阅读说明：本技术 抗原组合在制备用于肺癌相关自身抗体检测的试剂盒中的用途以及相应的试剂盒和检测方法 (Application of antigen combination in preparation of kit for detecting lung cancer related autoantibody, corresponding kit and detection method ) 是由 刘浩 王东 李宾 张涛 于 2019-11-13 设计创作，主要内容包括：本发明提供了一种抗原组合在制备用于肺癌相关自身抗体检测的试剂盒中的用途以及相应的试剂盒和检测方法,以能更准确地辅助早期肺癌筛查和诊断,其特征在于：该抗原组合包括以下抗原蛋白中的任意七个或七个以上的组合：P53、SOX2、GBU4-5、KRAS、P53-95、CK8、HuD、SSX1、P62、EGFR-vIII、P16、CK20、CAGE以及NY-ESO-1。(The invention provides an application of antigen combination in preparing a kit for detecting lung cancer related autoantibodies, a corresponding kit and a detection method, so as to more accurately assist early lung cancer screening and diagnosis, and is characterized in that: the antigen combination comprises any seven or more than seven combinations of the following antigen proteins: p53, SOX2, GBU4-5, KRAS, P53-95, CK8, HuD, SSX1, P62, EGFR-vIII, P16, CK20, CAGE, and NY-ESO-1.)

1. Use of an antigen combination for the preparation of a kit for the detection of lung cancer-associated autoantibodies for the diagnosis of lung cancer, characterized in that:

the antigen combination comprises any seven or more than seven combinations of the following antigen proteins:

p53, SOX2, GBU4-5, KRAS, P53-95, CK8, HuD, SSX1, P62, EGFR-vIII, P16, CK20, CAGE, and NY-ESO-1.

2. Use according to claim 1, characterized in that:

wherein the combination is one of the following:

P53、SOX2、GBU4-5、KRAS、CK8、HuD、SSX1；

P53、SOX2、GBU4-5、KRAS、P53-95、SSX1、CAGE；

P53、SOX2、GBU4-5、HuD、SSX1、EGFR-vIII、NY-ESO-1；

SOX2、GBU4-5、KRAS、P53-95、CK8、CK20、NY-ESO-1；

P53、GBU4-5、KRAS、P53-95、EGFR-vIII、CAGE、NY-ESO-1；

P53、SOX2、GBU4-5、P53-95、CK8、HuD、SSX1；

P53、SOX2、GBU4-5、P53-95、HuD、SSX1、NY-ESO-1；

P53、SOX2、P53-95、P16、CAGE、P62、CK20；

SOX2、GBU4-5、P53-95、CK8、SSX1、HuD、NY-ESO-1；

GBU4-5、KRAS、P53-95、CK8、HuD、SSX1、P62。

3. a kit for detecting lung cancer-related autoantibodies, characterized in that:

the antigen combination is used as a target for detecting the lung cancer related autoantibody,

wherein the antigen combination comprises any seven or more than seven of the following antigen proteins:

p53, SOX2, GBU4-5, KRAS, P53-95, CK8, HuD, SSX1, P62, EGFR-vIII, P16, CK20, CAGE, and NY-ESO-1.

4. The kit of claim 3, wherein:

wherein the combination is one of the following:

P53、SOX2、GBU4-5、KRAS、CK8、HuD、SSX1；

P53、SOX2、GBU4-5、KRAS、P53-95、SSX1、CAGE；

P53、SOX2、GBU4-5、HuD、SSX1、EGFR-vIII、NY-ESO-1；

SOX2、GBU4-5、KRAS、P53-95、CK8、CK20、NY-ESO-1；

P53、GBU4-5、KRAS、P53-95、EGFR-vIII、CAGE、NY-ESO-1；

P53、SOX2、GBU4-5、P53-95、CK8、HuD、SSX1；

P53、SOX2、GBU4-5、P53-95、HuD、SSX1、NY-ESO-1；

P53、SOX2、P53-95、P16、CAGE、P62、CK20；

SOX2、GBU4-5、P53-95、CK8、SSX1、HuD、NY-ESO-1；

GBU4-5、KRAS、P53-95、CK8、HuD、SSX1、P62。

5. the kit according to claim 3 or 4, characterized in that it comprises:

a solid carrier on which each of the antigen proteins constituting the antigen combination is immobilized,

wherein each of said respective antigenic proteins is immobilized to said solid support by direct or indirect immobilization,

the direct immobilization refers to the immobilization of the antigen protein directly on the solid support,

the indirect fixation refers to the indirect fixation of the antigen protein to the solid phase carrier through the specific reaction between biotin and streptavidin.

6. The kit of claim 5, further comprising:

a standard substance, a low-concentration quality control substance, a high-concentration quality control substance and an enzyme-labeled antibody,

the standard is used for obtaining detection standards with different concentrations so as to form corresponding logic curves.

7. The kit of claim 6, wherein:

wherein each antigen protein is a recombinant protein with His label obtained by expressing and purifying escherichia coli,

the solid phase carrier is also fixed on an empty carrier protein with His label expressed and purified from escherichia coli, the coating concentration and the coating condition of the empty carrier protein are consistent with the coating concentration and the coating condition of the antigen protein,

the empty carrier proteins are respectively fixed in different holes so as to be respectively used for corresponding blood samples, detection standard substances with different concentrations, high-concentration quality control substances and low-concentration quality control substances.

8. A method for detecting a lung cancer-associated autoantibody, characterized by:

detection of lung cancer-associated autoantibodies using a kit according to any of claims 3-7.

9. The detection method according to claim 8, comprising:

adding to each well immobilized with an antigen proteinDiluting the blood samples to obtain respective test sample ODs of different antigen proteins corresponding to each blood sample₆₅₀A value of adding the diluted blood sample to the hole for fixing the empty carrier protein corresponding to the blood sample to obtain each detection background OD corresponding to each blood sample₆₅₀Adding each of the detection standards at the corresponding concentration to each of the wells in which the empty carrier protein is immobilized corresponding to each of the detection standards to obtain a detection standard OD corresponding to each of the concentrations of the standard₆₅₀Adding the corresponding high concentration quality control substance into the hole for fixing the empty carrier protein corresponding to the high concentration quality control substance to obtain OD of the high concentration quality control substance₆₅₀And adding the corresponding low concentration quality control substance to the hole of the immobilized empty carrier protein corresponding to the low concentration quality control substance to obtain the OD of the low concentration quality control substance₆₅₀A value;

taking the logarithm with the base of 10 as the concentration value of each detection standard as the abscissa, and each detection standard OD₆₅₀Drawing a logical curve with the value of the ordinate and calculating a regression equation of the standard curve;

detection OD for detection of respective antigen proteins in the same serum sample₆₅₀Subtracting the detection background OD corresponding to the serum sample from the values₆₅₀Obtaining the absolute OD of the blood sample corresponding to different antigen proteins₆₅₀Value, individual absolute OD₆₅₀Substituting the values into the standard curve regression equation to calculate the autoantibody relative concentration value aiming at the antigen protein in the blood sample.

10. The detection method according to claim 9, characterized in that:

wherein, total 6 detection standard substances with different concentrations are respectively a detection standard substance 1, a detection standard substance 2, a detection standard substance 3, a detection standard substance 4, a detection standard substance 5 and a detection standard substance 6, and the detection standard substances with different concentrations are obtained as follows:

taking a standard substance in the kit as a detection standard substance 1, and carrying out gradient dilution on the detection standard substance by adopting a sample and an antibody diluent to respectively obtain detection standard substances 2-6, wherein the concentrations of the detection standard substances 2-6 are respectively as follows: 0.1111. mu.g/mL, 0.03704. mu.g/mL, 0.01235. mu.g/mL, 0.004115. mu.g/mL and 0.001372. mu.g/mL,

the standard substance is a humanized Anti-His antibody standard substance with the preset concentration of 0.3333 mug/mL, which is obtained by diluting a humanized Anti-His monoclonal antibody by a sample and an antibody diluent,

the sample and antibody diluent is a PBST buffer solution containing 0.1% casein by mass volume ratio.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of an antigen combination in preparation of a kit for detecting lung cancer-related autoantibodies, a corresponding kit and a detection method.

Background

In the early stage of tumor development, tumor cells produce genetic mutation, ectopy or recombination, and after release of the associated protein, the associated protein is recognized as a "non-self protein" by the immune system as an antigen (TAA) to generate an antibody against the antigen, i.e., a Tumor Autoantibody (TAB). Autoantibodies exist in circulation in the body either free or bound to the relevant antigen as antigen-antibody complexes. The use of autoantibodies for early diagnosis of cancer, efficacy assessment and prediction of recurrence has several advantages: autoantibodies can be detected during the asymptomatic phase of cancer; autoantibodies against tumor associated antigens are contained in the serum of cancer patients, and the serum is readily available and used for cancer screening; the autoantibody is not subjected to the enzymolysis of various proteins and can stably exist in serum for a long time; the immune system generates immune reaction to the antigen to play a role in signal amplification, so that the concentration of the autoantibody is higher than that of the corresponding antigen. Various reviews have reported that combinations of autoantibodies can be used as serum biomarkers for diagnosing early stage cancer or distinguishing benign nodules from malignant tumors. Autoantibody levels can also fluctuate in the course of disease progression and can be used to monitor disease progression and recurrence. For some anti-cancer therapies, increased levels of autoantibodies are associated with complete remission of the cancer. When anti-CTLA-4 antibodies are used to treat prostate cancer patients, increased levels of autoantibodies indicate that the patient has a better response to treatment. Some autoantibody assays are also associated with different disease subtypes, e.g., some autoantibodies in lung cancer are more sensitive to small cell lung cancer, while others are more sensitive to non-small cell lung cancer. The autoantibody is also applied to indicate recurrence of cancer, and the positive thyroglobulin autoantibody indicates that the disease has higher recurrence possibility within one year after the papillary thyroid cancer patient receives treatment, while the negative thyroglobulin autoantibody indicates that the patient has better treatment effect. Thus, determining autoantibody levels can identify to which populations cancer treatment is more effective, and monitoring these autoantibody levels during and after treatment can also aid in monitoring treatment efficacy and early detection of cancer recurrence.

The antigen corresponding to the tumor-associated autoantibody is used for detecting the tumor autoantibody, and the specificity and the sensitivity are higher than those of other diagnostic methods, so that the early cancer screening in clinic becomes possible. However, the immune system of different individuals reacts differently, and it is difficult for a single autoantibody to accurately predict the onset of all cancers. A comprehensive analysis of the expression profile of multiple autoantibodies binding is required to be able to determine a tumor autoantibody marker that matches a particular patient population.

In recent years, some technologies have adopted ELISA to detect a plurality of autoantibodies simultaneously to perform auxiliary diagnosis of lung cancer, for example, 6 (p53, NY-ESO-1, CAGE, GBU4-5, SOX2, Annexin I) and 7 (p53, NY-ESO-1, CAGE, GBU4-5, MAGE-A4, SOX2, Hu-D) antigens are respectively combined for detection, but due to the difference between Asian and European and American varieties, the combination of the antigens is not suitable for early detection of lung cancer of Asian people, and the detection sensitivity is less than 30%; for another example, the combination of seven antigens, namely P53, PGP9.5, SOX2, GAGE 7, GBU4-5, MAGE a1 and CAGE, is used for detection, and in this case, although the overall sensitivity of lung cancer detection can reach 50%, the sensitivity is low (about 30%) in early lung cancer detection, and the specificity is low (about 80%) in early lung cancer detection, and higher false positive results further increase the workload and economic burden of follow-up monitoring in later period.

Disclosure of Invention

The invention provides an application of antigen combination in preparing a kit for detecting lung cancer related autoantibodies, a corresponding kit and a detection method, so as to more accurately assist early lung cancer screening and diagnosis.

The invention provides an application of antigen combination in preparing a kit for lung cancer related autoantibody detection for lung cancer diagnosis, which is characterized in that: the antigen combination comprises any seven or more than seven combinations of the following antigen proteins: p53, SOX2, GBU4-5, KRAS, P53-95, CK8, HuD, SSX1, P62, EGFR-vIII, P16, CK20, CAGE, and NY-ESO-1.

The invention also provides an application, wherein the combination is one of the following:

P53、SOX2、GBU4-5、KRAS、CK8、HuD、SSX1；

P53、SOX2、GBU4-5、KRAS、P53-95、SSX1、CAGE；

P53、SOX2、GBU4-5、HuD、SSX1、EGFR-vIII、NY-ESO-1；

SOX2、GBU4-5、KRAS、P53-95、CK8、CK20、NY-ESO-1；

P53、GBU4-5、KRAS、P53-95、EGFR-vIII、CAGE、NY-ESO-1；

P53、SOX2、GBU4-5、P53-95、CK8、HuD、SSX1；

P53、SOX2、GBU4-5、P53-95、HuD、SSX1、NY-ESO-1；

P53、SOX2、P53-95、P16、CAGE、P62、CK20；

SOX2、GBU4-5、P53-95、CK8、SSX1、HuD、NY-ESO-1；

GBU4-5、KRAS、P53-95、CK8、HuD、SSX1、P62。

the invention also provides a kit for detecting the lung cancer related autoantibody, which is characterized in that: the method takes an antigen combination as a target for detecting the lung cancer related autoantibody, wherein the antigen combination comprises any seven or more than seven of the following antigen proteins:

p53, SOX2, GBU4-5, KRAS, P53-95, CK8, HuD, SSX1, P62, EGFR-vIII, P16, CK20, CAGE, and NY-ESO-1.

The kit provided by the invention is also characterized in that the combination is one of the following:

P53、SOX2、GBU4-5、KRAS、CK8、HuD、SSX1；

P53、SOX2、GBU4-5、KRAS、P53-95、SSX1、CAGE；

P53、SOX2、GBU4-5、HuD、SSX1、EGFR-vIII、NY-ESO-1；

SOX2、GBU4-5、KRAS、P53-95、CK8、CK20、NY-ESO-1；

P53、GBU4-5、KRAS、P53-95、EGFR-vIII、CAGE、NY-ESO-1；

P53、SOX2、GBU4-5、P53-95、CK8、HuD、SSX1；

P53、SOX2、GBU4-5、P53-95、HuD、SSX1、NY-ESO-1；

P53、SOX2、P53-95、P16、CAGE、P62、CK20；

SOX2、GBU4-5、P53-95、CK8、SSX1、HuD、NY-ESO-1；

GBU4-5、KRAS、P53-95、CK8、HuD、SSX1、P62。

the kit provided by the invention is also characterized by further comprising solid phase carriers on which the antigen proteins in the antigen combination are respectively fixed, wherein the antigen proteins are fixed to the solid phase carriers by direct fixation or indirect fixation, the direct fixation means that the antigen proteins are directly fixed on the solid phase carriers, and the indirect fixation means that the antigen proteins are indirectly fixed on the solid phase carriers by specific reaction between biotin and streptavidin, and the solid phase carriers are magnetic particles, microspheres, plastic beads, liquid phase chips, microporous plates (ELISA plates), microfluidic chips or affinity membranes.

The kit of the present invention is also characterized in that the coating concentration of the antigen protein is 50nM and 50. mu.L per well.

The kit provided by the present invention is also characterized in that the conditions for coating the antigen protein are as follows: incubation was carried out in the dark at room temperature for 14-18 h.

The kit provided by the invention is also characterized in that after being coated, the coated kit is sealed by adopting a sealing buffer solution, wherein the sealing buffer solution is as follows: PBS was added with 1% BSA, 0.02% Tween-20 and 0.05% sodium azide, pH 7.4.

The kit provided by the invention is also characterized by further comprising: the system comprises a standard substance, a low-concentration quality control substance, a high-concentration quality control substance and an enzyme-labeled antibody, wherein the standard substance is used for obtaining detection standard substances with different concentrations so as to form a corresponding logic curve.

The kit provided by the invention is also characterized in that each antigen protein is recombinant protein with His label obtained by expression and purification from escherichia coli, the solid phase carrier is also fixed with empty carrier protein with His label obtained by expression and purification from escherichia coli, the coating concentration and the coating condition of the empty carrier protein are consistent with those of the antigen protein, and the empty carrier proteins are respectively fixed in different holes to be respectively used for corresponding blood samples, detection standard products with different concentrations, high-concentration quality control products and low-concentration quality control products.

The kit provided by the invention is also characterized in that the standard substance is a humanized Anti-His antibody standard substance with a predetermined concentration of 0.3333 mu g/mL, which is obtained by diluting a humanized Anti-His monoclonal antibody by a sample and an antibody diluent, and the sample and the antibody diluent are PBST buffer solution containing 0.1% of casein in content volume ratio, mass volume ratio and volume ratio.

The kit provided by the invention is also characterized in that the high-concentration quality control product is a high-concentration humanized Anti-His antibody quality control product with the concentration of 0.05 mu g/mL, which is obtained by diluting a humanized Anti-His monoclonal antibody by a sample and an antibody diluent; the low-concentration quality control product is a low-concentration humanized Anti-His antibody quality control product with the concentration of 0.004 mu g/mL, which is obtained by diluting a humanized Anti-His monoclonal antibody by a sample and an antibody diluent, wherein the sample and the antibody diluent are PBST buffer solution containing casein with the content volume ratio, the mass volume ratio and the volume ratio of 0.1%.

The kit provided by the invention is also characterized in that the enzyme-labeled antibody is an anti-human IgG antibody labeled by horseradish peroxidase.

The kit provided by the invention is also characterized by further comprising: substrate solution, stop solution, sample and antibody dilution and concentrated wash.

The kit provided by the invention is also characterized in that the substrate solution is TMB substrate solution, the stop solution is NaF stop solution with the concentration of 1mg/mL, and the sample and antibody diluent is PBST buffer solution containing casein with the mass-volume ratio of 0.1%; the washing was concentrated to 10 XPBST buffer containing 0.5% Tween-20.

The invention also provides a detection method of the lung cancer related autoantibody, which is characterized by comprising the following steps: the kit is used for detecting the lung cancer related autoantibody.

The detection method provided by the invention is also characterized by comprising the following steps: adding the diluted blood samples to the respective antigen protein-immobilized wells to obtain respective detection sample ODs of different antigen proteins corresponding to each blood sample₆₅₀Value, adding diluted blood samples to the wells of the immobilized empty carrier protein corresponding to the blood samples to obtain respective detection background OD corresponding to the respective blood samples₆₅₀Adding each of the detection standards at the corresponding concentration to each well of the immobilized empty carrier protein corresponding to each of the detection standards to obtain a detection standard OD corresponding to each of the concentrations of the standard₆₅₀Adding the corresponding high concentration quality control substance into the hole for fixing the empty carrier protein corresponding to the high concentration quality control substance to obtain OD of the high concentration quality control substance₆₅₀And adding the corresponding low concentration quality control substance to the hole of the immobilized empty carrier protein corresponding to the low concentration quality control substance to obtain the OD of the low concentration quality control substance₆₅₀A value; taking the logarithm of the concentration value of each detection standard substance with 10 as the base as the abscissa, and each detection standard OD₆₅₀Drawing a logical curve with the value of the ordinate and calculating a regression equation of the standard curve; detection OD for detection of respective antigen proteins in the same serum sample₆₅₀Subtracting the detection background OD corresponding to the serum sample from the values₆₅₀Obtaining the absolute OD of the blood sample corresponding to different antigen proteins₆₅₀Value, individual absolute OD₆₅₀Substituting the values into a standard curve regression equation to calculate the autoantibody relative concentration value aiming at the antigen protein in the blood sample.

The detection method provided by the present invention is also characterized in that the blood sample is diluted 1:100 times with the sample and the antibody diluent.

The detection method provided by the present invention is also characterized in that the total of 6 detection standards with different concentrations are detection standard 1, detection standard 2, detection standard 3, detection standard 4, detection standard 5, and detection standard 6, and the detection standards with different concentrations are obtained as follows: taking a standard substance in the kit as a detection standard substance 1, and carrying out gradient dilution on the detection standard substance by adopting a sample and an antibody diluent to respectively obtain detection standard substances 2-6, wherein the concentrations of the detection standard substances 2-6 are respectively as follows: 0.1111. mu.g/mL, 0.03704. mu.g/mL, 0.01235. mu.g/mL, 0.004115. mu.g/mL and 0.001372. mu.g/mL.

Action and Effect of the invention

According to the antigen combination, the corresponding kit and the detection method provided by the invention, as the antigen combination consists of any seven antigen proteins of P53, SOX2, GBU4-5, KRAS, P53-95, CK8, HuD, SSX1, P62, EGFR-vIII, P16, CK20, CAGE and NY-ESO-1, the total sensitivity of the detection of the lung cancer related autoantibody can exceed 30 percent, the specificity can exceed 85 percent, and the total is far better than the results of the existing several antigen combinations; moreover, the false positive of the antigen combination consisting of any seven antigen proteins is lower than 15%, the result is more reliable, and the method can be more used for screening and diagnosing early lung cancer.

Drawings

FIG. 1 is a layout diagram of a 96-well microplate in which each of the antigen proteins and the empty carrier protein in example 1 of the present invention is immobilized;

fig. 2 is a logic diagram involved in embodiment 1 of the present invention.

Detailed Description

The following describes embodiments of the present invention with reference to the drawings. For the specific methods or materials used in the embodiments, those skilled in the art can make routine alternatives based on the existing technologies based on the technical idea of the present invention, and not limited to the specific descriptions of the embodiments of the present invention.