CN103383395A - Liquid chip kit for detecting lung cancer autoantibody - Google Patents
Liquid chip kit for detecting lung cancer autoantibody Download PDFInfo
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Abstract
The invention discloses a liquid chip kit for detecting a lung cancer autoantibody. The kit includes: microspheres coupling antigen proteins, a biotin labeled second antibody, streptavidin-phycoerythrin, a reaction buffer and a dilution buffer solution. The antigen proteins are at least two of p62, NY-ESO-1, p53 and CAGE, wherein the microspheres coupling different antigen proteins have different color codes. The second antibody is anti-human immunoglobulin G. The liquid chip kit for detecting the lung cancer autoantibody provided in the invention realizes joint detection of four antibodies in serum on a liquid chip technology platform, and takes the p62 antibody, the NY-ESO-1 antibody, the p53 antibody and the CAGE antibody as marks, has high detection accuracy, and provides a new idea for early diagnosis of lung cancer.
Description
Technical field
The present invention relates to the medicine bioengineering field, relate in particular to a kind of liquid phase chip reagent box for detection of the lung cancer autoantibody.
Background technology
In recent years, the incidence of China's cancer is obvious ascendant trend, accounts for more than 20% of the cause of death, occupies first of all kinds of causes of the death.World Health Organization's prediction to the year two thousand twenty, will have 2,000 ten thousand new cases of cancers, and wherein death toll reaches 1,200 ten thousand, and the overwhelming majority occurs in developing country.If do not take any effective prevention and control measure, expect the year two thousand twenty, kainogenesis cancer sum and the cancer mortality sum in China every year will reach 3,000,000 left and right, and ill sum will reach 6,600,000.
Lung cancer is the highest malignant tumour of M ﹠ M in global range at present.Over nearly 20 years, owing to carrying out energetically smoking cessation, the incidence of disease of western countries' male lung cancers such as Europe and the U.S. has begun to descend, but the incidence of disease of female lung cancer continues to rise.China is cigarette production and selling big country, no matter the male sex or women, the incidence of disease of lung cancer all is lasting ascendant trend, especially rises faster with women's incidence of disease.Clinical research shows, the carcinoma in situ cure rate is near 100%, and 5 years survival rates of I phase patients with lung cancer reach 60%～90%, and 5 years survival rates of IIIb and IV phase patient only 5%～20%, therefore, the early diagnosis early detection, early treatment is to reduce lung cancer mortality, the key that extends life cycle.Yet owing to lacking desirable method of early diagnosis, the early diagnostic rate of lung cancer is 14% left and right only.Therefore, how to improve lung cancer early diagnosis level and become the serious and urgent task that lung cancer preventing and controlling person faces.
The marks such as the tumour antigen in serum can induce body to produce autoantibody, tumour occur also to fail by the clinical examination means detect early stage, body immune system just can monitor the existence of the tumour antigen of low expression level, and initiation immune response, produce a large amount of antibody, play effective bio signal amplification.
Contrast clinical tumor marker albumen commonly used, the detection of autoantibody accounts for great advantage in diagnosing tumor.The first, can carry out early diagnosis to tumour, be convenient to early treatment, improve cure rate.Multinomially studies show that the existence that autoantibody can be detected before imaging examination is made a definite diagnosis solid carcinoma several months to the several years, even 2-10 just can detect the antibody that has in serum for tumour antigen before tumour is made a definite diagnosis.The second, autoantibody is higher than corresponding tumour antigen titre, increases in a large number by immune response for the autoantibody of single antigen, in the situation that in serum, a large amount of albumin exist and disturb, more easily detects than other mark.Therefore analyze the immune change of tumor patient, rather than oncoprotein itself becomes a very important research approach.The 3rd, sample easily obtains, and testing result is relatively stable.In a single day tumour antigen is secreted into blood, may be degraded soon or remove, and autoantibody is subject to the protease hydrolytic effect unlike other polypeptide, can be for a comparatively long period of time in serum stable, sustainable existence, and the half life period of autoantibody is very long, is approximately 7 days, and fluctuation hourly is very little, physicochemical property is stable, in-80C long preservation on its activity almost without affecting.The 4th, its operation reagent and technology used is simple and easy to do, good reproducibility, and enzyme linked immunosorbent assay (ELISA) or enzyme-linked immuno assay (EIA) by routine just can detect.
Tumour is the Carcinogenesis of a polygenes, multi-step; although the tumour autoantibody is the ideal candidates index of diagnosing tumor; only depend on an index to diagnose; usually can cause false positive and false negative; so; single tumour autoantibody detects and certainly exists some limitation, affects the clinical diagnosis of lung cancer.One, in specific tumors, the positive rate of single tumour autoantibody only has 10% usually, is only also 20-30% in optimal tumour colony; They are two years old, a lot of biological approaches in tumor invasion are that various diseases (comprising tumour and other autoimmune diseases) is common, generally, tumour of the same race can produce one or more tumour autoantibodies singularly, and the histological types of different tumours or tumour of the same race both can detect common tumour antibody, also can detect different tumour antibodies.
Research trend in recent years is in carrying out cancer detection for the associating antibody repertoire.The utilization ELISA such as Chapman detect the serum antibody for 7 kinds of tumour antigens (p53, c-myc, HER2, NY-ESO-1, CAGE, MUC1 and GBU4-5) in lung cancer (comprising non-small cell lung cancer and small-cell carcinoma of the lung) patient and normal control crowd serum, result shows that monospecific antibody detects positive rate between 5%-36%, the specificity of diagnosis reaches 96%-100%, and the susceptibility of 7 kinds of antibody combined detections can reach 76%, and specificity is 92%.This individual event that proves absolutely tumor markers detects, and specificity is higher, but susceptibility and accuracy are lower; After joint-detection, although specificity decreases, susceptibility and accuracy all improve a lot.
So relevant autoantibody of joint-detection kinds of tumors, form the exclusive autoantibody " finger-print " of tumour itself, specificity and the susceptibility of diagnosis and prognostic are brought up to the single individual level that is beyond one's reach, because these autoantibodies occur usually, thereby has important diagnosis and prognosis judgement value before clinical symptoms.
Summary of the invention
The invention provides a kind of liquid phase chip reagent box for detection of the lung cancer autoantibody, realized the joint-detection of Multiple Antibodies in serum, high to the detection accuracy of lung cancer.
A kind of liquid phase chip reagent box for detection of the lung cancer autoantibody, comprise the microballoon of coupled antigen albumen, biotin labeled two anti-, SA-PE, reaction buffer and dilution buffer liquid, antigen protein is p62, NY-ESO-1, p53 and CAGE, wherein, the microballoon of the different antigen proteins of coupling has different color codings; Two anti-are biotin labeled anti-human immunoglobulin G.
CAGE: tumor-related gene, belong to cancer-testis (cancer-testis CT) antigen family member, that the people such as Cho in 2002 adopt recombinant clone to express the serological analysis technology of antigen, a new Cancer-testis antigen encoding gene of finding during examination testis tissue cDNA library.
P62: nucleoporin is that relative molecular mass is about 1 tumor associated antigen of 62 * 103, belongs to IMA-IGF2BP3-001 (IGF2) mRNA in conjunction with a member of protein family, claims again MP2/IGF2BP2.
NY-ESO-1: be that chen in 1997 etc. utilize the SEREX technology to screen the tumour antigen that obtains from cancer of the esophagus cDNA library, belong to CTA(cancer-testis antigen) family, this family member only expresses in testis tissue and some tumor tissues.
The p53:p53 gene is a kind of antioncogene, is positioned human chromosomal 17p13.1, and the molecular weight that 393 amino acid of encoding form is the interior phosphorylated protein of the core of 53kD, is called as p53 albumen.
the present invention is with antigen protein p62, NY-ESO-1, the autoantibody that p53 and CAGE produce is as detected object, in liquid phase chip reagent box of the present invention, antigen protein and two anti-all can be special with corresponding Sera of Lung Cancer in antibody (p62 antibody, NY-ESO-1 antibody, p53 antibody or CAGE antibody) combination, and SA-PE can with the combination of biotin high degree of specificity, therefore, finally can form " microballoon-antigen protein+serum antibody+two resist+SA-PE " compound for Sera of Lung Cancer antibody, detect by instrument, definite reaction types different from the microballoon color, excite phycoerythrin with green laser, measure the quantity of the report fluorescence molecule of combination on microballoon, be used for indirectly determining the Sera of Lung Cancer Antibody concentration of combination on microballoon.
Described reaction buffer can be 1%PBSB.
Described dilution buffer liquid can be 1%PBSB.
Described antigen protein is p62, NY-ESO-1, p53 and CAGE.
Described antigen protein can pass through HaloTag
TMInterchangeable labelling technique is connected with microballoon.
The amino acid sequence of p62 albumen is as shown in SEQ ID NO.5; The gene order of coding p62 albumen is as shown in SEQ ID NO.1; The amino acid sequence of NY-ESO-1 albumen is as shown in SEQ ID NO.6, and the gene order of coding NY-ESO-1 albumen is as shown in SEQ ID NO.2; The amino acid sequence of p53 albumen is as shown in SEQ ID NO.7, and the gene order of coding p53 albumen is as shown in SEQ ID NO.3; The amino acid sequence of CAGE albumen is as shown in SEQ ID NO.8; , the gene order of coding CAGE albumen is as shown in SEQ ID NO.4.
Preferably, described two anti-are goat anti-human immunoglobulin G, and experiment finds that mouse source antibody can produce cross reaction, and the antibody of Yang Yuan can overcome this defective.
The microballoon that described microballoon can adopt conventional liquid-phase chip to use gets final product.
When preparing the microballoon of coupled antigen albumen, the addition of antigen protein is 5～10 μ g/6.25 * 10
6Individual microballoon is preferably 6.25 μ g/6.25 * 10
6Individual microballoon.
The concentration of the microballoon of every kind of coupled antigen albumen is 1.2～1.8 * 10
4Individual/μ l is preferably 1.2 * 10
4Individual/μ l.
Biotin labeled two anti-concentration are 0.1～0.125 μ g/ml, are preferably 0.1 μ g/ml.
Compared with prior art, beneficial effect of the present invention is:
The present invention is for detection of the liquid phase chip reagent box of lung cancer autoantibody, realized the joint-detection to four kinds of antibody in serum, and p62 antibody, NY-ESO-1 antibody, p53 antibody and CAGE antibody in the serum are as sign, detection accuracy is high, for the diagnosis and prognosis of lung cancer provides new instrument and thinking.
The present invention is for detection of the liquid phase chip reagent box of the lung cancer autoantibody technology platform based on liquid-phase chip, can high-throughout trace sample be detected fast, liquid phase environment more is conducive to keep the native conformation of protein, also more is conducive to reaction and carries out, highly sensitive, signal to noise ratio (S/N ratio) is good.
Description of drawings
Fig. 1 is the distribution of the unimolecule autoantibody in patients with lung cancer and normal healthy controls;
Fig. 2 is that 3 kinds of biostatistics algorithms (CCP, DLDA, BCCP) are analyzed the accuracy that liquid phase chip reagent box of the present invention detects.
Embodiment
Below in conjunction with specific embodiment, the present invention is done further explaination.
The preparation of embodiment 1 liquid phase chip reagent box
Reagent and solution
(1) 0.1M NaH
2PO
4, pH6.2: weighing 3.5814g NaH
2PO
4In 90mL ddH
2In O, NaOH is settled to 100mL, the 0.22um membrane filtration after regulating pH to 6.2;
(2) 0.05M MES, pH5.0: weighing 0.976g MES is in 90mL ddH
2In O, NaOH is settled to 100mL, the 0.22um membrane filtration after regulating pH to 5.0;
(3) 0.1M MES(2-(N-morpholino) ethyl sulfonic acid), pH6.0: weighing 1.952g MES is in 90mL ddH
2In O, NaOH is settled to 100mL, the 0.22um membrane filtration after regulating pH to 6.0;
(4) 0.1M MES, pH4.5: weighing 1.952g MES is in 90mL ddH
2In O, NaOH is settled to 100mL, the 0.22um membrane filtration after regulating pH to 4.5;
(5) PBS:NaCl, 137mM; KCl, 2.7mM; Na
2HPO
4, 8.1mM; KH
2PO
4, 1.5mM; PH7.2-7.4, the 0.22um membrane filtration;
(6) contain 0.02%Proclin300 in 1%PBSB:0.1%PBS, the 0.22um membrane filtration;
(7) contain 0.1%tween-20 in 0.1%PBST:0.1%PBS, the 0.22um membrane filtration;
(8) 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC);
(9) N-hydroxy thiosuccinimide (S-NHS).
1, for detection of the liquid phase chip reagent box of autoantibody, include:
1) coupling has 55#, 47#, 27#, the 33# microballoon of Halo amine (O2) ligand;
2) contain the fusion of Halo Tag: p62, NY-ESO-1, p53, CAGE;
3) SA-PE;
4) goat anti-human igg-biotin: concentration is 0.1 μ g/ml;
5) reaction buffer: 1%PBSB;
6) dilution buffer liquid: 1%PBSB;
7) sealed membrane;
8) 96 orifice plates.
Prepare above-mentioned liquid phase chip reagent box, comprise the steps:
1, recombinant protein abduction delivering and purifying
Reagent and solution:
KRX recombinant bacterium (containing respectively just like the gene shown in SEQ ID NO.1～4 ,-80 ℃ of preservations)
Dusty yeast (OXOID)
Peptone (OXOID)
Halo?tag?protein?purfication?resins（Promega）
Protease?inhibitor（Roche）
Lysozyme（TOPBIO）
DNase?I
TEV?protease（Promega）
His link resin (Promega)
Purification column (BIO-RAD)
CA-630（Sigma）
LB nutrient culture media (5.0gNaCl transfers PH to 7.0～7.5 to be settled to 1L, 120 ° of autoclaving 20min for 10.0g peptone, 5.0g dusty yeast)
Damping fluid 1(50mM Hepes, PH7.5,150mM NaCl, 1mM DTT)
Damping fluid 2(50mM Hepes, PH7.5,150mM NaCl, 1mM DTT, 0.5mM EDTA, 0.05%CA630)
Experimental procedure:
One, a large amount of abduction deliverings of recombinant bacterium
1, the amplification of spending the night
Take out the KRX recombinant bacterium of preservation from-80 degree refrigerators, join in the ammonia benzyl resistance LB nutrient culture media that 10ml contains 0.2% glucose, 37 ℃ of 275rpm amplification of spending the night.
2, enlarge cultivation
The thalline that will spend the night joins according to 1:100 that in the LB nutrient culture media that 1L contains ammonia benzyl resistance, 37 ℃ of 275rpm are cultured to OD=0.4-0, and 5, adding final concentration in nutrient culture media is 0.05% glucose and rhamnose, 25 ℃ of 225rpm overnight incubation.
3, the centrifugal 15min of 5000g collects thalline, and the 100ml/ pipe is carried out mark and is placed in-80 ° of preservations.
Two, KRX recombinant bacterium lysis
1, get 1 the pipe-80 ° of frozen KRX recombinant bacteriums, add resuspended rear 100ul Protease inhibitor, the 10ul Lysozyme(100mg/ml of adding respectively of 15ml damping fluid 1), 10ul DNase I(5mg/ml), 100ul1%CA-630; Resuspended thalline, after mixing, ice bath is placed 30min.
2, (super 5s stops 10s to the ultrasonication thalline, and 85% power 3min), is placed 10min on ice.
3,12000g, 4 ° of centrifugal 30min get supernatant, get 100ul supernatant (wherein 50ul is labeled as S, and the TEV enzyme labeling of 50ul+10ul1:100 dilution is S-TEV in addition) in the 1.5ml centrifuge tube.
4, the centrifugal Halo link that cleans simultaneously: get respectively the Halo link beads of 1 pipe 2ml, 3300rpm is centrifugal, and 5min abandons supernatant, add the 2ml lavation buffer solution in the beads after mixing the centrifugal 5min of 3300rpm abandon supernatant, repeated washing 2 times.
5, will clean complete Halo link and join in centrifugal bacterium liquid supernatant, the room temperature blending incubation was in conjunction with 2 hours.
6, above-mentioned mixed liquor is joined in purification column gravity and cross post, collect filtrate 100ul(FT); Then add lavation buffer solution 10ml, resuspended rear room temperature blending incubation 10min, after abandon filtrate, repeated washing 4 times.
7, close purification column, add the lavation buffer solution of 3ml, put to close after 1ml and add 5ul TEV enzyme rear enclosed purification column, 37 ° of reaction 1h.
8, cross post and collect filtrate (E1-1), add 2ml purifying damping fluid in pillar, envelope post mixing 10min, filter collection filtrate and be labeled as (E1-2), after add 10ul His link resin in the E1-1 that mixes and E1-2, incubated at room 1h, 1000g is centrifugal, and the collection supernatant is labeled as E2.
2, albumen and microballoon coupling
With p62, the NY-ESO-1 of purifying, p53, CAGE recombinant protein respectively with 55#, 47#, 27#, the coupling of 33# microballoon.
Reagent and solution:
(1) 0.1M NaH
2PO
4, pH6.2: weighing 3.5814g NaH
2PO
4In 90mL ddH
2In O, NaOH is settled to 100mL, the 0.22um membrane filtration after regulating pH to 6.2;
(2) 0.05M MES, pH5.0: weighing 0.976g MES is in 90mL ddH
2In O, NaOH is settled to 100mL, the 0.22um membrane filtration after regulating pH to 5.0;
(3) 0.1M MES(2-(N-morpholino) ethyl sulfonic acid), pH6.0: weighing 1.952g MES is in 90mL ddH
2In O, NaOH is settled to 100mL, the 0.22um membrane filtration after regulating pH to 6.0;
(4) 0.1M MES, pH4.5: weighing 1.952g MES is in 90mL ddH
2In O, NaOH is settled to 100mL, the 0.22um membrane filtration after regulating pH to 4.5;
(5) PBS:NaCl, 137mM; KCl, 2.7mM; Na
2HPO
4, 8.1mM; KH
2PO
4, 1.5mM; PH7.2-7.4, the 0.22um membrane filtration;
(6) contain 0.02%Proclin300 in 1%PBSB:0.1%PBS, the 0.22um membrane filtration;
(7) contain 0.1%tween-20 in 0.1%PBST:0.1%PBS, the 0.22um membrane filtration;
(8) 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC)
(9) N-hydroxy thiosuccinimide (S-NHS).
Experimental procedure:
(1) take out microballoon stoste, vortex 20s, ultrasonic 20s gets 50ul(approximately 6.25 * 10
6Individual microballoon) in Axygen EP pipe (mark microballoon model and title), 14000x g, 4mins;
(2) 14000g, the RT(room temperature), 4min;
(3) carefully suck supernatant, can residual a small amount of supernatant for reducing the loss;
(4) add 100ul dH
2O, vortex and ultrasonic approximately 20s respectively;
（5）14000g，RT，4min；
(6) carefully suck supernatant, can residual a small amount of supernatant for reducing the loss, add 80ul0.1MNaH
2PO
4, pH6.2, vortex and ultrasonic approximately 20s respectively;
(7) add rapidly 10ul S-NHS(ddH
2O is dissolved to 50mg/mL), the vortex mixing;
(8) add rapidly 10ul EDC(ddH
2O is dissolved to 50mg/mL), difference vortex and ultrasonic approximately 20s;
(9) RT, the lucifuge rotation is hatched 20min(every the 10min vortex once);
（10）14000g，RT，4min；
(11) carefully suck supernatant, add 0.05M MES, pH5.0 to 125 μ L, vortex and ultrasonic approximately 20s respectively;
（12）14000g，RT，4min；
(13) carefully suck supernatant, repeat above-mentioned washing step once;
(14) carefully suck supernatant, with 12.5ul 0.05M MES, the resuspended microballoon of pH5.0, vortex and ultrasonic approximately 20s respectively;
(15) add albumen that 6.25ug implementation step 1 purifying obtains in above-mentioned microballoon, to final volume be 250ul 0.05M MES, pH5.0, the about 20s of vortex respectively;
(16) RT, 2h is hatched in the lucifuge rotation;
（17）14000g，RT，4min；
(18) carefully suck supernatant, add 1mL0.01%PBST, respectively vortex and ultrasonic approximately 20s;
（19）14000g，RT，4min；
(20) carefully suck supernatant, repeat above-mentioned washing step once;
(21) add 1mL1%PBSB, respectively vortex and ultrasonic approximately 20s;
(22) RT, 30min is hatched in the lucifuge rotation, the activated carboxyl site of sealing microsphere surface remnants;
（23）14000g，RT，4min；
(24) carefully suck supernatant, add 1mL1%PBSB, respectively vortex and ultrasonic approximately 20s;
(25) carefully suck supernatant, adding 1%PBSB is 37ul to final volume, difference vortex and ultrasonic approximately 20s;
(26) get 2ul in 38ul1%PBSB, vortex 20s, after ultrasonic 20s, the blood counting chamber counting, ultimate density (individual/mL).
(27) mark microballoon concentration (1.2 * 10 on the Axygen centrifuge tube
4Individual/μ l), coupling time, then place 4 the degree keep in Dark Place.
Embodiment 2 lung cancer detection tests
1, detection method
(1) first take out all reagent before the use, place balance to room temperature;
(2)-80 ℃ of serum that take out patient's (trouble lung cancer) and Healthy People, after being placed on respectively thawing on ice, the vortex mixing is got V-type 96 hole blood-coagulation-boards (96 orifice plate) with 200 times of serum dilutions, and the rifle head is mixing up and down, does not produce bubble as far as possible;
(3) get the microballoon of 4 kinds of coupled antigen proteins in embodiment 1, vortex mixing 20s, ultrasonic 20s gets in right amount in 1000ul1%PBSB by 2500, every hole, vortex mixing 20s, ultrasonic 20s, final volume is the 10ul/ hole;
(5)---Prime----rinse(channel2)---Prime---places 96 orifice plates---Run5:MAGX3 that opens the plate machine of washing;
(6) add immediately the dilution serum of 200 times, the 30ul/ hole;
(7) wrap 96 orifice plates with aluminium-foil paper, room temperature, concussion incubation reaction 60min;
(8) 0.1%PBST washes three times, Run5:MAGX3;
(9) 1%PBSB dilution goat anti-human igg-biotin (1:5000) and SAPE(1:500), the 50ul/ hole;
(10) wrap 96 orifice plates with aluminium-foil paper, room temperature, concussion incubation reaction 60min;
(11) 1%PBST washes three times, Run5:MAGX3;
(12) the resuspended microballoon of 100ul/ hole 1%PBSB, room temperature, concussion 3-5min;
(13) reading result on Luminex series liquid-phase chip analyser, instrument is the drawing standard curve automatically, and calculates the measured value of sample to be tested.
2, interpretation of result
Fig. 1 has shown the distribution of the unimolecule autoantibody in 48 early stages of lung cancer and 48 normal healthy controls.
Utilize 3 kinds of biostatistics algorithm (CCP, DLDA, BCCP) commonly used to analyze, find that accuracy rate can reach 72% by the aborning p62 antibody of detection self, NY-ESO-1 antibody, p53 antibody and CAGE antibody.
Claims (8)
1. liquid phase chip reagent box for detection of the lung cancer autoantibody comprises the microballoon of coupled antigen albumen, biotin labeled two anti-, SA-PE, reaction buffer and dilution buffer liquid, it is characterized in that,
Antigen protein is at least two kinds in p62, NY-ESO-1, p53 and CAGE, and wherein, the microballoon of the different antigen proteins of coupling has different color codings;
Two anti-are anti-human immunoglobulin G.
2. liquid phase chip reagent box as claimed in claim 1, is characterized in that, described reaction buffer is 1%PBSB.
3. liquid phase chip reagent box as claimed in claim 1, is characterized in that, described dilution buffer liquid is thought 1%PBSB.
4. liquid phase chip reagent box as claimed in claim 1, is characterized in that, described antigen protein is p62, NY-ESO-1, p53 and CAGE.
5. liquid phase chip reagent box as claimed in claim 1, is characterized in that, described two anti-are goat anti-human immunoglobulin G.
6. when liquid phase chip reagent box as claimed in claim 1, the microballoon of preparation coupled antigen albumen, the addition of antigen protein is 5～10 μ g/6.25 * 10
6Individual microballoon.
7. liquid phase chip reagent box as claimed in claim 1, the concentration of the microballoon of every kind of coupled antigen albumen is 1.2～1.8 * 10
4Individual/μ l.
8. liquid phase chip reagent box as claimed in claim 1, biotin labeled two anti-concentration are 0.1～0.125 μ g/ml.
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| CN109142755A (en) * | 2018-10-09 | 2019-01-04 | 福建省立医院 | It is a kind of diagnose early stage esophageal squamous cell carcinoma four kinds of autoantibody combined detection kits and application |
| CN109470853A (en) * | 2017-09-08 | 2019-03-15 | 广州市丹蓝生物科技有限公司 | Diagnosing autoimmune diseases liquid phase protein chip, kit and production method |
| CN115267165A (en) * | 2022-08-15 | 2022-11-01 | 海仕兰(上海)生物科技有限公司 | Suspension chip system, application thereof and method for detecting tumor-associated diagnostic factor based on suspension chip system |
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| CN109142755A (en) * | 2018-10-09 | 2019-01-04 | 福建省立医院 | It is a kind of diagnose early stage esophageal squamous cell carcinoma four kinds of autoantibody combined detection kits and application |
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