CN103383395B - A liquid chip kit for detecting autoantibodies in lung cancer - Google Patents

Abstract

The invention discloses a kind of liquid phase chip reagent box for detection of lung cancer autoantibody, comprise the microballoon of coupled antigen albumen, biotin labeled two anti-, SA-PE, reaction buffer and dilution buffers, antigen protein is at least two kinds in p62, NY-ESO-1, p53 and CAGE, wherein, the microballoon of the different antigen protein of coupling has different color codings; Two resist for human immunoglobulins G.The present invention is used for the liquid phase chip reagent box of detection of lung cancer autoantibody, on the technology platform of liquid-phase chip, achieve the joint-detection to four kinds of antibody in serum, and with p62 antibody, NY-ESO-1 antibody, p53 antibody and CAGE antibody for mark, detection accuracy is high, and the early diagnosis for lung cancer provides new thinking.

Description

A liquid chip kit for detecting autoantibodies in lung cancer

technical field

The invention relates to the field of medical biology, in particular to a liquid-phase chip kit for detecting autoantibodies of lung cancer.

Background technique

In recent years, the incidence of cancer in my country has shown an obvious upward trend, accounting for more than 20% of the causes of death, ranking first among all causes of death. The World Health Organization predicts that by 2020, there will be 20 million new cancer cases and 12 million deaths, most of which will occur in developing countries. If no effective prevention and control measures are taken, it is estimated that by 2020, the total number of new cancers and cancer deaths in my country will reach about 3 million, and the total number of patients will reach 6.6 million.

Lung cancer is currently the malignant tumor with the highest morbidity and mortality worldwide. In the past 20 years, due to vigorous promotion of smoking cessation, the incidence of male lung cancer in Europe and the United States has begun to decline, but the incidence of female lung cancer has continued to rise. my country is a big country in the production and sales of cigarettes. Regardless of male or female, the incidence of lung cancer continues to rise, especially in females. Clinical studies have shown that the cure rate of carcinoma in situ is close to 100%, and the 5-year survival rate of patients with stage I lung cancer is 60% to 90%, while the 5-year survival rate of patients with stage IIIb and IV is only 5% to 20%. Early diagnosis and early treatment are the key to reducing the mortality rate of lung cancer and prolonging the survival period. However, due to the lack of ideal early diagnosis methods, the early diagnosis rate of lung cancer is only about 14%. Therefore, how to improve the level of early diagnosis of lung cancer has become a serious and urgent task for lung cancer prevention and treatment workers.

Markers such as tumor antigens in the serum can induce the body to produce autoantibodies. In the early stage of tumor occurrence that has not been detected by clinical examination methods, the body's immune system can monitor the existence of tumor antigens expressed at low levels and trigger an immune response , produce a large number of antibodies, and play an effective biological signal amplification role.

Compared with the commonly used clinical tumor marker proteins, the detection of autoantibodies has a great advantage in tumor diagnosis. First, tumors can be diagnosed early, which facilitates early treatment and improves the cure rate. Many studies have shown that the presence of autoantibodies can be detected months to years before the diagnosis of solid cancer by imaging examinations, and antibodies against tumor antigens can be detected in serum even 2-10 years before the diagnosis of tumors. Second, the titer of autoantibodies is higher than that of the corresponding tumor antigen, and autoantibodies against a single antigen are greatly amplified through immune reactions, and are easier to detect than other markers when there is interference from a large amount of albumin in serum. Therefore, analyzing changes in the immune system of cancer patients, rather than tumor proteins themselves, has become a very important research avenue. Third, the samples are easy to obtain and the test results are relatively stable. Once a tumor antigen is secreted into the blood, it may be degraded or cleared quickly, while autoantibodies are not susceptible to protease hydrolysis like other polypeptides, and can be stable and persistent in serum for a long period of time, and the half-life of autoantibodies It is very long, about 7 days, with little fluctuation per hour, stable physical and chemical properties, and long-term storage at -80C has little effect on its activity. Fourth, the reagents and techniques used in its operation are simple and easy, with good repeatability, and can be detected by conventional enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunoassay (EIA).

Tumor occurrence is a multi-gene, multi-step cancer process. Although tumor autoantibodies are ideal candidate indicators for tumor diagnosis, relying on only one indicator for diagnosis often leads to false positives and false negatives. Therefore, there must be a single tumor autoantibody detection Some limitations affect the clinical diagnosis of lung cancer. First, in a specific tumor, the positive rate of a single tumor autoantibody is usually only 10%, and in the most ideal tumor population, it is only 20-30%; (including tumors and other autoimmune diseases), in general, the same tumor can abnormally produce one or more tumor autoantibodies, and different tumors or different tissue types of the same tumor can detect common autoantibodies Tumor antibodies, different tumor antibodies can also be detected.

Recent studies have tended to target combined antibody profiles for cancer detection. Chapman et al. used ELISA to detect seven tumor antigens (p53, c-myc, HER2, NY-ESO-1, CAGE, MUC1 and GBU4- 5) Serum antibodies were detected, and the results showed that the positive rate of a single antibody was between 5% and 36%, and the diagnostic specificity reached 96% to 100%, while the sensitivity of the combined detection of 7 antibodies could reach 76%. The specificity was 92%. This fully shows that the single detection of tumor markers has high specificity, but low sensitivity and accuracy; after joint detection, although the specificity has decreased, the sensitivity and accuracy have greatly improved.

Therefore, the joint detection of various tumor-related autoantibodies forms a unique autoantibody "fingerprint" of the tumor itself, which improves the specificity and sensitivity of diagnosis and prognosis to a level that cannot be achieved by a single individual. Appears before clinical symptoms, so it has important diagnostic and prognostic value.

Contents of the invention

The invention provides a liquid-phase chip kit for detecting lung cancer autoantibodies, which realizes joint detection of multiple antibodies in serum and has high detection accuracy for lung cancer.

A liquid chip kit for the detection of lung cancer autoantibodies, including microspheres coupled to antigenic proteins, biotin-labeled secondary antibodies, streptavidin-phycoerythrin, reaction buffer and dilution buffer, antigen The proteins are p62, NY-ESO-1, p53 and CAGE, and the microspheres coupled with different antigen proteins have different color codes; the secondary antibody is biotin-labeled anti-human immunoglobulin G.

CAGE: Tumor-associated gene, a member of the cancer-testis CT antigen family, is a new cancer discovered by Cho et al. in 2002 when they screened the cDNA library of testis tissue by using the serological analysis technology of recombinant cloning and expressing antigens. - Genes encoding testis antigens.

p62: nucleoporin, a tumor-associated antigen with a relative molecular mass of about 62×103, and a member of the insulin-like growth factor 2 (IGF2) mRNA-binding protein family, also known as MP2/IGF2BP2.

NY-ESO-1: It is a tumor antigen screened from esophageal cancer cDNA library by Chen et al. using SEREX technology in 1997. It belongs to the CTA (cancer-testis antigen) family. Members of this family are only expressed in testis tissue and some tumor tissues.

p53: The p53 gene is an anti-cancer gene, located on human chromosome 17p13.1, encoding a nuclear phosphorylated protein with a molecular weight of 53kD consisting of 393 amino acids, called p53 protein.

In the present invention, the autoantibodies produced by antigenic proteins p62, NY-ESO-1, p53 and CAGE are used as detection objects. In the liquid phase chip kit of the present invention, both the antigenic proteins and the secondary antibodies can be specifically compared with the corresponding lung cancer serum. Antibodies (p62 antibody, NY-ESO-1 antibody, p53 antibody or CAGE antibody), and streptavidin-phycoerythrin can bind to biotin with high specificity, so serum antibodies against lung cancer can be finally formed The complex of "microsphere-antigen protein + serum antibody + secondary antibody + streptavidin-phycoerythrin" is detected by the instrument, and the reaction type is determined according to the color of the microsphere, and the phycoerythrin is excited by a green laser to measure the microsphere The number of reporter fluorescent molecules bound on the ball is used to indirectly determine the content of lung cancer serum antibody bound on the microsphere.

The reaction buffer can be 1% PBSB.

The dilution buffer can be 1% PBSB.

The antigen proteins are p62, NY-ESO-1, p53 and CAGE.

The antigenic protein can be connected with microspheres through HaloTag ^TM interchangeable labeling technology.

The amino acid sequence of p62 protein is shown in SEQ ID NO.5; the gene sequence encoding p62 protein is shown in SEQ ID NO.1; the amino acid sequence of NY-ESO-1 protein is shown in SEQ ID NO.6, encoding NY- The gene sequence of ESO-1 protein is shown in SEQ ID NO.2; the amino acid sequence of p53 protein is shown in SEQ ID NO.7, and the gene sequence encoding p53 protein is shown in SEQ ID NO.3; the amino acid sequence of CAGE protein As shown in SEQ ID NO.8; The gene sequence encoding CAGE protein is shown in SEQ ID NO.4.

Preferably, the secondary antibody is goat anti-human immunoglobulin G. Experiments have found that murine antibodies can produce cross-reactions, while goat-derived antibodies can overcome this defect.

The microspheres can be conventional microspheres used in liquid phase chips.

When preparing microspheres coupled with antigenic protein, the amount of antigenic protein added is 5-10 μg/6.25×10 ⁶ microspheres, preferably 6.25 μg/6.25×10 ⁶ microspheres.

The concentration of each antigen protein-coupled microsphere is 1.2-1.8×10 ⁴ /μl, preferably 1.2×10 ⁴ /μl.

The concentration of the biotin-labeled secondary antibody is 0.1-0.125 μg/ml, preferably 0.1 μg/ml.

Compared with prior art, the beneficial effect of the present invention is:

The liquid phase chip kit for detecting lung cancer autoantibodies of the present invention realizes the joint detection of four antibodies in serum, and is marked by p62 antibody, NY-ESO-1 antibody, p53 antibody and CAGE antibody in serum, The detection accuracy is high, which provides new tools and ideas for the diagnosis and prognosis of lung cancer.

The liquid phase chip kit for detecting lung cancer autoantibodies of the present invention is based on the technical platform of the liquid phase chip, which can quickly detect micro samples with high throughput, and the liquid phase environment is more conducive to maintaining the natural conformation of the protein, and is more efficient Facilitate the reaction, high sensitivity, good signal-to-noise ratio.

Description of drawings

Figure 1 is the distribution of monomolecular autoantibodies in lung cancer patients and healthy controls;

Fig. 2 shows the detection accuracy of the liquid phase chip kit of the present invention analyzed by three biostatistical algorithms (CCP, DLDA, BCCP).

Detailed ways

The present invention will be further explained below in conjunction with specific examples.

The preparation of embodiment 1 liquid phase chip kit

Reagents and Solutions

(1) 0.1M NaH ₂ PO ₄ , pH6.2: Weigh 3.5814g NaH ₂ PO ₄ in 90mL ddH ₂ O, adjust the pH to 6.2 with NaOH, dilute to 100mL, and filter through a 0.22um filter;

(2) 0.05M MES, pH5.0: Weigh 0.976g MES into 90mL ddH ₂ O, adjust the pH to 5.0 with NaOH, dilute to 100mL, filter with 0.22um membrane;

(3) 0.1M MES (2-(N-morpholino)ethanesulfonic acid), pH6.0: Weigh 1.952g MES in 90mL ddH ₂ O, adjust the pH to 6.0 with NaOH, then dilute to 100mL, 0.22 um membrane filtration;

(4) 0.1M MES, pH4.5: Weigh 1.952g MES into 90mL ddH ₂ O, adjust the pH to 4.5 with NaOH, dilute to 100mL, and filter through a 0.22um membrane;

(5) PBS: NaCl, 137mM; KCl, 2.7mM; Na ₂ HPO ₄ , 8.1mM; KH ₂ PO ₄ , 1.5mM; pH7.2-7.4, 0.22um membrane filtration;

(6) 1%PBSB: 0.1%PBS containing 0.02%Proclin300, 0.22um membrane filtration;

(7) 0.1%PBST: 0.1%PBS contains 0.1%tween-20, filtered by 0.22um filter membrane;

(8) 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC);

(9) N-hydroxysulfosuccinimide (S-NHS).

1. Liquid phase chip kit for detecting autoantibodies, including:

1) 55#, 47#, 27#, 33# microspheres coupled with Halo amine (O2) ligand;

2) Fusion proteins containing Halo Tag: p62, NY-ESO-1, p53, CAGE;

3) streptavidin-phycoerythrin;

4) Goat anti-human IgG-biotin: the concentration is 0.1 μg/ml;

5) Reaction buffer: 1% PBSB;

6) Dilution buffer: 1% PBSB;

7) Parafilm;

8) 96-well plate.

The preparation of the liquid phase chip kit includes the following steps:

1. Induced expression and purification of recombinant protein

Reagents and solutions:

KRX recombinant bacteria (respectively containing the genes shown in SEQ ID NO.1-4, stored at -80°C)

Yeast powder (OXOID)

Peptone (OXOID)

Halo tag protein purification resins (Promega)

Protease inhibitor (Roche)

Lysozyme (TOPBIO)

DNase I

TEV protease (Promega)

His link resin (Promega)

Purification column (BIO-RAD)

CA-630 (Sigma)

LB medium (10.0g peptone, 5.0g yeast powder, 5.0gNaCl, adjust the pH to 7.0-7.5 and set the volume to 1L, autoclave at 120° for 20min)

Buffer 1 (50mM Hepes, pH7.5, 150mM NaCl, 1mM DTT)

Buffer 2 (50mM Hepes, pH7.5, 150mM NaCl, 1mM DTT, 0.5mM EDTA, 0.05%CA630)

Experimental steps:

1. Large-scale induced expression of recombinant bacteria

1. Overnight amplification

Take out the preserved KRX recombinant bacteria from the -80°C refrigerator, add it to 10ml of ampicillin-resistant LB medium containing 0.2% glucose, and amplify overnight at 37°C and 275rpm.

2. Expand training

Add the overnight bacterial cells to 1L ampicillin-resistant LB medium at a ratio of 1:100 and culture at 275rpm at 37°C to OD=0.4-0,5, and add glucose and rhamnose at a final concentration of 0.05% to the medium. Cultivate overnight at 25°C and 225rpm.

3. Centrifuge at 5000g for 15 minutes to collect bacteria, 100ml/tube, mark and store at -80°.

2. Cell lysis of KRX recombinant bacteria

1. Take 1 tube of KRX recombinant bacteria frozen at -80°, add 15ml buffer 1 to resuspend, then add 100ul Protease inhibitor, 10ul Lysozyme (100mg/ml), 10ul DNase I (5mg/ml), 100ul1%CA- 630; resuspend the bacteria, mix well and place in ice bath for 30min.

2. Ultrasonic disrupt the bacteria (over 5s, stop for 10s, 85% power, 3min), place on ice for 10min.

3. Centrifuge at 12000g at 4° for 30min, take the supernatant, and put 100ul of the supernatant into a 1.5ml centrifuge tube (50ul of which is marked as S, and the other 50ul+10ul of 1:100 diluted TEV enzyme is marked as S-TEV).

4. Centrifuge and wash Halo link at the same time: take 1 tube of 2ml Halo link beads, centrifuge at 3300rpm for 5min, discard the supernatant, add 2ml washing buffer to the beads, mix well, centrifuge at 3300rpm for 5min, discard the supernatant, and repeat washing twice.

5. Add the washed Halo link to the supernatant of the centrifuged bacterial liquid, mix and incubate at room temperature for 2 hours.

6. Add the above mixture to the purification column and pass through the column by gravity, collect the filtrate 100ul (FT); then add 10ml of washing buffer, resuspend, mix and incubate at room temperature for 10min, discard the filtrate, and repeat washing 4 times.

7. Close the purification column, add 3ml of washing buffer, put 1ml, close the purification column after adding 5ul TEV enzyme, and react at 37° for 1h.

8. Collect the filtrate (E1-1) through the column, add 2ml of purification buffer to the column, seal the column and mix for 10min, filter and collect the filtrate as (E1-2), then add 10ul His link resin to the well-mixed E1- 1 and E1-2, incubate at room temperature for 1 h, centrifuge at 1000 g to collect the supernatant and mark it as E2.

2. Coupling of proteins and microspheres

The purified p62, NY-ESO-1, p53, and CAGE recombinant proteins were coupled to 55#, 47#, 27#, and 33# microspheres, respectively.

Reagents and solutions:

(1) 0.1M NaH ₂ PO ₄ , pH6.2: Weigh 3.5814g NaH ₂ PO ₄ in 90mL ddH ₂ O, adjust the pH to 6.2 with NaOH, dilute to 100mL, and filter through a 0.22um filter;

(2) 0.05M MES, pH5.0: Weigh 0.976g MES into 90mL ddH ₂ O, adjust the pH to 5.0 with NaOH, dilute to 100mL, filter with 0.22um membrane;

(3) 0.1M MES (2-(N-morpholino)ethanesulfonic acid), pH6.0: Weigh 1.952g MES in 90mL ddH ₂ O, adjust the pH to 6.0 with NaOH, then dilute to 100mL, 0.22 um membrane filtration;

(4) 0.1M MES, pH4.5: Weigh 1.952g MES into 90mL ddH ₂ O, adjust the pH to 4.5 with NaOH, dilute to 100mL, and filter through a 0.22um membrane;

(5) PBS: NaCl, 137mM; KCl, 2.7mM; Na ₂ HPO ₄ , 8.1mM; KH ₂ PO ₄ , 1.5mM; pH7.2-7.4, 0.22um membrane filtration;

(6) 1%PBSB: 0.1%PBS containing 0.02%Proclin300, 0.22um membrane filtration;

(7) 0.1%PBST: 0.1%PBS contains 0.1%tween-20, filtered by 0.22um filter membrane;

(8) 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC)

(9) N-hydroxysulfosuccinimide (S-NHS).

Experimental steps:

(1) Take out the microsphere stock solution, vortex for 20s, sonicate for 20s, take 50ul (about 6.25× ¹⁰⁶ microspheres) into an Axygen EP tube (mark the microsphere model and name), 14000x g, 4mins;

(2) 14000g, RT (room temperature), 4min;

(3) Carefully suck off the supernatant, and a small amount of supernatant can be left to reduce loss;

(4) Add 100ul dH ₂ O, vortex and sonicate for about 20s;

(5) 14000g, RT, 4min;

(6) Carefully suck off the supernatant. In order to reduce the loss, a small amount of supernatant can be left, add 80ul 0.1M NaH ₂ PO ₄ , pH 6.2, vortex and sonicate for about 20s;

(7) Quickly add 10ul S-NHS (ddH ₂ O dissolved to 50mg/mL), vortex and mix;

(8) Quickly add 10ul EDC (ddH ₂ O dissolved to 50mg/mL), vortex and sonicate for about 20s respectively;

(9) RT, incubate in the dark for 20 minutes (vortex every 10 minutes);

(10) 14000g, RT, 4min;

(11) Carefully suck off the supernatant, add 0.05M MES, pH 5.0 to 125μL, vortex and sonicate for about 20s;

(12) 14000g, RT, 4min;

(13) Carefully suck off the supernatant, and repeat the above washing steps once;

(14) Carefully suck off the supernatant, resuspend the microspheres with 12.5ul 0.05M MES, pH 5.0, vortex and sonicate for about 20s;

(15) Add 6.25ug of the protein purified in Step 1 to the above-mentioned microspheres to a final volume of 250ul 0.05M MES, pH 5.0, and vortex for about 20s;

(16) RT, incubate for 2 hours in the dark;

(17) 14000g, RT, 4min;

(18) Carefully suck off the supernatant, add 1mL0.01%PBST, vortex and sonicate for about 20s;

(19) 14000g, RT, 4min;

(20) Carefully suck off the supernatant, and repeat the above washing steps once;

(21) Add 1mL1%PBSB, vortex and sonicate for about 20s;

(22) RT, incubate in the dark for 30 minutes, and block the remaining activated carboxyl sites on the surface of the microspheres;

(23) 14000g, RT, 4min;

(24) Carefully suck off the supernatant, add 1mL1%PBSB, vortex and sonicate for about 20s;

(25) Carefully suck off the supernatant, add 1% PBSB to a final volume of 37ul, vortex and sonicate for about 20s;

(26) Take 2 ul in 38 ul of 1% PBSB, vortex for 20 s, and after ultrasonication for 20 s, count on a hemocytometer, the final concentration (unit/mL).

(27) Mark the microsphere concentration (1.2×10 ⁴ /μl) and coupling time on the Axygen centrifuge tube, and then store it at 4 degrees in the dark.

Example 2 Lung cancer detection test

1. Detection method

(1) Take out all the reagents before use, and let them equilibrate to room temperature;

(2) Take out the serum of patients (suffering from lung cancer) and healthy people at -80°C, put them on ice to melt, vortex and mix well, take V-type 96-well hemagglutination plate (96-well plate) and dilute the serum by 200 times, Mix the tip of the pipette up and down, try not to generate air bubbles;

(3) Take the four kinds of microspheres coupled with antigenic protein in Example 1, vortex and mix for 20 s, sonicate for 20 s, take an appropriate amount according to 2500 microspheres per well, and put it in 1000 ul1% PBSB, vortex and mix for 20 s, sonicate for 20 s, The final volume is 10ul/well;

(5) Turn on the plate washer---Prime----rinse (channel2)----Prime---place the 96-well plate---Run5: MAGX3;

(6) Immediately add serum diluted 200 times, 30ul/well;

(7) Wrap the 96-well plate with aluminum foil, and incubate with shaking for 60 minutes at room temperature;

(8) Wash three times with 0.1% PBST, Run5: MAGX3;

(9) Dilute goat anti-human IgG-biotin (1:5000) and SAPE (1:500) in 1% PBSB, 50ul/well;

(10) Wrap the 96-well plate with aluminum foil, and incubate with shaking for 60 minutes at room temperature;

(11) Wash three times with 1% PBST, Run5: MAGX3;

(12) 100ul/well 1%PBSB resuspended microspheres, room temperature, shaking for 3-5min;

(13) Read the results on the Luminex series liquid chip analyzer, the instrument can automatically draw the standard curve, and calculate the measured value of the sample to be tested.

2. Result analysis

Figure 1 shows the distribution of monomolecular autoantibodies in 48 early-stage lung cancers and 48 healthy controls.

Using 3 commonly used biostatistical algorithms (CCP, DLDA, BCCP) to analyze, it is found that the accuracy rate can reach 72% by detecting the p62 antibody, NY-ESO-1 antibody, p53 antibody and CAGE antibody produced by itself.

Claims (1)

1., for a liquid phase chip reagent box for detection of lung cancer autoantibody, comprise the microballoon of coupled antigen albumen, biotin labeled two anti-, SA-PE, reaction buffer and dilution buffers, it is characterized in that,

Antigen protein is p62, NY-ESO-1, p53 and CAGE, and wherein, the microballoon of the different antigen protein of coupling has different color codings;

Two resist for goat anti-human immunoglobulin G;

When preparing the microballoon of coupled antigen albumen, the addition of antigen protein is 5 ~ 10 μ g/6.25 × 10 ⁶individual microballoon;

The concentration of the microballoon of often kind of coupled antigen albumen is 1.2 ~ 1.8 × 10 ⁴individual/μ l;

Biotin labeled two concentration resisted are 0.1 ~ 0.125 μ g/ml.