CN103389375B - A kind of liquid phase chip reagent box for pulmonary cancer diagnosis - Google Patents
A kind of liquid phase chip reagent box for pulmonary cancer diagnosis Download PDFInfo
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Abstract
The invention discloses a kind of liquid phase chip reagent box for pulmonary cancer diagnosis, comprising: microballoon, biotin labeled detection antibody, the coupling Halotag of the captured antibody of bag? Amine (O
2) microballoon of ligand, antigen protein, SA-PE and biotin labeled human immunoglobulins G; Wherein, capture antibody is at least one in CRP, Prolactin, CEA, NSE, CK19, Axl and ADAM8 capture antibody; Detecting antibody is at least one that CRP, Prolactin, CEA, NSE, CK19, Axl and ADAM8 detect in antibody, and corresponding with capture antibody; Antigen protein is at least one in p62, CTAG, p53 and CAGE; Microballoon with different capture antibody or antigen protein has different color codings.The present invention is to antigen and autoantibody parallel detection, and accuracy rate is high.
Description
Technical Field
The invention relates to the field of medical biology, in particular to a liquid chip kit for lung cancer diagnosis.
Background
Lung cancer is currently the most common malignant tumor with the highest incidence and mortality worldwide. Over the last 20 years, the incidence of lung cancer in men in western countries such as europe and the united states has begun to decline as smoking cessation has been strongly pursued. The incidence of lung cancer in women continues to rise. China is a big country for cigarette production and sale, whether male or female. The incidence of lung cancer is on the rising trend, especially in women. Clinical studies show that the cure rate of carcinoma in situ approaches 100%. The 5-year survival rate of stage I lung cancer patients reaches 60-90%, while the 5-year survival rate of IIIb and IV patients is only 5-20%. It is suggested that early diagnosis is the key to improve lung cancer prognosis. According to the statistical data of national cancer institute, if the lung cancer can be diagnosed in stage I, the five-year survival rate can reach more than 90%; after the second period, the five-year survival rate is sharply reduced to below 20 percent; in the third and fourth stages, the operation is not only useless, but also can promote the tumor to spread rapidly and die. Therefore, how to improve the early diagnosis level of lung cancer has become a serious and urgent task for lung cancer prevention and treatment workers.
The detection means of lung cancer at present depend on image detection instruments such as CT, MR, PET and the like, but the image detection means can only detect tumors with the size of more than 1-2cm generally, and the tumor grows to 1-2cm from lesion, generally, 2-5 years or more are needed, so the early stage missed diagnosis rate of cancer is as high as 80-90%.
At present, the early diagnosis of cancer is usually performed by searching for tumor markers, especially protein markers therein, through blood test. The serum tumor marker can be determined by radioimmunoassay, enzyme immunoassay, chemiluminescence immunoassay, electrochemiluminescence immunoassay, etc. However, these methods are single index detection methods: however, the detection of a single marker for a tumor always has the defects of low specificity, low sensitivity and the like, and particularly the detection rate of early tumors is low, so that the missed diagnosis of part of patients may be caused.
The markers such as tumor antigen in serum can induce the organism to generate autoantibody, and in the early stage that the tumor generation can not be detected by clinical examination means, the immune system of the organism can monitor the existence of the tumor antigen expressed at low level, and trigger immune reaction, generate a large amount of antibodies, and play an effective biological signal amplification role.
Compared with the tumor marker protein which is commonly used in clinic, the detection of the autoantibody is greatly advantageous in tumor diagnosis. For example, the early diagnosis of the tumor can be carried out, the early treatment is convenient, and the cure rate is improved. Several studies have shown that the presence of autoantibodies can be detected months to years before solid cancer is confirmed by imaging, and that antibodies against tumor antigens can be detected in serum even 2-10 years before tumor diagnosis, with autoantibodies having higher titers than the corresponding tumor antigens.
Nevertheless, the detection of autoantibodies has its limitations, such as in general, the abnormal production of one or more tumor autoantibodies by the same tumor, and the detection of either a common tumor antibody or different tumor antibodies by different tumors or different tissue types of the same tumor.
Therefore, how to combine the detection of the autoantibody and the tumor antigen to screen a suitable tumor marker combination has important significance for improving the sensitivity and the accuracy of tumor diagnosis.
Disclosure of Invention
The invention provides a liquid chip kit for lung cancer diagnosis, which is used for detecting antigens and antibodies in serum in parallel, has no cross reaction, high accuracy and can realize early lung cancer diagnosis with high flux and rapidness.
A liquid chip kit for lung cancer diagnosis, comprising: microspheres coated with capture antibody, biotin-labeled detection antibody, coupled Halotag Amine (O)2) Microspheres of ligand, antigenic protein, streptavidin-phycoerythrin and biotin-labeled anti-human immunoglobulin G; wherein,
the capture antibody is at least one of CRP capture antibody, Prolactin capture antibody, CEA capture antibody, NSE capture antibody, CK19 capture antibody, Axl capture antibody and ADAM8 capture antibody;
the detection antibody is at least one of a CRP detection antibody, a Prolactin detection antibody, a CEA detection antibody, an NSE detection antibody, a CK19 detection antibody, an Axl detection antibody and an ADAM8 detection antibody, and corresponds to the capture antibody;
the antigen protein is at least one of p62, CTAG, p53 and CAGE;
microspheres with different capture antibodies or antigenic proteins have different color codes.
NSE: neural specific enolase, enolase is a glycolytic enzyme that is ubiquitous in mammalian tissues, in the form of dimers of a series of isoenzymes (α α, α β, β β, and γ).
CK 19: cyfra21-1, a cytokeratin 19 fragment, is one of the soluble components of epithelial cytokeratin with the molecular weight of about 30kDa, is a very good marker for identifying benign and malignant lung diseases, and has a better detection effect on lung squamous cell carcinoma.
CEA: carcinoembryonic antigen is a tumor antigen existing in various tumor cells, and lung cancer cells can directly produce CEA.
CRP: the C-reactive protein, as an acute phase protein, is less affected by factors such as age, immune status, drugs and the like, and has been recognized to have significance in diagnosis, observation of curative effect and prognosis of clinical diseases such as acute and chronic infections, tissue injuries and the like.
Axl: also known as UFO, ARK, or Tyro7, was first discovered as a transforming gene in leukemia. The protein consists of 894 amino acids, has a molecular weight of 140KD, and is approximately uniformly distributed on two sides of a cell membrane.
ADAM 8: disintegrin-metalloproteinases (ADAMs) are a family of glycoproteins found in recent years that bind to cell membranes and are involved in cell-cell adhesion, cell-matrix adhesion, cell fusion, degradation of extracellular matrix, signal transduction, and other processes.
Prolactin: prolactin, a polypeptide protein hormone secreted by the anterior pituitary, is also known as Prolactin (PRL).
CAGE: the tumor related gene belongs to a cancer-testis (cancer-testis CT) antigen family member, and is a new cancer-testis antigen coding gene discovered in Cho et al 2002 by screening a testis tissue cDNA library by adopting a serological analysis technology of recombinant clone expression antigen.
p 62: nucleoporin, having a relative molecular mass of about 62X 103Belonging to the insulin-like growth factor 2(IGF2) mRNA binding protein familyA member of the family, also known as MP2/IGF2BP 2.
CTAG: NY-ESO-1, a tumor antigen screened from esophageal cancer cDNA library by SEREX technology in 1997 chen et al, belongs to CTA (cancer-testins antigen) family, and the family members are only expressed in testis tissues and some tumor tissues.
In the liquid phase chip kit, the capture antibody and the detection antibody can be specifically combined with corresponding lung cancer serum markers (namely CRP, Prolactin, CEA, NSE, CK19, Axl or ADAM8), and streptavidin-phycoerythrin can be highly specifically combined with biotin, so that a microsphere-capture antibody + serum marker + detection antibody + streptavidin-phycoerythrin composite for the lung cancer serum markers can be formed; in addition, the antigen protein and the anti-human immunoglobulin G can be specifically combined with corresponding antibodies (p62 antibody, NY-ESO-1 antibody, p53 antibody or CAGE antibody) in the lung cancer serum, and the anti-human immunoglobulin G is marked by phycoerythrin, so that a microsphere-antigen protein + serum antibody + anti-human immunoglobulin G + streptavidin-phycoerythrin compound aiming at the lung cancer serum antibody can be formed,
through instrument detection, the reaction type is determined according to different colors of the microspheres, phycoerythrin is excited by green laser, and the number of the reporter fluorescent molecules combined on the microspheres is determined, so that the content of the lung cancer serum marker and the serum antibody combined on the microspheres can be indirectly determined.
The microspheres can be microspheres used for conventional liquid phase chips.
The capture antibody is at least two of a CRP capture antibody, a Prolactin capture antibody, a CEA capture antibody, an NSE capture antibody, a CK19 capture antibody, an Axl capture antibody and an ADAM8 capture antibody.
The capture antibody is CRP capture antibody and Prolactin capture antibody.
When preparing the coated microspheres, catchingThe addition amount of the obtained antibody is 20-30 mu g/6.25 multiplied by 106And (3) microspheres.
Preferably, the antigenic protein is CTAG.
The concentration of each microsphere coated with the capture antibody is 1.2-1.8 multiplied by 104Mu.l, preferably 1.2X 104Mu.l/l.
Coupling Halotag Amine (O)2) The concentration of the microspheres of ligand is 1.2-1.8 multiplied by 104Mu.l, preferably 1.2X 104Mu.l/l.
The concentration of each biotin-labeled detection antibody is 0.8-1.2. mu.g/ml, preferably 1. mu.g/ml.
The concentration of the biotin-labeled anti-human immunoglobulin G is 0.8 to 1.2. mu.g/ml, preferably 1. mu.g/ml.
Compared with the prior art, the invention has the beneficial effects that:
the liquid chip kit for lung cancer diagnosis is based on a liquid chip technology platform, can carry out combined detection on seven serum markers of CRP, Prolactin, CEA, NSE, CK19, Axl and ADAM8 and four serum antibodies of p62 antibody, CTAG antibody, p53 antibody and CAGE antibody in serum, has the accuracy of lung cancer diagnosis up to 91 percent, the false positive reaction less than or equal to 2 percent and the accuracy of lung cancer diagnosis by carrying out combined detection on the CTAG antibody, CRP and Prolactin.
The liquid chip kit for lung cancer diagnosis has the advantages of no side effect, high flux, high sensitivity, good repeatability, quick and accurate detection, low cost and the like.
Drawings
FIG. 1 is a schematic diagram of a standard curve for detecting CRP;
FIG. 2 is a schematic diagram of a standard curve for detecting Prolactin;
FIG. 3 is a schematic diagram of a standard curve for detection of CTAG autoantibodies.
Detailed Description
For better understanding of the technical solutions of the present invention, the following embodiments are further described, but those skilled in the art should recognize that the present invention is not limited to these embodiments.
Example 1 preparation of CTAG antibody, CRP and Prolactin marker detection kit for lung cancer serum sample
1. Reagents and solutions
(1)0.1M NaH2PO4pH 6.2: 3.5814g of NaH were weighed out2PO4In 90mL ddH2In O, NaOH is used for regulating the pH value to 6.2, the volume is fixed to 100mL, and the solution is filtered by a 0.22um filter membrane;
(2)0.05M MES, pH 5.0: weigh 0.976g MES in 90mL ddH2In O, NaOH is used for regulating the pH value to 5.0, the volume is fixed to 100mL, and the solution is filtered by a 0.22um filter membrane;
(3)0.1M MES, pH 6.0: weigh 1.952g MES in 90mL ddH2In O, NaOH is used for regulating the pH value to 6.0, the volume is fixed to 100mL, and the solution is filtered by a 0.22um filter membrane;
(4)0.1M MES, pH 4.5: weigh 1.952g MES in 90mL ddH2In O, NaOH is used for regulating the pH value to 4.5, the volume is fixed to 100mL, and the solution is filtered by a 0.22um filter membrane;
(5)PBS：NaCl，137mM；KCl，2.7mM；Na2HPO4，8.1mM；KH2PO41.5 mM; filter membrane filtration of 0.22um with pH7.2-7.4;
(6)1% PBSB: 0.02% Proclin300 in 0.1% PBS, and 0.22um membrane filtration;
(7)0.1% PBST: 0.1% PBS containing 0.1% tween-20, 0.22um filter membrane filtration;
(8) EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride);
(9) S-NHS (N-hydroxythiosuccinimide).
2. A liquid chip kit for early diagnosis of lung cancer comprises:
(1)2-plex coated microspheres: comprises 34# microsphere coated with CRP capture antibody and 45# microsphere coated with Prolactin capture antibody; the concentration of different kinds of coating microspheres is 1.2 multiplied by 104Mu/l;
(2) coupled with HaloTag (O)2) amine ligand 55# and 47# microspheres coupled with goat anti-human IgG 42# microspheres;
(3)2-plex biotin-labeled detection antibody: respectively using mixed liquor of CRP detection antibody marked by biotin and Prolactin detection antibody;
(4) CTAG and Halo antigen proteins;
(5) streptavidin-phycoerythrin;
(6) goat anti-human igg-biotin and human igg;
(7) reaction buffer: 1% PBSB;
(8) dilution buffer: 1% PBSB;
(9) sealing films;
(10) a 96-well plate;
(11) quality control liquid (CRP standard protein, prolactin standard protein).
3. The preparation of the liquid phase chip kit comprises the following steps:
3.1 according to the composition of the kit, each capture antibody coats the corresponding microsphere, and the preparation method is the same:
(1) taking 45#, 34#, 42# microsphere (magnetic microsphere, bio-rad) suspensions, respectively vortexing and sonicating for 20s, and respectively sucking 200ul (6.25 × 10)6Microspheres) into a centrifuge tube;
(2) 14000g, centrifuging for 4min, and sucking the supernatant;
(3) 100ul ddH was added2O, respectively vortexing and sonicating for 20 s;
(4) 14000g, centrifuging for 4min, removing supernatant, adding 160ul0.1M, pH6.2NaH2PO4Vortexing and sonicating for 20s, respectively;
(5) rapidly adding 20ul of S-NHS with the concentration of 50mg/mL, and uniformly mixing by vortex;
(6) rapidly adding 20ul50mg/mL EDC, and separately vortexing and sonicating for about 20 s;
(7) performing rotary incubation in dark for 20min, and performing vortex mixing once every 10 min;
(8) 14000g, centrifuging for 4min, carefully sucking off the supernatant, adding 250 μ L0.05M MES (pH5.0), and vortexing and sonicating for about 20 s;
(9) 14000g, centrifuging for 4min, carefully sucking off the supernatant, and repeating the above washing steps once;
(10) carefully aspirate the supernatant and resuspend the microspheres in 50ul0.05M MES pH5.0, vortexe and sonicate separately for about 20 s;
(11) adding 25ug CRP capture antibody (R & D Systems, product number KXL0211061), Prolactin capture antibody (R & D Systems, product number DJJ0111061) and goat anti-human immunoglobulin G (Jackson Immunoresearch Laboratories, product number 109-;
(12) rotationally incubating for 2h in a dark place;
(13) 14000g, centrifuging for 4min, carefully removing the supernatant, adding 1ml of 0.01% PBST, and respectively performing vortex and ultrasonic treatment for about 20 s;
(13) 14000g, centrifuging for 4min, carefully sucking off the supernatant, and repeating the above washing steps once;
(14) adding 1mL of 1% PBSB, and respectively vortexing and sonicating for about 20 s;
(15) rotationally incubating for 30min in a dark place, and sealing the residual activated carboxyl sites on the surface of the microsphere;
(16) 14000g, centrifuging for 4min, carefully sucking off the supernatant, adding 1mL of 1% PBSB, and respectively vortexing and sonicating for about 20 s;
(17) carefully aspirate the supernatant, add 1% PBSB to a final volume of 200ul, vortex and sonicate for about 20s, respectively;
(18) 2ul of the suspension was placed in 38ul of 1% PBSB, and after vortexing and sonication for about 20s, the blood count plates were counted;
(19) marking the concentration of the microspheres and the coupling date on a centrifugal tube, and then placing the centrifugal tube at 4 ℃ in a dark place for storage;
3.2 Halo Tag (O) according to the composition of the above-mentioned kit2) Amine ligand is coupled with corresponding microspheres, and the preparation method is the same as that of the microspheres:
taking out 55# and 47# microsphere stock solutions, respectively performing vortex and ultrasonic treatment for about 20s, and taking 200ul (about 6.25 × 10)6One) in a centrifuge tube, 14000g and centrifuging for 4 min;
gently sucking off the supernatant, adding 1ml of 0.1M MES with the pH value of 6.0, and respectively carrying out vortex and ultrasonic treatment for about 20 s;
14000g, centrifuging for 4min, and removing supernatant;
adding 40ul5mg/mLHalo Tag (O)2) Amine ligand, supplementing 0.1M MES with pH6.0 to the final volume of 450ul, and mixing by vortex;
adding 50ul50mg/mL EDC (for use in preparation, 0.1M, dissolved by MES with pH 6.0), and respectively performing vortex and ultrasonic treatment for about 20 s;
sixthly, rotationally incubating for 2 hours in a dark place;
seventhly, adding 500ul of 0.1M MES with the pH value of 4.5, whirling for 20s and 14000g, and centrifuging for 4 min;
eighthly, lightly sucking the supernatant, and washing twice by using 500ul0.1M MES with the pH value of 4.5;
ninthly 14000g, centrifuging for 4min, carefully sucking the supernatant, adding 0.1M MES with pH4.5 to a final volume of 200ul, and respectively carrying out vortex and ultrasonic treatment for about 20 s;
collecting 2ul fraction in 38ul fraction 1% PBSB, vortexing and sonicating for 20s, and counting with blood counting plate;
marking the microsphere concentration and the ligand coupling date on a centrifugal tube, and then placing the centrifugal tube at 4 ℃ in a dark place for storage;
example 2 application of the liquid chip kit of the invention to detection of Lung cancer
(1) All reagents are taken out before use and are placed and balanced to room temperature;
(2) taking 2-plex Halo Tag (O)2) Amine ligand is coupled with microspheres, vortex and uniformly mixed for 20s, ultrasonic treatment is carried out for 20s, and a proper amount of 2500 microspheres in each hole is taken and placed in 500ul1% PBSB;
(3) sealing in dark for 1 h;
(4) 14000g, centrifuging for 4min, sucking the supernatant, adding corresponding protein, wherein CTAG (creative Biomart, Cat. CTAG1B-1512H) and Halo are respectively 0.2 ug/hole, and 1% PBSB is supplemented until the final volume is 500 ul;
(5) incubating for 1h at room temperature in a dark place;
(6) the microspheres are subjected to vortex and ultrasonic treatment for about 20s, and then are subpackaged into a 96-pore plate, wherein each pore is 10 ul;
(7) preparing CRP (R & D Systems, product number 1229006), Prolactin (R & DSystems, product number 1175435) and human IgG standard protein, marking 8 centrifuge tubes with S1, S2, S3, S4, S5, S6, S7 and S8 respectively, and diluting by 2-fold gradient series, specifically as shown in Table 1:
TABLE 1
Wherein the standard protein is diluted with 1% PBSB and the human IgG is diluted with serum matrix without IgG by 4 times.
(9) Opening a plate washing machine, namely Prime, ring (channel2), Prime, placing a 96-hole plate, Run 5: MAGX 3;
(10) taking 2-plex capture antibody coated microspheres, vortexing and uniformly mixing for 20s, and adding 2500 microspheres in each hole into 1% PBSB;
(11) immediately adding the diluted standard substances, each 30 ul/hole;
(12) wrapping the flat plate with aluminum foil paper, and performing shaking incubation reaction for 60 min;
(13) 0.1% PBST washed three times, Run 5: MAGX 3;
(14) mixing the raw materials in a ratio of 1: 180, diluting 2-plex biotin-labeled detection antibodies (biotin-labeled CRP detection antibodies with the product numbers of DY2648, R & D system and biotin-labeled Prolactin detection antibodies with the product numbers of DY682 and R & D system) by using 1% PBSB, wherein the concentration of each detection antibody is 1ug/ml, and the detection antibodies are respectively packaged into 50 ul/hole;
(15) wrapping the flat plate with aluminum foil paper, and performing shaking incubation reaction for 60 min;
(16) 0.1% PBST washed three times, Run 5: MAGX 3;
(17) diluting anti-human IgG-biotin (concentration of 1ug/ml) and SAPE (concentration of 1ug/ml) with 1% PBSB, and subpackaging into 50 ul/well;
(18) wrapping the flat plate with aluminum foil paper, and performing shaking incubation reaction for 60 min;
(19) 0.1% PBST washed three times, Run 5: MAGX 3;
(20) resuspending microspheres in 100ul/well1% PBSB, RT, shaking for 3-5 min;
(21) and reading the result on the liquid phase chip analyzer, and automatically drawing a standard curve by the analyzer and calculating the measured value of the sample to be measured.
3.4 analysis of results
When a standard curve is prepared, the predicted concentration, the actually measured concentration and the MFI value of the CRP, the Prolactin and the human IgG standard substance are shown in a table 2, and the detection curves of the CRP, the Prolactin and the autoantibody are shown in figures 1-3.
The calculation result shows that the content of the compound,
the standard curve for CRP is: FI =99.8135+ (4893.65-99.8135)/((1+ (Conc/13.9421) ^ 0.878012)) ^ 1.02009;
the standard curve for Prolactin is: FI =7.03791+ (852.475-7.03791)/((1+ (Conc/25.6551) ^ 1.09938)) ^ 0.920591;
the standard curve for autoantibodies is: FI =15.1731+ (12382-15.1731)/((1+ (Conc/517.673) ^ -1.2711)) ^ 0.857029.
TABLE 2 preparation of calibration curves for standards
As can be seen from the figure and the table, the linearity of standard curves of each index of CRP, Prolactin and CTAG in the detected concentration range R2More than or equal to 0.99, the relative error of the external quality control product measured by the kit is less than or equal to +/-5 percent, the accuracy of lung cancer diagnosis can reach 91 percent by combining the four indexes, and the false positive reaction is less than or equal to 2 percent. The amount of serum required for the assay is very small (2 microliters) and can be completed within three hours.
Claims (1)
1. A liquid chip kit for lung cancer diagnosis, comprising: microspheres coated with capture antibody, biotin-labeled detection antibody, coupled Halotag Amine (O)2) Microspheres of ligand, antigenic protein, streptavidin-phycoerythrin and biotin-labeled anti-human immunoglobulin G; wherein,
the capture antibody is CRP capture antibody and Prolactin capture antibody;
the detection antibody is CRP detection antibody, Prolactin detection antibody, and is corresponding to the capture antibody;
microspheres with different capture antibodies or antigenic proteins have different color codes;
when the coated microspheres are prepared, the addition amount of the capture antibody is 20-30 mu g/6.25 multiplied by 106A plurality of microspheres;
the antigen protein is CTAG;
the concentration of each microsphere coated with the capture antibody is (1.2-1.8) multiplied by 104Mu/l;
coupling Halotag Amine (O)2) The concentration of the microspheres of the ligand is (1.2-1.8) multiplied by 104Mu/l;
the concentration of each biotin-labeled detection antibody is 0.8-1.2 mu g/ml.
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| CN105510586B (en) * | 2015-12-22 | 2017-09-12 | 湖北鹊景生物医学有限公司 | Kit and its application method for pulmonary cancer diagnosis |
| CN105548547A (en) * | 2016-02-18 | 2016-05-04 | 山东信力科生物科技有限公司 | Flow type array immunoassay kit for detecting lung cancer markers based on flow cytometry |
| CN106680515B (en) * | 2016-10-21 | 2018-06-12 | 杭州金式麦生物科技有限公司 | It is combined for the polymolecular marker of pulmonary cancer diagnosis |
| CN109682969A (en) * | 2019-02-18 | 2019-04-26 | 四川大学华西医院 | A kind of liquid phase chip reagent box detecting intractable epilepsy disease |
| CN110286220A (en) * | 2019-06-04 | 2019-09-27 | 郑州大学第一附属医院 | A kind of application of the molecular marker group and its capture albumen of the early stage cancer of the esophagus in kit |
| CN111474355A (en) * | 2020-04-23 | 2020-07-31 | 北京唯公医疗技术有限公司 | Liquid phase chip for lung cancer diagnosis and use method thereof |
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