CN112730837A

CN112730837A - Antibody chip and kit for simultaneously and quantitatively detecting multiple lung cancer markers

Info

Publication number: CN112730837A
Application number: CN202011612570.8A
Authority: CN
Inventors: 张卓; 黄若磐
Original assignee: Reboo Guangzhou Biotechnology Co ltd
Current assignee: Reboo Guangzhou Biotechnology Co ltd; Raybiotech Inc
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2021-04-30

Abstract

The invention relates to a chip for simultaneously detecting a plurality of lung cancer markers, which comprises a solid phase carrier and a capture antibody of the tumor markers fixed on the solid phase carrier, wherein the lung cancer markers are at least three selected from a CA125 antibody, a CEA antibody, an NSE antibody, an SCCA1 antibody, a CY21-1 antibody and a pro-GRP antibody. By optimizing to a proper antibody combination (especially the combination of a primary antibody and a secondary antibody), the invention can realize the high-specificity and high-sensitivity one-time detection of a plurality of (one to six) biomarkers (CA125, CEA, NSE, SCCA1, CY21-1 and pro-GRP) of the lung cancer, can greatly improve the detection efficiency of the lung cancer, realizes early diagnosis, and effectively reduces the cost and brings good news to human health under a detection platform of a high-throughput chip.

Description

Antibody chip and kit for simultaneously and quantitatively detecting multiple lung cancer markers

Technical Field

The invention belongs to the technical field of in-vitro immunoassay, and particularly relates to an antibody chip and a kit for simultaneously and quantitatively detecting multiple lung cancer markers.

Background

Lung cancer is the most common malignant tumor, and is the first leading cause of death of cancer in China to all over the world. According to the global statistics report published by the world health organization in 2012, the number of newly-added lung cancer patients reaches 180 million every year in the world, and more than 140 million people die of lung cancer every year; the lung cancer death rate is also the first in China. Early symptoms of lung cancer are not obvious, about 80 percent of lung cancer is in middle and advanced stages once diagnosed, the survival rate is only 7.5 percent, but the survival rate can reach more than 80 percent in five years after early diagnosis and treatment. Therefore, the lung cancer diagnosis product meeting the clinical application requirements is developed and marketed, provides detection service for early-stage lung cancer patients, realizes early discovery and early treatment, brings benefits to lung cancer patients, and has very important value.

As tumor cells develop, certain proteins in tumor patients change, or new abnormal proteins associated with tumors are produced. These chemical species that reflect the presence of tumors are collectively referred to as tumor markers. Because the tumor markers are not present in normal adult tissues but only present in tumor tissues, or the content of the tumor markers in the tumor tissues is greatly higher or lower than that in normal tissues, the existence or the quantity of the tumor markers can indicate the nature of the tumor, so the detection of the abnormal proteins can be used for diagnosing the occurrence of the tumor and monitoring the treatment progress of the medicine on the tumor, and the diagnosis and the treatment of the tumor play an important role. Tumor markers for clinical diagnosis include 6 major classes, such as carcinoembryonic antigen, enzyme, hormone, glycoprotein, oncogene and cell surface tumor antigen. The following are the common blood lung-heat-clearing cancer markers for auxiliary diagnosis in clinical diagnosis: carcinoembryonic antigen (CEA), cancer antigen CA125, keratin 19(CY21-1), human squamous cell carcinoma antigen 1(SCCA1), gastrin releasing precursor (pro-GRP), Neuron Specific Enolase (NSE). Carcinoembryonic antigen (CEA) and cancer antigen CA125 are cancer broad-spectrum biomarkers, and have high expression in lung cancer, pancreatic cancer, breast cancer, ovarian cancer and the like. Human squamous cell carcinoma antigen 1(SCCA1) is highly expressed in lung and ovarian cancers. Keratin 19(CY21-1) is used mainly as a marker for distinguishing non-small cell lung cancer. Neuron-specific enolase (NSE) is a marker for neuroblastoma and small cell lung cancer.

The biochip is based on a mature enzyme-linked immunosorbent assay (ELISA) technology, realizes high-throughput screening and detection of samples, and is a very important technical support and a basic platform for future application for a multi-index diagnosis mode of lung cancer.

Clinical diagnosis of lung cancer markers mostly depends on an immunological method mainly based on double antibody sandwich ELISA. Conventional ELISA can only detect one tumor marker from a sample at a time. The detection of multiple tumor markers using conventional ELISA is not only time consuming and laborious, but also requires many patient samples.

Disclosure of Invention

Based on the above, in order to solve the above technical problems, the present invention provides a lung cancer chip and a kit capable of simultaneously and quantitatively detecting six tumor markers.

The technical scheme for achieving the purpose is as follows.

A chip for simultaneously detecting a plurality of lung cancer markers comprises a solid phase carrier and a capture antibody of the tumor markers fixed on the solid phase carrier, wherein the lung cancer markers are at least three selected from a CA125 antibody, a CEA antibody, an NSE antibody, an SCCA1 antibody, a CY21-1 antibody and a pro-GRP antibody, and the solid phase carrier is an aminated glass slide or an aldehyde glass slide.

In some embodiments, the solid support is an aminated glass slide or an aldehyde glass slide, and more preferably an aldehyde glass slide.

A kit for simultaneously and quantitatively detecting a plurality of lung cancer markers comprises at least 6 chips for simultaneously detecting the plurality of lung cancer markers and detection antibodies of tumor markers, wherein the lung cancer markers are at least three antibodies selected from a CA125 antibody, a CEA antibody, an NSE antibody, an SCCA1 antibody, a CY21-1 antibody and a pro-GRP antibody, and the detection antibodies are marked with immune signal amplification markers; the amount of the corresponding capture antibodies coated between the chips for simultaneously detecting the multiple lung cancer markers is different.

In some of these embodiments, the CA125 antibody is at least one of 118-11248 from Riboao Biotechnology, Guangzhou and Sc-52095 from Santa Cruz Biotechnology.

In some of these embodiments, the CEA antibody is at least one of an antibody having a CTA-1003 or a CTA-1004 from Zhengzhou Saikang Biotechnology Ltd, and an antibody having a NBP2-52673 from Novus Biologicals.

In some embodiments, the NSE antibody is at least one of the antibodies with the cargo numbers 9602-.

In some embodiments, the SCCA1 antibody is at least one of 130-10459, and SCCA1-103 or SCCA1-104, of tibo biotechnology limited, cantonese.

In some embodiments, the CY21-1 antibody is at least one of 130-.

In some of these embodiments, the pro-GRP antibody is at least one of the antibodies of the ficoll bio-part ltd, cat # 4 and cat # 5.

In some of these embodiments, the capture antibody is a CA125 antibody, a CEA antibody, an NSE antibody, an SCCA1 antibody, a CY21-1 antibody, and a pro-GRP antibody.

In some of these embodiments, the solid support further comprises a protein standard, preferably biotin-labeled bovine IgG.

In some of these embodiments, a phosphate buffer containing 0.5% ± 0.01 sucrose at a pH of 7.3-7.5 is also included.

In some of these examples, the capture antibody is the CA125 antibody from Santa Cruz Biotechnology, Cat # Sc-52095, CEA antibody from Zhengzhou Saikang Biotechnology Inc., Cat # CTA-1003, CEA antibody from Novus Biologicals, Cat # NBP2-52673, NSE antibody from Guangzhou Ribo Biotechnology Inc., Cat # 9602-100116, NSE antibody from Shenzhen Sheng Yuan Biotechnology Inc., Cat # C191, SCCA1 antibody from Guangzhou Ribo Biotechnology Inc., Cat # 130-10459, CY21-1 antibody from Guangzhou Ribo Biotechnology Inc., Cat # 130-10317, CY21-1 antibody from Shanghai Huichi Chaochen Biotechnology Inc, Cat # L1C00706, and GRP # Proc # 5 from Guangzhou Biotechnology Inc.

In some of these examples, the detection antibody is CA125 antibody of 118- & ltd & gt 11248 from Guangzhou Riboao Biotechnology, Inc., CEA antibody of CTA-1003 from Zhengzhou Sakakong Biotechnology, Inc., CEA antibody of CTA-1004 from Zhengzhou Sakakong Biotechnology, Inc., NSE antibody of 9602- & ltd & gt 100116 from Guangzhou Riboao Biotechnology, Inc., NSE antibody of C191 from Shenzhen Sheng Yuan Biotechnology, antibodies of SCCA1-103 and SCCA1-104 from Guangzhou Chong Biotechnology, CY21-1 antibody of 130- & ltd & gt 10317 from Guangzhou Riboao Biotechnology, and proGRP antibody of 4#, from Fengco Biotechnology, Inc.

In one embodiment, the immune signal amplification system is a biotin-avidin system, and the detection antibody is labeled with biotin.

In one embodiment, there are 6-10, 6-12, 6-16, or 16 chips for simultaneously detecting multiple lung cancer markers according to any one of claims 1-4, wherein the capture antibody of each tumor marker on each chip is coated in the following amount:

CA125 antibody: 140-; CEA antibody: 190-;

NSE antibody: 580-620 ug/ml; SCCA1 antibody: 580-620 ug/ml;

CY21-1 antibody: 190-; pro-GRP antibody: 780-820 ug/ml.

In one preferred embodiment, the coating amount of the capture antibody of the tumor marker on the chip is as follows:

CA125 antibody: 150 ug/ml; CEA antibody: 200 ug/ml; NSE antibody: 600 ug/ml;

SCCA1 antibody: 600 ug/ml; CY21-1 antibody: 200 ug/ml; pro-GRP antibody: 800 ug/ml.

In one preferred embodiment, the solubility of the detection antibody is: 22-26ng/ml CA125 antibody, 12.5-13.5ng/ml CEA antibody, 38-42ng/ml NSE antibody, 14-16ng/ml SCCA1 antibody, 12.5-13.5ng/ml CY21-1 antibody and 48-52ng/ml pro-GRP antibody.

The invention can realize high specificity and high sensitivity and one-time detection of a plurality of (one to six) biomarkers (CA125, CEA, NSE, SCCA1, CY21-1 and pro-GRP) of the lung cancer by optimizing to a proper antibody combination (especially the combination of a primary antibody and a secondary antibody), on the basis, the chip processing is optimized, the detection efficiency of the lung cancer is greatly improved, the early diagnosis is realized, and simultaneously, the cost is effectively reduced under the detection platform of a high-throughput chip, and the invention brings good news to the human health.

Drawings

FIG. 1 is a reference diagram of the usage steps of the chip for simultaneously and quantitatively detecting multiple lung cancer markers according to the present invention.

FIG. 2 is a diagram showing the results of two different chips in example 3.

FIG. 3 is a standard graph of 6 lung cancer markers.

FIG. 4 is a schematic diagram showing the correlation analysis result between the detection indexes of 6 lung cancer markers and the detection result of Roche reagent.

Detailed Description

In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It is to be understood that the experimental procedures in the following examples, where specific conditions are not noted, are generally in accordance with conventional conditions, or with conditions recommended by the manufacturer. The various reagents used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The invention provides a lung cancer chip kit capable of simultaneously and quantitatively detecting six tumor markers, which comprises: (1) a slide glass fixed with a plurality of specific antibodies (capture antibodies) at the same time, wherein the specific antibodies and tumor markers can generate antibody-antigen reaction, and each specific antibody is respectively fixed on the slide glass to form a plurality of independent recognition sites; and (2) a reactant and a detection agent, wherein the detection antibody capable of generating antibody-antigen reaction by the tumor marker is used for detecting whether a substance capable of generating antibody-antigen reaction with the specific antibody exists in the sample to be detected by a sandwich ELISA method.

Preferably, a single concentration of one antibody is immobilized on each of the individual recognition sites on each chip, and one or more concentrations of one antibody are immobilized on the slide to form one or more individual recognition sites.

Preferably, any one of the specific capture antibodies is spotted at a content of 200ug to 1000ug onto the independent recognition sites.

Preferably, the kit contains at least 6 of the above antibody chips, preferably 6-16, and can contain 8, 9, 10, 11, 12, 13, 14, 15 and 16, and the coating amount of the same specific capture antibody on each antibody chip is the same. Preferably, the kit comprises a chip set consisting of 16 chips, the coating amount of the same specific capture antibody on each chip is the same, the concentration ranges from 150ug to 1000ug/ml (preferably, CA125 antibody: 140-. The preparation of the chip set can be referred to the applicant's patent CN 201420604204.1: for example, the hard frame is divided into 2 × 8 cells, and 16 reaction holes can be formed. A standard glass slide, a soft silica gel pad and a 16-hole (2 multiplied by 8-hole) hard frame are sequentially assembled into a 16-hole independent reaction container by two U-shaped clamps; multiple antibodies can be spotted in each hole to form an antibody chip.

In a preferred embodiment of the present invention, the reagent includes an antibody mixture, the antibody mixture is a mixture of the specific antibodies, and the specific antibodies in the antibody mixture are labeled with biotin. More preferably, the reagent further comprises streptavidin labeled with recognition biotin, and the streptavidin is labeled with fluorescent dye, and the fluorescent dye is Cy3 or fluorescent dye with similar absorption wavelength.

In a preferred embodiment of the invention, the reagent comprises a protein standard which is a mixture of said several recombinant proteins with stepped concentrations. Different kinds of recombinant proteins are mixed together according to a certain quantitative ratio, and are dried by a freezing and pumping method after being subpackaged.

In another aspect, the present invention also provides a method for preparing a protein chip kit for quantitatively detecting a tumor marker, the method comprising a step of immobilizing a specific antibody on a glass slide, the step comprising:

(1) prepared antibodies (POS1, POS2 (biotin-labeled bovine IgG), CA125, CEA, NSE, SCCA1, CY21-1 and pro-GRP) are spotted in an entire column (6 x 8, row) of FIG. 1 by using a spotting instrument, and the specific concentration of the capture antibody is 150ug/ml of the CA 125-coated antibody; CEA coated antibody 200 ug/ml; NES coating antibody 600 ug/ml; SCCA1 coated antibody 600 ug/ml; CY21-1 coating antibody 200 ug/ml; the pro-GRP coated antibody was 800 ug/ml.

(2) The spotted slide is placed at room temperature and kept overnight, and then stored at 2-8 ℃ for later use.

Wherein, the sample application operation is finished by adopting a full-automatic sample application instrument in the step (1), and the specific protein chip lattices are arranged on the glass sheet.

In one embodiment of the present invention, the spotting process of step (1) is performed by using a fully automated spotting apparatus manufactured by Perkin Elmer (Perkin Elmer) of the United states of America. The specific antibody protein chip lattices are arranged on the glass slide, in the specific operation process, the arrangement of the specific antibodies can be adjusted according to the experimental design requirements, and the full-automatic sample spotting instrument is controlled according to the arrangement arrays of different antibody protein chips to prepare the required intermediate product.

Generally, the difficulty in the biochip preparation process is how to dissolve the antibody by using a specially treated solid phase and an adapted buffer solution, and the reagent not only needs to be capable of recognizing the target substance, but also needs to be capable of not reacting with other target substances, and simultaneously needs to keep the stability of the reaction, so that the effective period of the biochip is not less than one year. The buffer solution which is screened for many times is combined with the specific antibody to finally form the qualified solid-phase chip.

Example 1

The chip for simultaneously and quantitatively detecting a plurality of lung cancer markers is prepared as follows.

Firstly, production of antibody chips:

1. and (3) condition control:

temperature: (15-20) DEG C, humidity: 45% -55%.

2. Preparing an antibody chip by spotting:

the prepared antibodies (POS1, POS2, CA125, CEA, NSE, SCCA1, CY21-1, pro-GRP) at a concentration of 150 ug-1000 ug/ml (CA 125-coated antibody 150 ug/ml; CEA-coated antibody 200 ug/ml; NES-coated antibody 600 ug/ml; SCCA 1-coated antibody 600 ug/ml; CY 21-1-coated antibody 200 ug/ml) were spotted with a spotting instrument to the entire column (6 × 8, row) of FIG. 1.

3. Combining:

the binding was carried out overnight at the preparation temperature and humidity set under the above-mentioned condition control.

4. And (3) drying:

drying at 37 ℃ for 3 hours.

5. Assembling:

and assembling the chip frame and the kit semi-finished product.

Second, experimental procedure (one-step method)

1. Pretreatment of slide chip

Taking out the slide chip from the box, balancing at room temperature for 20-30min, opening the packaging bag, and uncovering the sealing strip.

2. And (3) sealing: add 100. mu.l of blocking solution to each well, seal the plate, incubate for 30min on a shaker at room temperature, block the quantitative antibody chip.

3. And cleaning: washing with washing solution I at 1 × for 5 times, shaking for 10 seconds each time, and draining the washing solution after the plate washing is completed, wherein each well is 300 μ l.

4. Loading and incubating: 75ul of the detection antibody mixture was added to each well, and 25ul of the standard or sample was added thereto, followed by incubation for 1 hour at 37 ℃ and 500rad/min with a plate.

5. Cleaning: step 3 is the same.

6. Color developing solution (Cy 3-streptavidin)

Mu.l of developing solution was added to each well, and the wells were sealed and incubated at 37 ℃ and 500rad/min for 45 minutes.

7. Cleaning: step 3 is the same.

8. Fluorescence detection

1) The slide frame was removed, taking care not to touch the antibody-printed side of the slide by hand.

2) The residual wash solution of the slide was removed.

3) The signal was scanned using a laser scanner from beijing boao using either Cy3 or a green channel (excitation frequency of 532 nm).

9. Data extraction of the chip and data analysis with analysis software.

1) And establishing a least square method curve through the logarithmic relation of the standard substance signal and the concentration to obtain a standard curve of 6 items of the kit.

2) And substituting the logarithm of the sample signal into a standard curve, and calculating each index concentration of the detected sample.

The reference antibodies used in this example are shown in table 1, where the suppliers involved are as follows: guangzhou Ribo ao Biotechnology Co., Ltd (Ribo ao), Zhengzhou Satukang Biotechnology Co., Ltd (Satukang), Invitrogen, Novus, supraHill collar tide Biotech Co., Ltd (Shanghai collar tide), Hangzhou Hua Zymujin culture Biotech Co., Ltd (Hua Zymujin culture), Santa Cruz, Fei Peng Bio-Ltd (Fei Peng), Shen constant medical engineering Co., Ltd (Shen constant), Guangzhou Angstrom Biotech Co., Ltd (Guangzhou Angstrom), R constant&D. Origene, Shenzhen shenyuan biotechnologies Limited (Shenzhen shenyuan), Guangzhou shenyuan biotechnologies Limited (Chunhuan organism). At 37 ℃ with Thermo Scientific^TM Wellwash^TMAnd washing the plate machine to make each reference antibody respectively generate immunoreaction with CA125, CEA, NSE, SCCA1, CY21-1 and pro-GRP antigens so as to carry out the pairing detection of the reference antibody and the antigens.

The experimental parameters of the pair-wise assay were: vector, 96-well plate; antigen diluent, 1% BSA + PBS; blocking solution, 2% BSA + PBS; antibody diluent, 1% BSA + PBS; wash, 0.1% tween-20+ Tris.Cl.

TABLE 1 reference antibody information

2. The result of the detection

The results of the pairwise test of some of the reference antibodies of this example are shown in tables 2-7.

TABLE 2 antigen-pairing detection response Signal values for CA 125-specific antibodies

TABLE 3 antigen-pairing detection response Signal values for CEA-specific antibodies

TABLE 4 antigen-pairing detection response Signal values for NSE-specific antibodies

TABLE 5 antigen-pairing detection response Signal values for SCCA 1-specific antibodies

TABLE 6 antigen-pairing test response Signal values for CY 21-1-specific antibodies

TABLE 7 antigen-pairing detection response Signal values for pro-GRP-specific antibodies

It was concluded from the detection of the pairability of anti-CA 125-specific antibodies that the second, fourth, and sixth CA125 antibodies cross-reacted with other antigens.

It can be seen from the detection of the pairability of anti-CEA-specific antibodies that the fourth CEA antibody has a low response signal value and the third and fifth CEA antibodies cross-react with other antigens.

From the pairwise detection of anti-NSE-specific antibodies, it can be concluded that the second, fourth and sixth NSE antibodies cross-react with other antigens.

As can be seen from the paired detection of anti-SCCA 1-specific antibodies, the second, fifth and sixth SCCA1 antibodies cross-reacted with other antigens.

It can be concluded from the pairwise detection of anti-CY 21-1-specific antibodies that the fourth, fifth and sixth CY21-1 antibodies cross-react with other antigens.

From the pairwise detection of the anti-pro-GRP specific antibodies, it can be concluded that the third, fourth and sixth pro-GRP antibodies cross-react with other antigens.

In conclusion, the first, third and fifth CA125 antibodies, the first, second and sixth CEA antibodies, the first, third and fifth NSE antibodies, the first, third and fourth SCCA1 antibodies, the first, second and third CY-21-1 antibodies and the first, second and fifth pro-GRP antibodies are suitable for manufacturing the detection chip for simultaneously realizing the multi-flux detection of CA125, CEA, NSE, SCCA1, CY-21-1 and pro-GRP.

Example 2

1. Reference antibody

The following antibodies from example 1 were selected as the reference antibodies of this example:

a first CA125 antibody, a third CA125 antibody, a fifth CA125 antibody;

a first CEA antibody, a second CEA antibody, a sixth CEA antibody;

a first NSE antibody, a third NSE antibody, a fifth NSE antibody;

a first SCCA1 antibody, a third SCCA1 antibody, a fourth SCCA1 antibody;

a first CY-21-1 antibody, a second CY-21-1 antibody, a third CY-21-1 antibody;

a first pro-GRP antibody, a second pro-GRP antibody, a fifth pro-GRP antibody.

2. Experimental setup mode

The method comprises the following steps of respectively taking reference antibodies with the same lung cancer tumor marker as specific antigens as capture antibodies (primary antibodies) and detection antibodies (secondary antibodies), correspondingly combining the capture antibodies (primary antibodies) and the detection antibodies (secondary antibodies) one by one, and carrying out pairing experiments among the antibodies, wherein the experimental parameters of the pairing experiments among the antibodies are as follows: a carrier, an aldehydized glass sheet; primary anti-diluent, PBS + 1% BSA; blocking solution, 2% BSA + PBS; antigen diluent, 1% BSA + PBS; secondary antibody diluent, PBS; wash, 0.1% tween-20+ Tris.Cl.

3. The result of the detection

The results of the pairing experiments between the antibodies are shown in tables 8-13, and the kit for detecting lung cancer tumor, which is designed according to the ELISA double-antibody sandwich method, can be constructed by selecting the antibody pair with high response signal value.

TABLE 8 detection of response Signal values in the inter-antibody pairing assay for anti-CA 125-specific antibodies

TABLE 9 detection of response Signal values for the inter-antibody pairing experiments for anti-CEA-specific antibodies

TABLE 10 detection of response signal values for the inter-antibody pairing experiments against NSE-specific antibodies

TABLE 11 detection of response signal values in the inter-antibody pairing experiment for anti-SCCA 1-specific antibodies

TABLE 12 detection of response Signal values for the inter-antibody pairing assay for CY 21-1-specific antibodies

TABLE 13 detection of response Signal values in the inter-antibody pairing experiments for anti-pro-GRP-specific antibodies

Example 3

1. Experimental setup mode

In this example, two sets of treatment groups were set with the solid phase carrier of the chip for preparing the capture antibody mixture as a variable, and the treatment groups were set in the following manner: treating a, taking an aldehyde glass slide as a solid phase carrier of the chip; and b, treating by using the aldehyde glass slide as a solid phase carrier of the chip.

The primary antibody combination used in this example is that of example 1: a third CA125 antibody, a first CEA antibody, a sixth CEA antibody, a first NSE antibody, a third NSE antibody, a first SCCA1 antibody, a second CY21-1 antibody, a fourth CY21-1 antibody, and a first pro-GRP antibody.

The secondary antibody combination adopted in the embodiment is the combination of the secondary antibodies in the embodiment 1: a first CA125 antibody, a second CEA antibody, a first CEA antibody, a third NSE antibody, a first NSE antibody, a fourth SCCA1 antibody, a third SCCA1 antibody, a first CY21-1 antibody, and a second pro-GRP antibody.

Dissolving the antibodies comprised by the primary antibody combination in a phosphate buffer containing 0.5% sucrose at pH 7.4 to form an antibody mixture; a full-automatic sample application instrument (platinum Elmer) is adopted, each antibody is respectively fixed in sample application holes of an amination slide and an aldehyde slide in an antibody mixture, a specific chip lattice schematic diagram is shown in figure 1, and bovine IgG marked by biotin is used as a positive control. Biotin-labeled bovine IgG there were two positive controls at different concentrations (both preferably 1mg/ml,0.3mg/ml in the examples of the invention) in each chip, as well as 6 replicates of the capture antibody. Secondary antibodies (i.e., 0.01-0.5mg/ml detection antibody, in this example, 25ng/ml CA125 antibody, 13.3ng/ml CEA antibody, 40ng/ml NSE antibody, 15ng/ml SCCA1 antibody, 13.3ng/ml CY21-1 antibody and 50ng/ml pro-GRP antibody) and substrate were then added for detection. Chips prepared by using a phosphate buffer solution with a treatment pH of 7.4 as a reference buffer solution were numbered as chip a and chip b, respectively.

2. Test results

As shown in FIG. 2, the background of both chip a and chip b is clear, and compared with the background of chip a and chip b, the chip prepared by using the aldehyde glass as a solid phase carrier has more excellent test performance.

Example 4

1. Experimental setup mode

Example two treatment groups were set up with the composition of the buffer in which the capture antibody mixture was formulated as a variable, in a manner that:

1.1 treatment A

Reference buffer: phosphate buffer at pH 7.4;

antibody combination:

the primary antibody combinations used were those of example 1: a third CA125 antibody, a first CEA antibody, a sixth CEA antibody, a first NSE antibody, a third NSE antibody, a first SCCA1 antibody, a second CY21-1 antibody, a fourth CY21-1 antibody, and a first pro-GRP antibody.

The secondary antibody combination used was that of example 1: a first CA125 antibody, a second CEA antibody, a first CEA antibody, a third NSE antibody, a first NSE antibody, a fourth SCCA1 antibody, a third SCCA1 antibody, a first CY21-1 antibody, and a second pro-GRP antibody.

1.2 treatment B

Reference buffer: pH 7.4 phosphate buffer containing 0.5% sucrose.

Antibody combination:

As in example 3, the antibodies included in the two primary antibody combinations were dissolved in the reference buffers of treatment a and treatment B, respectively, to form antibody mixtures; a full-automatic sample application instrument (platinum Elmer) is adopted, each antibody content is fixed in a sample application hole of an aldehyde-based slide in a manner of 0.01-0.5 ng (25 ng/ml of CA125 antibody, 13.3ng/ml of CEA antibody, 40ng/ml of NSE antibody, 15ng/ml of SCCA1 antibody, 13.3ng/ml of CY21-1 antibody and 50ng/ml of pro-GRP antibody) in an antibody mixture, a specific chip lattice schematic diagram is shown in figure 1, and biotin-labeled bovine IgG is used as a positive control. Biotin-labeled bovine IgG was present at two different concentrations, with six replicates in each chip as were the capture antibodies. Then adding the same group of secondary antibody and substrate for detection. And numbering the chips prepared by the solid phase carriers and the antibody combinations of the treatment A and the treatment B one by one correspondingly to obtain the chip A and the chip B.

2. Test results

The test results of chip a and chip B are shown in table 14 and table 15, respectively. In the liquid environment provided by chip A, B, no cross reaction occurred in each antibody pair, but it was found that in the liquid environment provided by chip A, the positive signal was lower relative to chip B and the background was higher relative to chip B; and the pairing response value between CY21-1 antibodies in the liquid environment provided by the chip B is low. It follows from this that: when a test chip was prepared, phosphate buffer containing 0.5% sucrose at pH 7.4 was used as a buffer for primary antibody dissolution, and an ideal liquid reaction environment was provided for multi-throughput detection of CA125, CEA, NSE, SCCA1, CY21-1, and pro-GRP by combining with an aldehyde chip.

TABLE 14 response signal values for chip A

TABLE 15 response signal values for chip B

Example 5

1) The chip for simultaneously and quantitatively detecting a plurality of lung cancer markers as described in embodiment 3 is composed of 16 chips for simultaneously and quantitatively detecting a plurality of lung cancer markers by using an aldehyde glass slide as a solid carrier of the chip, wherein 6 chips are added with protein standard substances as a standard and the other 10 chips are added with a blood sample to detect the concentration of a target protein in the blood sample. The concentration of the coated capture antibody on each chip was the same: 150ug/ml of CA125 coated antibody; CEA coated antibody 200 ug/ml; NES coating antibody 600 ug/ml; SCCA1 coated antibody 600 ug/ml; CY21-1 coating antibody 200 ug/ml; the pro-GRP coated antibody was 800 ug/ml.

The concentration of the detection antibody was: CA125 antibody 25ng/ml, CEA antibody 13.3ng/ml, NSE antibody 40ng/ml, SCCA1 antibody 15ng/ml, CY21-1 antibody 13.3ng/ml, and pro-GRP antibody 50 ng/ml.

Phosphate buffer at pH 7.4 with 0.5% sucrose was used as buffer to dissolve the primary antibody.

2) Sensitivity and specificity data for the kit

S1, preprocessing a sample

The RIPA cell lysate, the protease inhibitor and the phosphatase inhibitor are used for preparing a sample processing solution, the sample is diluted by the sample processing solution, and plasma, serum, cell supernatant, cell or tissue lysate are all suitable for being used as samples to be detected of the detection kit in the embodiment. Note: the sample pretreatment modes of different samples are different: plasma and serum sample treatment solution 1: 1, diluting; cell supernatant can be used as stock solution; after the protein concentration of the cell or tissue lysate is measured, the sample processing solution is used for preparing a sample diluent with the protein concentration of 50-500 mug/mL.

S2, completely drying the detection chip

And taking the detection chip out of the box, balancing at room temperature for 20-30 minutes, opening the packaging bag, uncovering the sealing strip, and then placing the chip in a vacuum drier or drying at room temperature for 1-2 hours.

S3, sealing and incubating

S3.1 Add 100. mu.L of sample dilution to each well, incubate for 30min on a shaker at room temperature, block the quantitative antibody chip.

S3.2 remove the buffer from each well, add 100. mu.L of standard and sample to the wells, and incubate overnight at 4 ℃ on a shaker.

S3.3 cleaning

The standard or sample in each well was aspirated, 1 wash from wash I5 times, shaking at room temperature for 5min each time, 150 μ L of 1 × wash I per well, with each wash being aspirated, and 20 × wash I diluted with deionized water.

And (3) pumping out the 1 Xwashing liquid I in each hole, adding the 1 Xwashing liquid II to wash for 2 times, shaking the 1 Xwashing liquid II in a shaking table at room temperature for 5min every time, pumping out the washing liquid II in 150 mu L of each hole, and diluting the 20 Xwashing liquid II with deionized water.

S3.4 incubation of detection antibody mixtures

The test antibody mixture vials were centrifuged and then 1.4ml of sample diluent was added, mixed well and then centrifuged quickly again. Add 80. mu.l of detection antibody to each well and incubate for 2 hours on a shaker at room temperature.

S3.5 cleaning

And (3) extracting the detection antibody in each hole, washing with 1 Xwashing liquid I for 5 times, shaking in a shaking table at room temperature for 5min every time, extracting 150 mu L of 1 Xwashing liquid I in each hole, cleaning the washing liquid completely in each washing, then adding 1 Xwashing liquid II for washing for 2 times, shaking in a shaking table at room temperature for 5min every time, extracting 150 mu L of 1 Xwashing liquid II in each hole, and cleaning the washing liquid completely in each washing.

S3.6 incubation of Cy 3-streptavidin

The Cy 3-streptavidin vial was centrifuged, then 1.4mL of sample diluent was added, mixed well and centrifuged quickly again. Add 80. mu.L of Cy 3-streptavidin to each well, wrap the slide with aluminum foil and incubate in the dark for 1 hour on a shaker at room temperature.

S3.7 cleaning

Cy 3-streptavidin was removed from each well and the 1 XWash I was washed 5 times, 5min at room temperature with shaking, 150. mu.l of 1 XWash I per well, and the wash was removed for each wash.

S3.8 fluorescence detection

2) The slide was placed in a slide wash tube, and about 30ml of 1 XWash I was added to cover the slide entirely, shaking for 15min on a room temperature shaker, discarding 1 XWash I, adding about 30ml of 1 XWash II, and shaking for 5min on a room temperature shaker.

3) The residual wash solution of the slide was removed. The slides were placed in slide wash/dry tubes without lid and centrifuged at 1000rpm for 3 min.

4) The signal is scanned with a laser scanner, e.g. Axon GenePix, using either Cy3 or the green channel (excitation frequency 532 nm).

S3.9 data extraction of chip and data analysis with analysis software

1) The fluorescence values of the biochips were read with GenePix software.

2) The value selected after reading is the Median reading (F532 media-Local Background) excluding the Local Background. The standard curve of each tumor marker was calculated using conventional quantitative chip calculation software.

The standard curve can be drawn as follows:

add 500. mu.L of sample treatment solution to the vial of tumor marker standard mixture and redissolve the standard. Before opening the tubule, rapidly centrifuging, gently pumping up and down to dissolve the powder, and marking the tubule as Std 1; labeling 6 clean centrifuge tubes as Std2, Std3, Std4, Std5, respectively, and adding 200 μ L of sample treatment solution to each vial; 100 μ L of diluted sample is extracted from Std1 and added to Std2 for gentle mixing, and then 100 μ L of diluted sample is extracted from Std2 and added to Std3, and the diluted sample is subjected to gradient dilution to Std 5; draw 100 μ L of sample dilution into another new centrifuge tube, labeled CNTRL, as a negative control. The fluorescence intensities measured by the detection chip of the kit were respectively determined by Std 1-Std 5, and a standard curve was obtained by fitting the standard concentration values and the fluorescence intensity values corresponding thereto (see FIG. 3).

Linear range:

sensitivity:

calculating the average value of the corresponding concentration of 10 blank tests according to the standard curve; see the following Table

Specificity:

a single antigen (500U/ml CA125, 100ng/ml CEA, 100ng/ml NSE, 30ng/ml SCCA1, 20ng/ml CY21-1, 1000pg/ml pro-GRP, 100ng/ml AFP, 500pmol/ml HE4, 500U/ml CA199, 100U/ml CA724) was added to each well separately to test the cross-specificity under this system; see table below.

3) Stability test of kit

Preparing two quality control products, placing the two quality control products in a refrigerator at 2-8 ℃, placing the kit in a refrigerator at 37-3 days, 7 days and 8 days, then using the kit at 2-8 ℃ as a control group, respectively detecting the two quality control products (protein standard products) stored at 2-8 ℃, and calculating the relative deviation of detection results before and after acceleration; see table below. And the kit is placed at 37 ℃, and the quality control products stored in the refrigerator are respectively detected every 0, 3, 7 and 8 days after the kit is placed for 0, 3, 7 and 8 days, so that the stability result is obtained. The results showed good stability, with a deviation of within 6% even after 8 days at 37 ℃.

High concentration quality control product

High concentration quality control product

4) Clinical serum implementation of the kit (in contrast to other kits)

The correlation between each index and the detection result of the Roche reagent was analyzed as follows.

Kits were prepared as in example 3 with antibodies specific for the 6 proteins of CA125, CEA, NSE, SCCA1, CY21-1, proGRP. Antibodies specific for 6 proteins were spotted as shown in FIG. 1. 30 clinical samples (the detection value of the clinical samples is known and is measured by an electrochemiluminescence method) are detected by using the kit, and the detection method is the same as that described in example 5. Comparing the detection value of the clinical sample with the detection value of the kit to draw a scatter diagram, wherein the correlation is as follows: the electrochemical luminescence detection result is basically equivalent to the detection result of the kit. The results are shown in FIG. 4 (X-axis represents the detection signal value of Roche kit, and Y-axis represents the detection signal value of the kit).

Fig. 4 shows the correlation between the CEA antibody in the kit and the detection result in the electrochemiluminescence method kit, where the trend equation is y-0.9774 x-0.2893, and the correlation coefficient is 0.9808. The correlation between the CA125 antibody and the detection result of the electrochemiluminescence kit is as follows, and the trend equation is that y is 1.0488x-27.724, and the correlation coefficient is 0.966. The correlation between the NSE antibody and the detection result of the electrochemiluminescence kit is as follows, the trend equation is that y is 0.9966x-1.3364, and the correlation coefficient is 0.9672. The correlation between the SCCA1 antibody and the detection result of the electrochemiluminescence kit is as follows, and the trend equation is that y is 1.1348x +0.0452, and the correlation coefficient is 0.9911. The correlation between the pro-GRP antibody and the detection result of the electrochemiluminescence kit is as follows, the trend equation is that y is 1.0338x +1.2336, and the correlation coefficient is 0.9933. The correlation between the CY21-1 antibody and the detection result of the electrochemiluminescence kit is as follows, the trend equation is that y is 0.9793x +0.9794, and the correlation coefficient is 0.9802.

5) Repeatability of other experimental results: preparing a quality control product, and repeatedly detecting for 10 times to obtain a signal repetitive CV; the result shows that the kit has 10 percent of CV and good repeatability in the repeatability analysis of disease sample detection. See table below.

Example 6

The following 16 sera were tested using the kit described in example 5 and the clinical fluorescence signal values obtained were as follows.

The detection method was according to examples 3 to 5.

16 clinical sera confirmed by hospital were selected and the results were as follows.

The result obtained by detection is highly consistent with the clinical specimen, which shows that the kit for simultaneously detecting a plurality of lung cancer markers has good detection effect and meets the requirement of actual detection.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

Claims

1. A chip for simultaneously detecting a plurality of lung cancer markers is characterized by comprising a solid phase carrier and a capture antibody of the tumor markers fixed on the solid phase carrier, wherein the lung cancer markers are at least three antibodies selected from a CA125 antibody, a CEA antibody, an NSE antibody, an SCCA1 antibody, a CY21-1 antibody and a pro-GRP antibody.

2. The chip for simultaneously detecting multiple lung cancer markers according to claim 1, wherein the capture antibody is a CA125 antibody, a CEA antibody, an NSE antibody, an SCCA1 antibody, a CY21-1 antibody, or a pro-GRP antibody.

3. The chip for simultaneously detecting multiple lung cancer markers according to claim 1, wherein the solid phase carrier further comprises a protein standard, preferably a biotin-labeled bovine IgG, coated with an immune signal amplification marker as a positive control immobilized thereon.

4. The chip for simultaneously detecting multiple lung cancer markers according to any one of claims 1-3, wherein the solid support is an aminated glass slide or an aldehyde-based glass slide, preferably the solid support is an aldehyde-based glass slide.

5. A kit for simultaneously detecting a plurality of lung cancer markers, which comprises at least 6 chips for simultaneously detecting a plurality of lung cancer markers according to claim 1 or 2, and detection antibodies, wherein the detection antibodies are labeled with immune signal amplification markers, and the amounts of the same capture antibodies coated on the at least 6 chips for simultaneously detecting a plurality of lung cancer markers are the same.

6. The kit for simultaneously detecting multiple lung cancer markers according to claim 5, wherein the CA125 antibody is at least one of 118-11248 antibody of Riboao Biotechnology, Guangzhou and Sc-52095 antibody of Santa Cruz Biotechnology.

7. The kit for simultaneously and quantitatively detecting a plurality of lung cancer markers according to claim 5, wherein the CEA antibody is at least one of antibodies of Zhengzhou Satukang Biotechnology Ltd, having a CTA-1003 or CTA-1004, and antibodies of Novus Biologicals, having a NBP 2-52673.

8. The kit for simultaneously and quantitatively detecting a plurality of lung cancer markers as claimed in claim 5, wherein the NSE antibody is at least one of antibodies with the cargo numbers of 9602-.

9. The kit for simultaneously and quantitatively detecting a plurality of lung cancer markers according to claim 5, wherein the SCCA1 antibody is at least one of antibodies having a product number of 130-10459 of Riboao Biotechnology Ltd, Guangzhou, Chunhuan Biotechnology Ltd, a product number of SCCA1-103 or SCCA 1-104.

10. The kit for simultaneously and quantitatively detecting a plurality of lung cancer markers as claimed in claim 5, wherein the CY21-1 antibody is at least one of antibodies with the cargo numbers of 130-10317 and 130-10426 of Riboao Biotechnology Limited, Guangzhou and antibodies with the cargo number of L1C00706 of Shanghai tide Biotechnology Limited.

11. The kit for simultaneously and quantitatively detecting multiple lung cancer markers according to claim 5, wherein the pro-GRP antibody is at least one of antibodies of Fipeng biological corporation under the trade designations 4# and 5 #.

12. The kit for simultaneously and quantitatively detecting multiple lung cancer markers according to any one of claims 5 to 11, which further comprises a phosphate buffer containing 0.5% ± 0.01 sucrose and having a pH of 7.3 to 7.5.

13. The kit for simultaneously and quantitatively detecting multiple lung cancer markers according to any one of claims 5 to 11, wherein the capture antibody is CA125 antibody of Santa Cruz Biotechnology with the catalog number Sc-52095, CEA antibody of Zhengzhou Seikang Biotechnology Ltd with the catalog number CTA-1003, CEA antibody of Novus Biologicals with the catalog number NBP2-52673, NSE antibody of Guangzhou Rubo-ao Biotechnology Ltd with the catalog number 9602 100116, NSE antibody of Shenzhen Sheng Biotechnology Ltd with the catalog number C191, SCCA1 antibody of Guangzhou Rubo-ao Biotechnology Ltd with the catalog number 130-10459, CY21-1 antibody of Guangzhou Rubo-ao Biotechnology Ltd with the catalog number 130-10317, CY21-1 antibody of Shanghai Yoghi Biotechnology Ltd, CY 00735 of Yoghe Biotechnology Ltd with the catalog number L1C 06, CY 00735-00735 of Yoghe Biotechnology Ltd, pro-GRP antibody, cat # 5.

14. The kit for simultaneously and quantitatively detecting multiple lung cancer markers according to any one of claims 5 to 11, wherein the detection antibody comprises CA125 antibody of 118-, pro-GRP antibody, cat # 4.

15. The kit for simultaneously and quantitatively detecting a plurality of lung cancer markers according to any one of claims 5 to 11, wherein the immune signal amplification marker is biotin.

16. The kit for simultaneously and quantitatively detecting a plurality of lung cancer markers according to any one of claims 5 to 11, which comprises 16 chips for simultaneously detecting a plurality of lung cancer markers according to any one of claims 1 to 4, wherein the coating concentration of the capture antibody of each tumor marker on each chip is as follows:

CA125 antibody: 140-;

CEA antibody: 190-;

NSE antibody: 580-620 ug/ml;

SCCA1 antibody: 580-620 ug/ml;

CY21-1 antibody: 190-;

pro-GRP antibody: 780-820 ug/ml.

17. The kit for simultaneously and quantitatively detecting a plurality of lung cancer markers according to claim 16, wherein the coating concentration of the capture antibody of the tumor marker on the chip is as follows:

CA125 antibody: 150 ug/ml;

CEA antibody: 200 ug/ml;

NSE antibody: 600 ug/ml;

SCCA1 antibody: 600 ug/ml;

CY21-1 antibody: 200 ug/ml;

pro-GRP antibody: 800 ug/ml.

18. The kit for simultaneous quantitative detection of multiple lung cancer markers according to any one of claims 5 to 11,

the solubility of the detection antibody is: 22-26ng/ml CA125 antibody, 12.5-13.5ng/ml CEA antibody, 38-42ng/ml NSE antibody, 14-16ng/ml SCCA1 antibody, 12.5-13.5ng/ml CY21-1 antibody and 48-52ng/ml pro-GRP antibody.