and mankind's asparagus fern amine acyl group B-hydroxylase make respectively.
4. according to claim 3ly it is characterized in that for lung cancer early metaphase quick diagnosis reagent kit, the step that the preparation method of described capture antibody comprises is as follows:
(1) preparation of antigen: the Gene cloning of galactose agglutinin, cytokeratin fragment antigen 21-1 and mankind's asparagus fern amine acyl group B-hydroxylase, to carrier for expression of eukaryon, and is realized to the expression of albumen in mammalian cell, obtain antigen after purifying;
(2) preparation of capture antibody: by above-mentioned antigen immune mammal, obtaining corresponding monoclonal antibody is capture antibody.
5. according to claim 3ly it is characterized in that for lung cancer early metaphase quick diagnosis reagent kit, the step that the preparation method of described detection antibody comprises is as follows:
(1) preparation of antigen: the Gene cloning of galactose agglutinin, cytokeratin fragment antigen 21-1 and mankind's asparagus fern amine acyl group B-hydroxylase, to carrier for expression of eukaryon, and is realized to the expression of albumen in mammalian cell, obtain antigen after purifying;
(2) preparation of detection antibody: by above-mentioned antigen immune mammal, obtain corresponding polyclonal antibody for detecting antibody;
(3) HRP mark: described polyclonal antibody is carried out to HRP mark.
6. arbitrary described for lung cancer early metaphase quick diagnosis reagent kit according to claim 1-5, it is characterized in that, described galactose agglutinin is CBP-35.
7. according to claim 6ly it is characterized in that for lung cancer early metaphase quick diagnosis reagent kit, described capture antibody is coated in the hole of microtiter plate in advance.
8. the preparation method for lung cancer early metaphase quick diagnosis reagent kit according to claim 6, its feature in, it comprises the following steps:
(1) preparation of antigen: by the Gene cloning of galactose agglutinin, cytokeratin fragment antigen 21-1 and mankind's asparagus fern amine acyl group B-hydroxylase to carrier for expression of eukaryon, and in mammalian cell, realize the expression of albumen, after purifying, obtain required antigen;
(2) preparation of capture antibody: above-mentioned antigen immune mammal is obtained to corresponding monoclonal antibody;
(3) preparation of detection antibody: above-mentioned antigen immune mammal is obtained to corresponding polyclonal antibody, described polyclonal antibody is carried out to HRP mark;
(4) capture antibody is coated:
1.. be the hole of the coated microtiter plate of capture antibody of 1 μ g/ml by concentration with carbonate/bicarbonate damping fluid; 2.. capping microtiter plate overnight incubation at 4 ℃; 3.. discard coating buffer, and with cleansing solution washing microtiter plate twice, in micropore, add 200 μ l PBST at every turn, remove cleansing solution and remaining drop, dry and be put in 4 ℃ of environment;
(5) sealing: 200 μ l sealing damping fluids, remaining protein binding site in the coated hole of sealing are added in every hole.
9. the detection method for lung cancer early metaphase quick diagnosis reagent kit according to claim 6, its feature in, comprise the following steps:
(1) preparation of antigen: by the Gene cloning of galactose agglutinin, cytokeratin fragment antigen 21-1 and mankind's asparagus fern amine acyl group B-hydroxylase to carrier for expression of eukaryon, and in mammalian cell, realize the expression of albumen, after purifying, obtain required antigen;
(2) preparation of capture antibody: above-mentioned antigen immune mammal is obtained to corresponding monoclonal antibody;
(3) preparation of detection antibody: above-mentioned antigen immune mammal is obtained to corresponding polyclonal antibody, described polyclonal antibody is carried out to HRP mark;
(4) capture antibody is coated:
1.. be the hole of the coated microtiter plate of capture antibody of 1 μ g/ml by concentration with carbonate/bicarbonate damping fluid;
2.. capping microtiter plate overnight incubation at 4 ℃;
3.. discard coating buffer, and with cleansing solution washing microtiter plate twice, in micropore, add 200 μ l PBST at every turn, remove cleansing solution and remaining drop, dry and be put in 4 ℃ of environment;
(5) sealing
1.. add 200 μ l sealing damping fluids in each hole of microtiter plate, remaining protein binding site in the coated hole of sealing;
2.. capping microtiter plate is also hatched 1 hour at 37 ℃;
(6) application of sample
1.. the sample of 100 μ l is added to each hole of microtiter plate, at 37 ℃, hatch 60 minutes;
2.. discard sample, and wash microtiter plate three times, in micropore, add 200 μ l PBST at every turn;
3.. the detection antibody that is 0.5 μ g/ml by 100 μ l concentration adds each hole to;
4.. capping microtiter plate is also hatched 1 hour at 37 ℃;
5.. with PBST washing microtiter plate four times;
(7) detect
1.. add TMB solution to each hole, hatch 15-30 minute, add isopyknic stop buffer, then read optical density at 450nm place.
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|---|---|---|---|---|
| CN107202890A (en) * | 2017-05-31 | 2017-09-26 | 湖州华远生物技术有限公司 | Fast diagnosis reagent and the preparation containing the kit of the reagent and the kit and detection method for brain injury |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101363051A (en) * | 2008-08-29 | 2009-02-11 | 芮屈生物技术（上海）有限公司 | Kit for GALECTIN-3 gene hybridization in situ, detection method and application thereof |
| CN101377501A (en) * | 2007-08-31 | 2009-03-04 | 北京科美东雅生物技术有限公司 | Cell keratin 19 fragments chemiluminescence immune analysis determination reagent kit and preparing method thereof |
| WO2012115885A1 (en) * | 2011-02-22 | 2012-08-30 | Caris Life Sciences Luxembourg Holdings, S.A.R.L. | Circulating biomarkers |
| CN103033619A (en) * | 2013-01-08 | 2013-04-10 | 河南生生医疗器械有限公司 | Protein chip reagent kit and method for comprehensively detecting lung cancer marker |
| CN103163293A (en) * | 2012-06-19 | 2013-06-19 | 中国医学科学院肿瘤医院 | Test kit of auxiliary diagnosis of non-small cell lung cancer patients |
-
2014
- 2014-03-13 CN CN201410093875.0A patent/CN103823066B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101377501A (en) * | 2007-08-31 | 2009-03-04 | 北京科美东雅生物技术有限公司 | Cell keratin 19 fragments chemiluminescence immune analysis determination reagent kit and preparing method thereof |
| CN101363051A (en) * | 2008-08-29 | 2009-02-11 | 芮屈生物技术（上海）有限公司 | Kit for GALECTIN-3 gene hybridization in situ, detection method and application thereof |
| WO2012115885A1 (en) * | 2011-02-22 | 2012-08-30 | Caris Life Sciences Luxembourg Holdings, S.A.R.L. | Circulating biomarkers |
| CN103163293A (en) * | 2012-06-19 | 2013-06-19 | 中国医学科学院肿瘤医院 | Test kit of auxiliary diagnosis of non-small cell lung cancer patients |
| CN103033619A (en) * | 2013-01-08 | 2013-04-10 | 河南生生医疗器械有限公司 | Protein chip reagent kit and method for comprehensively detecting lung cancer marker |
Non-Patent Citations (2)
| Title |
|---|
| 张爱霞等: "血清肿瘤标记物CEA和CYFRA21-1在肺癌临床诊断中的意义", 《放射免疫学杂志》 * |
| 梁勇等: "半乳糖凝集素3及骨桥蛋白在非小细胞肺癌亚临床转移中的意义", 《中华外科杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107202890A (en) * | 2017-05-31 | 2017-09-26 | 湖州华远生物技术有限公司 | Fast diagnosis reagent and the preparation containing the kit of the reagent and the kit and detection method for brain injury |
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Denomination of invention: Rapid diagnostic kit for early and middle stage lung cancer and its detection method Effective date of registration: 20201208 Granted publication date: 20180130 Pledgee: Bank of China Limited by Share Ltd. Guangzhou Panyu branch Pledgor: GUANGZHOU HENGTAI BIOTECHNOLOGY Co.,Ltd. Registration number: Y2020440000389 |
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Date of cancellation: 20220927 Granted publication date: 20180130 Pledgee: Bank of China Limited by Share Ltd. Guangzhou Panyu branch Pledgor: GUANGZHOU HENGTAI BIOTECHNOLOGY CO.,LTD. Registration number: Y2020440000389 |
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Denomination of invention: Rapid diagnostic kit for lung cancer in early and middle stages and its detection method Effective date of registration: 20221014 Granted publication date: 20180130 Pledgee: Bank of China Limited by Share Ltd. Guangzhou Panyu branch Pledgor: GUANGZHOU HENGTAI BIOTECHNOLOGY CO.,LTD. Registration number: Y2022980018084 |
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