CN109884303B - Multiple immunohistochemical analysis kit for liver cancer and use method and application thereof - Google Patents
Multiple immunohistochemical analysis kit for liver cancer and use method and application thereof Download PDFInfo
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Abstract
The invention provides a multiple immunohistochemical analysis kit for liver cancer and a use method and application thereof, relating to the technical field of multiple immunohistochemical analysis, wherein a monoclonal antibody group is limited to comprise a first monoclonal antibody group (panel 1) and a second monoclonal antibody group (panel 2); the first monoclonal antibody group comprises a PanCK monoclonal antibody, a PDL1 monoclonal antibody, a CD8 monoclonal antibody, a CD66B monoclonal antibody and a CD163 monoclonal antibody; the second monoclonal antibody group includes PanCK monoclonal antibody, PDL1 monoclonal antibody, CD8 monoclonal antibody, PD1 monoclonal antibody and CD68 monoclonal antibody. The multiple immunohistochemical analysis kit provided by the invention takes the paraffin section of the in vitro human liver tissue as a sample for detection, so that the effect effectiveness of the immune checkpoint inhibitor on the liver cancer is predicted.
Description
Technical Field
The invention relates to the technical field of multiple immunohistochemistry, in particular to a multiple immunohistochemical analysis kit for liver cancer and a using method and application thereof.
Background
Liver cancer is the second leading cause of cancer-related death worldwide. It is estimated that 78.2 ten thousand new liver cancer cases and 74.5 ten thousand liver cancer death cases worldwide in 2012, of which about 50% occur in china. Hepatocellular carcinoma (HCC), which accounts for 90% of primary liver cancer, can be caused by chronic infection with Hepatitis B Virus (HBV) or Hepatitis C Virus (HCV), alcohol abuse, and metabolic syndrome associated with diabetes and obesity. In the past decade, significant research progress has been made in the molecular pathogenesis of HCC, but current treatment methods remain very limited. Only a few patients with liver cancer are diagnosed at an early stage, and the treatment methods such as surgical resection, transplantation or local ablation are effective for the patients with early stage liver cancer. In advanced patients, the only systemic treatments capable of improving survival are the polytyrosine kinase inhibitors Sorafenib (Sorafenib, first line) and Regorafenib (Regorafenib, second line), although the average life expectancy of patients is less than 2 years.
In recent years, immune checkpoint inhibitors release the body's own immune response to attack tumors by targeting regulatory pathways of T cells, showing significant efficacy in different solid tumors. 8 immune checkpoint inhibitors, such as Ipilimumab (anti-CTLA-4), Nivolumab (anti-PD1), Pembrolizumab (anti-PD1), have been approved by regulatory agencies, altering the prospects for cancer therapy. The prognosis of liver cancer patients is generally poor, and the 5-year survival rate of symptomatic liver cancer patients is less than 5%. Furthermore, these tumors have proven to be rather resistant to radiotherapy and chemotherapy, and immunotherapy is an attractive option in hepatocellular carcinoma. Based on the CheckMate-040 study, Nivolumab, an anti-programmed death receptor 1(PD1) monoclonal antibody, was approved by FDA in 2017 with accelerated approval for the treatment of advanced HCC patients who had received sorafenib treatment. The objective remission rate of the patient receiving the Nivolumab treatment was 20%, the disease control rate was 64%, the duration of remission reached 17 months, progression-free survival was 4 months, median total survival had not been reached, median total survival in the dose escalation group was 15 months, and at the same time the safety was good. New experimental data indicate that the presence of intratumoral T cell infiltration, Interferon (IFN) signaling, checkpoint molecules (PD1, PDL1 expression) or high tumor mutational burden may be beneficial for clinical remission. Unfortunately, little is known about the immunological features of hepatoma tumors and how to use this information to maximize the response rate to immunotherapy. Therefore, the detection of immune cell subsets and immune checkpoint molecules in the microenvironment of liver cancer tumors is very beneficial for predicting whether the application of immune checkpoint inhibitor drugs to patients is effective.
The principle behind the color development of conventional immunohistochemistry with diaminobenzidine (i.e., 3, -diaminobenzidine, DAB) is that diaminobenzidine is a chromogenic substrate for peroxidase and exhibits accumulation of color change upon electron loss in the presence of hydrogen peroxide, forming a tan insoluble product. DAB is mainly combined with NH2 or SH groups of proteins to form stable nn bonds or ns bonds, and then the chromogenic groups in DAB can display colors and mark the exposed proteins so as to display the protein distribution, the protein types and the like of target cells. The prior art typically labels a molecule on a slide. Although there are some currently multi-labeled immunohistochemical staining methods, these multi-labeled immunohistochemical staining methods may affect the staining formed in the previous round during elution, often require the addition of a staining enhancer, etc., and the detection result is not accurate enough.
Disclosure of Invention
The invention aims to provide a multiple immunohistochemical analysis kit for liver cancer, a using method and application thereof, the kit can effectively predict the effectiveness of an immune checkpoint inhibitor, and has the advantages of high sensitivity and good specificity.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a multiple immunohistochemical analysis kit for liver cancer, which comprises a monoclonal antibody group, an antigen retrieval solution, a horseradish peroxidase-labeled secondary antibody, hydrogen peroxide, a nuclear staining agent and a fluorescent group-labeled tyrosine salt, wherein the number of types of fluorescent groups in the fluorescent group-labeled tyrosine salt is consistent with the number of types of monoclonal antibodies in the monoclonal antibody group;
the monoclonal antibody groups include a first monoclonal antibody group and a second monoclonal antibody group; the first monoclonal antibody group comprises a PanCK monoclonal antibody, a PDL1 monoclonal antibody, a CD8 monoclonal antibody, a CD66B monoclonal antibody and a CD163 monoclonal antibody; the second monoclonal antibody group includes PanCK monoclonal antibody, PDL1 monoclonal antibody, CD8 monoclonal antibody, PD1 monoclonal antibody and CD68 monoclonal antibody.
Preferably, the kit further comprises an anti-quenching agent, a detergent and a blocking solution.
Preferably, the washing solution is TBST buffer.
Preferably, the nuclear stain is a DAPI stain.
Preferably, the fluorescent groups include 520-FITC, 570-Cy3, 620-Cy3.5, 650-Cy5, and 690-Cy5.5.
The invention also provides a use method of the multiple immunohistochemical analysis kit for the liver cancer, which comprises the following steps:
(1) mixing a sample to be detected with any monoclonal antibody in the monoclonal antibody group, incubating and washing to obtain an antigen-primary antibody compound;
(2) mixing the antigen-primary antibody complex with a secondary antibody marked by horseradish peroxidase, incubating and washing to obtain an antigen-primary antibody-secondary antibody complex;
(3) mixing the antigen-primary anti-secondary antibody compound, any one of fluorescent group labeled tyrosine salt and hydrogen peroxide for fluorescent staining, incubating and washing to obtain a first fluorescent labeled compound;
(4) mixing the first fluorescence labeling compound with an antigen repairing solution, performing microwave treatment, and washing to obtain a second fluorescence labeling compound;
the microwave treatment conditions include: treating for 1-3 min at 750-850 w, and treating for 12-20 min at 200-300 w;
(5) repeating the steps (1) to (4) by taking the second fluorescence labeling compound as a sample to be detected until all the monoclonal antibodies in the monoclonal antibody group are combined with the sample to be detected once, so as to obtain a multiple labeling compound;
wherein, the monoclonal antibodies adopted for the first time and each time in the step (1) are different from each other, and the tyrosine salts marked by the fluorescent groups adopted for the first time and each time in the step (3) are different from each other;
(6) and (3) adding a nuclear stain into the multi-labeled compound obtained in the step (5), incubating, washing, sealing, imaging by continuous spectrum and detecting.
Preferably, in the step (1), when the incubated monoclonal antibody is a CD66B monoclonal antibody, a PanCK monoclonal antibody, a CD163 monoclonal antibody, a CD8 monoclonal antibody, a PD1 monoclonal antibody or a CD68 monoclonal antibody, the incubation temperature is 35-42 ℃; when the incubated monoclonal antibody is a PDL1 monoclonal antibody, the incubation temperature is 2-6 ℃; the incubation sequence of the antibodies in the first monoclonal antibody group is as follows: CD66B monoclonal antibody, PanCK monoclonal antibody, PDL1 monoclonal antibody, CD163 monoclonal antibody, CD8 monoclonal antibody; the incubation sequence of the antibodies in the second monoclonal antibody group is as follows: PD-1 monoclonal antibody, PanCK monoclonal antibody, PDL1 monoclonal antibody, CD8 monoclonal antibody and CD68 monoclonal antibody.
Preferably, the dilution factor of the monoclonal antibody in the step (1) is 50-500 times; in the step (2), the dosage of the horseradish peroxidase-labeled secondary antibody is 50-200 mu L per sample.
The invention also provides application of the multiple immunohistochemical analysis kit for liver cancer in the scheme in predicting the effectiveness of the immune checkpoint inhibitor on liver cancer treatment, and the application is characterized by comprising the following steps:
s1, detecting CD8 in a sample to be detected by using the kit in the scheme+PDL1-，CD8+PD1-，CD68+PD1-，CD68+PDL1-，CD68+And CD8+The number of (2);
s2, calculating CD68 from the measurement result of S1+/CD8+The ratio of (A) to (B);
s3, according to the results of the measurement and calculation of S1 and S2, when the CD8 in the sample to be tested is+PDL1-＞2.14、CD8+PD1-When the sample is more than 2.83, judging that the treatment effectiveness of the immune checkpoint inhibitor on the patient from which the sample to be detected is obtained is high;
or when CD68 is present in the sample to be tested+/CD8+＜5.49、CD68+PD1-< 8.15 and CD68+PDL1-When the number is less than 8.15, the effectiveness of the immune checkpoint inhibitor in treating the patient from which the sample to be detected is obtained is judged to be high.
The invention has the following beneficial effects:
the invention provides a multiple immunohistochemical analysis kit for liver cancer, wherein a monoclonal antibody group is limited to comprise a first monoclonal antibody group and a second monoclonal antibody group; the first monoclonal antibody group comprises a PanCK monoclonal antibody, a PDL1 monoclonal antibody, a CD8 monoclonal antibody, a CD66B monoclonal antibody and a CD163 monoclonal antibody; the second monoclonal antibody group includes PanCK monoclonal antibody, PDL1 monoclonal antibody, CD8 monoclonal antibody, PD1 monoclonal antibody and CD68 monoclonal antibody. The multiple immunohistochemical analysis kit provided by the invention uses the paraffin section of the isolated human liver tissue as a sample for detection, can simultaneously mark a plurality of immune cell markers, tumor cell markers and immune related molecules in the same tissue sample, and utilizes the kit to detect CD8 in a sample to be detected+PDL1-，CD8+PD1-，CD68+PD1-，CD68+PDL1-And CD68+，CD8+And calculates the CD68+/CD8+The ratio of (A) to (B); the higher the probability that an immune checkpoint inhibitor will achieve a good therapeutic effect in the patient from whom the test sample is derived, is judged by whether each index is above or below a threshold.
The invention also provides a use method of the multiple immunohistochemical analysis kit for liver cancer, which comprises the following steps of firstly, marking a sample to be detected by using one monoclonal antibody in a monoclonal antibody group to form an antigen-anti compound; binding a secondary antibody labeled by horseradish peroxidase (HRP) with the primary antibody to form an antigen-primary antibody-secondary antibody complex; after adding hydrogen peroxide and one of the fluorescent group labeled tyrosine salts, in the presence of hydrogen peroxide, the fluorescent group labeled tyrosine salt forms an enzymatic product (with a fluorescent group) containing a covalent bond bonding site under the catalysis of HRP, and is combined with surrounding protein residues (tryptophan, histidine, tyrosine salt residues and the like) on antigen, primary antibody and secondary antibody, so that a large amount of the fluorescent group labeled enzymatic product is gathered at the antigen-primary antibody bonding site, and the more the antigen (primary antibody recognizes an immune check point), the more the enzymatic product is combined, the more the fluorescent group is contained, and the stronger the detection signal is.
Carrying out microwave treatment on the fluorescent label obtained by enzymatic reaction to separate the combination of the antigen and the primary antibody (the monoclonal antibody in the monoclonal antibody group), and removing the monoclonal antibody after elution; the enzyme-catalyzed product is combined with the antigen by a covalent bond, so that the microwave treatment has little influence on the combination of the enzyme-catalyzed product and the antigen, and the intensity of a fluorescence signal marked on the antigen cannot be influenced, thereby realizing the purpose of thoroughly removing the antibody in the previous round without losing the fluorescence marking signal in the previous round and causing no interference on the marking of the monoclonal antibody in the next round. Different monoclonal antibodies and different fluorescent group labeled casamino salts are adopted and repeated according to the method, so that a plurality of molecules can be labeled on the same sample to be detected, cross reaction is not needed to be worried about, the detection result is accurate, and at most, six different immune markers can be labeled simultaneously. And dyeing and sealing the multi-marked sample to be detected, and detecting the concentration of the immune marker in the sample to be detected by utilizing multispectral imaging.
Therefore, the use method provided by the invention can realize the marking of a plurality of molecules on the same sample to be detected, eliminates the interference of the polyclonal antibody in the previous round, and has high detection accuracy and high detection efficiency.
Drawings
FIG. 1 is a case display diagram showing the good and bad effects of immunotherapy in the panel1 group in example 1; wherein, FIG. 1A is the case spectrum of poor curative effect of the panel1 group immunotherapy, and FIG. 1B is the case spectrum of good curative effect of the panel1 group immunotherapy;
FIG. 2 is a case display diagram showing the good and bad effects of immunotherapy in the panel2 group in example 1; wherein, FIG. 2A is the case spectrum of poor curative effect of the panel2 group immunotherapy, and FIG. 2B is the case spectrum of good curative effect of the panel2 group immunotherapy;
FIG. 3 is a diagram of an A-Factorrimport forest;
FIG. 4 is a ROC graph.
Detailed Description
The invention provides a multiple immunohistochemical analysis kit for liver cancer, which comprises a monoclonal antibody group, an antigen retrieval solution, a horseradish peroxidase-labeled secondary antibody, hydrogen peroxide, a nuclear staining agent and a fluorescent group-labeled tyrosine salt, wherein the number of types of fluorescent groups in the fluorescent group-labeled tyrosine salt is consistent with the number of types of monoclonal antibodies in the monoclonal antibody group;
the monoclonal antibody groups include a first monoclonal antibody group and a second monoclonal antibody group; the first monoclonal antibody group comprises a PanCK monoclonal antibody, a PDL1 monoclonal antibody, a CD8 monoclonal antibody, a CD66B monoclonal antibody and a CD163 monoclonal antibody; the second monoclonal antibody group includes PanCK monoclonal antibody, PDL1 monoclonal antibody, CD8 monoclonal antibody, PD1 monoclonal antibody and CD68 monoclonal antibody.
In the present invention, the kit further comprises an anti-quenching agent, a detergent and a blocking solution.
In the present invention, each monoclonal antibody in the monoclonal antibody panel is used to separately recognize an immune checkpoint to be detected. In the invention, the multiple immunohistochemical analysis kit has the advantages of strong specificity and high sensitivity.
In the present invention, the monoclonal antibodies in the monoclonal antibody panel are preferably murine monoclonal antibodies and/or rabbit monoclonal antibodies; in the specific embodiment of the invention, the secondary antibodies used by PanCK, CD163, PD-1 and CD68 are PV-6002 (enzyme-labeled goat anti-mouse IgG polymer) produced by China fir Jinqiao; the secondary antibodies used by the CD66B, the PDL1 and the CD8 are PV-6001 (enzyme-labeled goat anti-rabbit IgG polymer) produced by China fir Jinqiao, the preparation method of various monoclonal antibodies in the monoclonal antibody group is not particularly limited, and the monoclonal antibodies can be prepared by commercial products or conventional methods.
In the invention, the antigen repairing liquid is used for repairing antigen and preventing complete marking during immunohistochemical staining. The source of the antigen retrieval solution is not particularly limited in the invention, and any commercially available product or known formula in the field can be adopted, and in the specific embodiment of the invention, Opal7-colorManual IHC Kit produced by Perkin Elmer is adopted as the antigen retrieval solution.
In the invention, a secondary antibody marked by horseradish peroxidase is used for being combined with the monoclonal antibody in the monoclonal antibody group, wherein the horseradish peroxidase catalyzes a fluorescent group marked tyrosine salt to generate an enzymatic product with a covalent binding site under the condition that hydrogen peroxide exists, and the enzymatic product can be combined with a protein residue in a sample to be detected, so that the fluorescent group is marked on the sample to be detected. The source of the secondary antibody marked by the horseradish peroxidase is not specially limited, and the secondary antibody can be obtained by adopting a commercial product.
In the present invention, the hydrogen peroxide provides conditions for an enzymatic reaction of horseradish peroxidase. The source of the hydrogen peroxide is not particularly limited in the present invention.
In the invention, the tyrosine salt marked by the fluorescent group is used as a reaction substrate, so that an enzymatic reaction is generated, an enzymatic product is combined with a protein residue in an antigen, the fluorescent group carried by the enzymatic product is marked on a sample to be detected, and the number of the marked fluorescent group is in direct proportion to the number of the markers recognized by the adopted primary antibody, so that the quantitative detection of the antigen to be recognized is realized. Preferably, the fluorescent groups comprise 520-FITC, 570-Cy3, 620-Cy3.5, 650-Cy5 and 690-Cy5.5.
In the present invention, the nuclear stain is preferably a DAPI stain. In the present invention, the nuclear stain is used to label the nucleus of a cell.
In the present invention, the kit further comprises an anti-quenching agent which plays a role in anti-fluorescence decay and prevention of fluorescence quenching. The type of the anti-quenching agent is not particularly limited in the invention, and a commercially available product in the field can be adopted. In a specific embodiment of the invention, the anti-quenching agent is preferably a Boble Ryder anti-fluorescence decay blocking tablet.
In the invention, the sealing liquid is used for sealing the antigen, so that the detection accuracy is improved. The type of the blocking solution is not particularly limited, and in the specific embodiment of the invention, an Antibody Diluen/Block produced by Perkin Elmer is used as the blocking solution.
In the present invention, the detergent is used for the washing step in the detection process, and the detergent for the present invention is preferably a TBST buffer.
The multiple immunohistochemical liver cancer analysis kit provided by the invention is designed aiming at the inhibition condition of the immune check point of the liver cancer, provides a corresponding monoclonal antibody group for identification, and has strong specificity and high sensitivity.
The invention also provides a use method of the multiple immunohistochemical analysis kit for the liver cancer, which comprises the following steps:
(1) mixing a sample to be detected with any monoclonal antibody in the monoclonal antibody group, incubating and washing to obtain an antigen-primary antibody compound;
(2) mixing the antigen-primary antibody complex with a secondary antibody marked by horseradish peroxidase, incubating and washing to obtain an antigen-primary antibody-secondary antibody complex;
(3) mixing the antigen-primary anti-secondary antibody compound, any one of fluorescent group labeled tyrosine salt and hydrogen peroxide for fluorescent staining, incubating and washing to obtain a first fluorescent labeled compound;
(4) mixing the first fluorescence labeling compound with an antigen repairing solution, performing microwave treatment, and washing to obtain a second fluorescence labeling compound;
the microwave treatment conditions include: treating for 1-3 min at 750-850 w, and treating for 12-20 min at 200-300 w;
(5) repeating the steps (1) to (4) by taking the second fluorescence labeling compound as a sample to be detected until all the monoclonal antibodies in the monoclonal antibody group are combined with the sample to be detected once, so as to obtain a multiple labeling compound;
wherein, the monoclonal antibodies adopted for the first time and each time in the step (1) are different from each other, and the tyrosine salts marked by the fluorescent groups adopted for the first time and each time in the step (3) are different from each other;
(6) and (3) adding a nuclear stain into the multi-labeled compound obtained in the step (5), incubating, washing, sealing, imaging by continuous spectrum and detecting.
In the present invention, the sample to be tested is preferably a tissue section of liver cancer, and more preferably, the tissue section is a paraffin section. In the invention, when the sample to be detected is a liver cancer tissue paraffin section, the sample to be detected is dewaxed and hydrated before being mixed with the monoclonal antibody, and the method specifically comprises the following steps:
A. baking the paraffin sections of the liver cancer tissues at 50-70 ℃ for 100-150 min to obtain baked sections;
B. and sequentially extracting the baked slices twice by using dimethylbenzene, extracting the slices twice by using absolute ethyl alcohol, extracting the slices once by using an ethanol solution with the mass fraction of 95%, extracting the slices once by using an ethanol solution with the mass fraction of 90%, extracting the slices once by using an ethanol solution with the mass fraction of 85%, extracting the slices once by using an ethanol solution with the mass fraction of 80% and extracting the slices once by using an ethanol solution with the mass fraction of 75%, so as to obtain the dewaxed and hydrated liver cancer tissue as a sample to be detected.
Preferably, the time for extracting the xylene in the step B is 8-12 min each time; the time for each extraction of the absolute ethyl alcohol is preferably 3-8 min; the extraction time of the 95% ethanol solution, the 90% ethanol solution, the 85% ethanol solution, the 80% ethanol solution and the 75% ethanol solution is preferably 3-8 min.
In the invention, if the multiple immunohistochemical analysis kit further comprises a blocking solution, the sample to be detected is blocked by the blocking solution and then mixed with the monoclonal antibody, wherein the dosage of the blocking solution is preferably 50-150 muL/sample, and more preferably 100 muL/sample. In the invention, the sealing time of the sealing liquid is preferably 8-15 min, and more preferably 10 min.
The invention mixes the sample to be tested with any monoclonal antibody in the monoclonal antibody group, incubates and washes to obtain the antigen-primary antibody compound. Preferably, in order to further improve the prediction accuracy of the kit, the incubation sequence of the antibodies in the first monoclonal antibody group in the monoclonal antibody group of the invention is as follows: CD66B monoclonal antibody, PanCK monoclonal antibody, PDL1 monoclonal antibody, CD163 monoclonal antibody, CD8 monoclonal antibody; the incubation sequence of the antibodies in the second monoclonal antibody group in the monoclonal antibody group of the invention is as follows in sequence: PD-1 monoclonal antibody, PanCK monoclonal antibody, PDL1 monoclonal antibody, CD8 monoclonal antibody and CD68 monoclonal antibody.
In the present invention, when the monoclonal antibody to be incubated is a CD66B monoclonal antibody, a PanCK monoclonal antibody, a CD163 monoclonal antibody, a CD8 monoclonal antibody, a PD1 monoclonal antibody or a CD68 monoclonal antibody, the incubation temperature is preferably 35 to 42 ℃, more preferably 37 ℃. In the present invention, the incubation time of the monoclonal antibody is preferably 1 hour.
In the invention, when the incubated monoclonal antibody is PDL1 monoclonal antibody, the incubation temperature is preferably 2-6 ℃, more preferably 3-5 ℃. In the present invention, the incubation time of the monoclonal antibody is preferably 12 to 16 hours.
In the invention, the monoclonal antibody is a purchased commercial monoclonal antibody stock solution, and is diluted by 50-500 times for use; in the present invention, it is preferable to use an antibody dilution/blocking solution (opal)TMKit) to dilute the monoclonal antibody. In the invention, the dosage of the monoclonal antibody is 100-300 mu L per sample.
In the present invention, when a washing solution is included in the multiplexed immunoassay kit, the washing is preferably washing with a washing solution; in the present invention, the washing is preferably repeated 2 to 3 times. In the present invention, the washing time is preferably 3 to 10min, and more preferably 5min each time. The washing steps in the following steps of the present invention are the same and will not be described again.
After the antigen-primary antibody complex is obtained, the antigen-primary antibody complex and a horseradish peroxidase-labeled secondary antibody are mixed, incubated and washed to obtain the antigen-primary antibody-secondary antibody complex.
In the invention, the horseradish peroxidase-labeled secondary antibody is preferably an HRP-labeled goat anti-rabbit IgG polymer or an enzyme-labeled goat anti-mouse IgG polymer. In the present invention, the horseradish peroxidase-labeled secondary antibody is a commercially available commercial product. In the invention, the use amount of the horseradish peroxidase-labeled secondary antibody is preferably 50-200 mu L/sample, and more preferably 100 mu L/sample.
In the invention, the incubation time of the horseradish peroxidase-labeled secondary antibody is preferably 8-15 min, and more preferably 10 min. In the invention, the incubation temperature is preferably 35-38 ℃, and more preferably 37 ℃.
After the antigen-primary anti-secondary antibody compound is obtained, the antigen-primary anti-secondary antibody compound, any one of the fluorescent group marked tyrosine salt and hydrogen peroxide are mixed for fluorescent staining, incubation and washing to obtain a first fluorescent marked compound.
In the present invention, the hydrogen peroxide is preferably provided in the form of a signal amplification solution, which is a commercially available product in which hydrogen peroxide is mixed. In the present invention, the commercially available signal amplification solution is preferably diluted 80 to 120 times, and more preferably 100 times. In the present invention, the fluorophore-labeled tyrosine salt is preferably commercially available, and in a specific embodiment of the present invention is a fluorophore-labeled tyrosine salt in a commercially available Opal (TM) 7-color fluorescent staining kit; in the invention, preferably, the tyrosine salt marked by each fluorescent group is diluted by 50-150 times and then used, and more preferably diluted by 100 times. Preferably, the using amount of the fluorescent group marked tyrosine salt is preferably 80-150 mu L/sample of the diluted solution, and more preferably 100 mu L/sample.
In the invention, the incubation time is preferably 8-15 min, and more preferably 10 min. In the invention, the incubation temperature is preferably 35-38 ℃, and more preferably 37 ℃.
After the first fluorescence labeling compound is obtained, the first fluorescence labeling compound is mixed with the antigen repairing solution, and then the mixture is subjected to microwave treatment and washing to obtain a second fluorescence labeling compound; the microwave treatment conditions include: treating at 750-850 w for 1-3 min, and treating at 200-300 w for 12-20 min. In the present invention, the microwave treatment conditions preferably include: preheating at 80w for 5min, treating at 800w for 2min, and treating at 240w for 15 min.
In the present invention, the antigen retrieval solution is commercially available from Opal, which is used in the specific examples of the present inventionTM7-color fluorescent staining of the antigen retrieval solution in the kit; the dosage of the antigen retrieval liquid is preferably 150-300 mL/sample, and more preferably 200 mL/sample.
In the invention, the first fluorescence labeling compound is dissociated with the monoclonal antibody combined with the antigen under the microwave treatment, and the dissociated monoclonal antibody and the secondary HRP labeled antibody can be removed through washing, so that the next reaction can not be influenced, and the mutual interference during multiple labeling can be prevented.
After the second fluorescence labeling compound is obtained, the invention takes the obtained second fluorescence labeling compound as a sample to be detected, repeats the step of identifying the monoclonal antibody to obtain the fluorescence labeling compound, and repeats until all the monoclonal antibodies in the monoclonal antibody group are combined with the sample to be detected once, so as to obtain the multiple labeling compound.
In the repeating process of the invention, the monoclonal antibody adopted in each round of repetition is different from the monoclonal antibody adopted in any other round of repetition; accordingly, the fluorophore in the caseinate labeled with the fluorophore used in each repetition cycle is also different from the fluorophore used in any of the other cycles. Thereby realizing the purpose of respectively labeling different monoclonal antibodies by adopting different fluorescent groups.
After obtaining the multiple-labeled compound, the invention adds a nuclear staining agent into the multiple-labeled compound for incubation, washing, sealing, continuous spectrum imaging, image processing and observation and analysis.
In the invention, the addition amount of the nuclear staining agent is preferably 80-150 mu L/sample, and more preferably 100 mu L/sample.
In the invention, the incubation time is preferably 5-10 min, and more preferably 8 min.
In the present invention, when the multiplex immunoassay kit further comprises an anti-quencher, the anti-quencher is added after the incubation and washing, and then a cover slip is added for mounting.
In the invention, the multiple immunohistochemical analysis kit can meet the requirement that the existing histological spectral imaging instrument simultaneously images a plurality of molecules, namely multispectral imaging.
Multispectral imaging relies on two processes of spectral data acquisition and spectral splitting computation:
collecting spectral data: there are many technical means for multispectral data collection, such as grating spectroscopy, prism spectroscopy, liquid crystal tunable filter spectroscopy, etc. The spectral signals of specific wave bands are collected by filtering with a Vectra system (liquid crystal tunable filter, LCTF) of Perkinelmer company. The LCTF is made of liquid crystal material, changes the optical path of light in the crystal by adjusting additional voltage, selectively outputs optical signals with specific wavelength, and achieves the purpose of light splitting. The CCD exposure is matched with the continuous filtering of the LCTF, so that the image signals of different wavelength bands can be accurately recorded.
Spectrum splitting: each pixel point signal of the spectral image is the superposition of different fluorescent dyes and a sample spontaneous signal, the spectral characteristic curve of each dye is taken as a standard, and reduction operation is carried out on the superposed signals in the spectral image by a mathematical method, so that the process of obtaining a single-channel image is called spectral splitting calculation. The spectrum splitting calculation is an indispensable important link of the whole spectrum imaging, and the accuracy of a data result is directly influenced.
The 'pure spectrum splitting algorithm' can be used for splitting color signals with up to 10 superposed colors, and the 'pure' dye signals hidden in the spectrum image are accurately resolved to obtain the specific distribution of each dye in the image. And the real target signal can be extracted from the autofluorescence background to obtain an image with ultrahigh signal-to-noise ratio, so that the weakly expressed fluorescence signal can be shown from the background.
The analysis software was able to co-localize at least 4 molecules at the same coordinate position on the tissue and check if any antigenic molecules were expressed on one cell at the same time.
The invention also provides application of the multiple immunohistochemical analysis kit for liver cancer in the scheme in predicting the effectiveness of the immune checkpoint inhibitor on liver cancer treatment, and the application is characterized by comprising the following steps:
s1, detecting CD8 in a sample to be detected by using the kit in the scheme+PDL1-，CD8+PD1-，CD68+PD1-，CD68+PDL1-，CD68+And CD8+The number of (2);
s2, calculating CD68 from the measurement result of S1+/CD8+The ratio of (A) to (B);
s3, according to the results of the measurement and calculation of S1 and S2, when the CD8 in the sample to be tested is+PDL1-> 2.14 and CD8+PD1-When the sample is more than 2.83, judging that the treatment effectiveness of the immune checkpoint inhibitor on the patient from which the sample to be detected is obtained is high;
or when CD68 is present in the sample to be tested+/CD8+＜5.49、CD68+PD1- < 8.15 and CD68+PDL1-When the number is less than 8.15, the effectiveness of the immune checkpoint inhibitor in treating the patient from which the sample to be detected is obtained is judged to be high.
In the invention, when the multi-grouping immunoassay kit is used for predicting the effectiveness of the immune checkpoint inhibitor, the paraffin section of the human isolated liver tissue is used as a sample to be detected.
Specifically, the present invention uses paraffin sections of isolated human liver tissues as samples, and the multiplex immunohistochemical assay kit according to the above technical scheme is used for detection according to the method shown in the above technical scheme, so as to obtain the contents of PanCK (Pan-cytokine, broad-spectrum cytokeratin), PDL1 (immune checkpoint molecule), CD66B (neutrophil marker), CD163 (M2-type macrophage marker) and CD8(T cell marker) in the sample to be detected, or to obtain the contents of PanCK, PDL1, PD-1 (immune checkpoint molecule), CD68 (macrophage marker) and CD8 in the sample to be detected.
Calculating CD68 from the above measurement results+/CD8+The ratio of (A) to (B);
according to the above determination and calculation results, CD8 in the sample to be tested+PDL1-> 2.14, and CD8+PD1-When the sample is more than 2.83, judging that the treatment effectiveness of the immune checkpoint inhibitor on the patient from which the sample to be detected is obtained is high;
or when CD68 is present in the sample to be tested+/CD8+＜5.49、CD68+PD1-< 8.15 and CD68+PDL1-When the number is less than 8.15, the effectiveness of the immune checkpoint inhibitor in treating the patient from which the sample to be detected is obtained is judged to be high. If the above conditions are not met, it is determined that the effectiveness of the immune checkpoint inhibitor in treating the patient from which the sample to be tested is derived is low.
The multiple immunohistochemical analysis kit provided by the invention has strong specificity and high sensitivity on the effectiveness prediction of the immune checkpoint inhibitor, and provides conditions for realizing accurate medication.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
20 patients with liver cancer were selected from 10 patients with poor tumor tissue immunotherapy efficacy (Overall Survival (OS) is shorter (nos. 1-10)) and good immunotherapy efficacy (OS is longer (nos. 11-20)), and the specific Survival is shown in table 1.
Survival time of 120 patients with liver cancer
Multiplex immunohistochemical analysis of individual proteins in the tumor microenvironment was performed as follows. 2 sections (panel 1 and panel 2) per patient, each labeled with 5 molecules. The dyeing sequence is as follows: panel 1: CD66B, PanCK, PDL1, CD163, CD 8; panel2 PD-1, PanCK, PDL1, CD8, CD 68. Information of primary antibody, secondary antibody and fluorescent dye in the experimental process is shown in table 2:
TABLE 2 Primary antibody, Secondary antibody and fluorescent dye information
The operation steps of the embodiment are as follows:
1. placing 20 paraffin sections in a constant temperature oven at 60 deg.C, and baking for 120 min;
2. dewaxing and hydrating: xylene (10min) → absolute ethanol (5min × 2 → 95% ethanol (5min × 2) → 90% ethanol (5min) → 85% ethanol (5min) → 80% ethanol (5min) → 75% ethanol (5 min);
3. washing with distilled water for 2 times (5 min/time);
4. antigen retrieval: performing microwave repairing with antigen repairing solution in Opal TM 7-color fluorescent staining Kit (Opal 7-color Manual IHC Kit, PerkinElmer), preheating at 80w for 5min, preheating at 800w for 2min, and preheating at 240w for 15min (microwave oven M1-231A);
5. naturally cooling at room temperature for 15 min;
6. washing: washing with TBST buffer solution for 3 times (5 min/time);
7. and (3) sealing: blocking with blocking solution (Perkin Elmer; trade name Antibody Diluent/Block) at room temperature for 10 min;
8. primary antibody incubation: dropwise adding primary antibody (100-300 mu L of primary antibody working solution), and incubating at 37 ℃ for 1h under the incubation conditions of CD66B, PanCK, CD163, CD8, CD68 and PD 1; the incubation conditions for PD-L1 were 4 ℃ overnight; the staining of the primary antibody was divided into two panels, in the order of staining: panel 1: CD66B, PanCK, PDL1, CD163, CD 8; panel2 PD-1, PanCK, PDL1, CD8, CD 68;
9. washing: washing with TBST buffer solution for 3 times (5 min/time);
10. and (3) secondary antibody incubation: dripping the secondary antibody in the kit, and incubating for 10min at 37 ℃; PanCK, CD163, PD-1, CD 68; the secondary antibody used is PV-6002 (enzyme-labeled goat anti-mouse IgG polymer) produced by China fir Jinqiao; the secondary antibodies used for CD66B, PDL1 and CD8 are PV-6001 (enzyme-labeled goat anti-rabbit IgG polymer) produced by China fir Jinqiao;
11. washing: washing with TBST buffer solution for 3 times (5 min/time);
12. fluorescence development: dripping opal fluorescent staining after 100 times of TSA dilution, and keeping the temperature for 10 min;
13. washing: washing with TBST buffer solution for 3 times (5 min/time);
14. antibody staining in sequence: after the first antibody is dyed, repeating the steps 4 to 13 for each subsequent antibody, and sequentially marking all the antibodies; the antibody staining sequence was: panel 1: CD66B, PanCK, PDL1, CD163, CD 8; panel2 PD-1, PanCK, PDL1, CD8, CD 68;
15. microwave treatment: repeating the steps 4) to 6);
dyeing with DAPI for 5-10 min at room temperature;
17. washing: TBST washing for 3 times, 5 min/time;
18. sealing: anti-quencher seals (Boble Ryder anti-fluorescence decay seals);
19. continuous spectral imaging, image processing and observational analysis
According to the above operation steps, multiple labeling of immune cell markers and immune related molecules of 20 liver cancer tumor tissue sections is completed. Continuous spectrum acquisition was performed using the Vectra system from PerkinElmer, Inc., and image processing and observation analysis were performed. The cases with poor and good immunotherapy efficacy showed 1 case each, each showing 1 picture, as shown in fig. 1(a/B) and fig. 2(a/B), and different colors in fig. 1 represent different markers, as shown in table 3 and table 4.
TABLE 3 color of the panel1 marker shown in FIG. 1
| Marker substance | PanCK | CD8 | PDL1 | CD66B | CD163 |
| Colour(s) | Green colour | Purplish red color | Orange colour | Watery red color | Blue green color |
TABLE 4 color of the panel2 marker shown in FIG. 2
| Marker substance | PanCK | CD8 | PDL1 | CD68 | PD1 |
| Colour(s) | Green colour | Purplish red color | Orange colour | Blue green color | Magenta color |
Statistical analysis of each molecule in the tumor region, the statistical results of the percentage of positive detected by 20 patients' immune cells and immune checkpoint molecules are shown in tables 5-8:
TABLE 5 statistics of the percentage of positive expression (%)
TABLE 6 statistics of the percentage of positive expression (%)
| Numbering | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| CD8+ | 0.47 | 2.69 | 2.32 | 7.05 | 3.88 | 14.85 | 2.14 | 0.34 | 0.14 | 0.22 |
| PDL1+ | 0.05 | 0.01 | 0 | 0 | 0.01 | 3.14 | 0.07 | 0 | 0.01 | 0.17 |
| PanCK+ | 0.01 | 40.45 | 0 | 0 | 5.99 | 2.13 | 0.01 | 55.43 | 11.57 | 5.22 |
| CD8+PDL1+ | 0 | 0 | 0 | 0 | 0 | 0.75 | 0 | 0 | 0 | 0 |
| CD8+PDL1- | 0.47 | 2.69 | 2.32 | 7.05 | 3.88 | 14.11 | 2.14 | 0.34 | 0.14 | 0.22 |
| PDL1+PanCK+ | 0 | 0.01 | 0 | 0 | 0 | 0.12 | 0 | 0 | 0.01 | 0 |
| CD66B+ | 0.3 | 1.06 | 0 | 0.66 | 0 | 20.54 | 0.38 | 0.05 | 9.2 | 1.59 |
| CD163+ | 6.05 | 3.83 | 2.56 | 2.6 | 6.28 | 21.2 | 0.13 | 2.87 | 11.82 | 1 |
| CD66B+/CD8+ | 0.85 | 0.59 | 0 | 0.1 | 0 | 1.48 | 0.26 | 0.21 | 51.7 | 93.24 |
| CD163+/CD8+ | 17.01 | 2.13 | 2 | 0.39 | 4.11 | 1.53 | 0.09 | 12.1 | 66.4 | 58.76 |
| PDL1+CD163+ | 0 | 0 | 0 | 0 | 0 | 0.58 | 0 | 0 | 0.03 | 0 |
| PDL1-CD163+ | 6.05 | 3.83 | 2.56 | 2.6 | 6.28 | 20.62 | 0.13 | 2.87 | 11.8 | 1 |
TABLE 7 statistics of the percentage of positive expression (%)
TABLE 8 statistics of the percentage of positive expression (%)
| Numbering | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| CD66B+PDL1- | 0.3 | 1.06 | 0 | 0.66 | 0 | 20.13 | 0.38 | 0.05 | 9.18 | 1.59 |
| CD66B+PDL1+ | 0 | 0 | 0 | 0 | 0 | 0.41 | 0 | 0 | 0.03 | 0 |
| PD1+ | 0 | 0 | 0 | 0 | 0 | 0.21 | 0 | 0 | 0 | 0 |
| CD68+ | 1.44 | 6.74 | 4.7 | 5.97 | 0.92 | 14.82 | 0.08 | 3.65 | 4.52 | 0.39 |
| CD68+/CD8+ | 2.45 | 1.88 | 1.4 | 0.81 | 0.15 | 0.93 | 0.03 | 8.15 | 44.74 | 0.93 |
| CD8+PD1- | 0.59 | 3.58 | 3.36 | 7.35 | 6.23 | 15.84 | 2.84 | 0.45 | 0.1 | 0.42 |
| CD8+PD1+ | 0 | 0 | 0 | 0 | 0 | 0.02 | 0 | 0 | 0 | 0 |
| CD68+PDL1+ | 0 | 0 | 0 | 0 | 0 | 2.8 | 0 | 0 | 0 | 0 |
| CD68+PDL1- | 1.44 | 6.74 | 4.7 | 5.97 | 0.92 | 12.02 | 0.08 | 3.65 | 4.52 | 0.39 |
| CD68+PD1+ | 0 | 0 | 0 | 0 | 0 | 0.08 | 0 | 0 | 0 | 0 |
| CD68+PD1- | 1.44 | 6.74 | 4.7 | 5.97 | 0.92 | 14.73 | 0.08 | 3.65 | 4.52 | 0.39 |
And performing multi-factor analysis on the group with poor immunotherapy curative effect and the group with good immunotherapy curative effect by adopting a random forest algorithm. For each decision tree, the corresponding out-of-bag data is selected to calculate out-of-bag data error, which is noted as errOOB 1. Noise interference is added to the characteristic X of all samples of the data outside the bag randomly, and the error of the data outside the bag is calculated again and is marked as errOOB 2. The importance of feature X ═ Σ (errOOB2-errOOB 1)/k. The determination method of the important factors comprises the following steps: the importance of each feature is calculated and sorted in descending order. The important factor is the characteristic number top 20%.
And (3) drawing an ROC curve: for each tree, the true and false positive rates of the samples were determined. The ROC curve was plotted with the true positive rate (% sensitivity) as ordinate and the false positive rate (% 1-specificity) as abscissa. The results of the multi-factor analysis of the group with poor and better immunotherapy effects: the Factor import forest graph is shown in FIG. 3, and the ROC curve is shown in FIG. 4.
From the multi-factor analysis results, 5 important factors for distinguishing the group with poor immunotherapy curative effect from the group with good immunotherapy curative effect are respectively CD8 in the tumor region+PDL1-，CD8+PD1-，CD68+PD1-，CD68+PDL1-And CD68+/CD8+The ratio of (a) to (b).
In the ROC curve, an orange point indicates that the Youden index at this point is the maximum (Youden index ═ sensitivity + specificity-1), i.e., the best sensitivity and specificity. AUC is 0.89 and P is 0.002, indicating statistical significance. When a subject detected the indices of the 2 panels above and the associated indices and their ratios met or exceeded the corresponding thresholds, the probability of future clinical benefit from immunotherapy for that subject would be much higher than for other non-compliant subjects.
Therefore, the method for simultaneously marking multiple immune cell markers, tumor cell markers and immune checkpoint molecules in the tissue paraffin section by adopting multiple immunohistochemistry is very beneficial to simultaneously predicting whether the application of immune checkpoint inhibitor drugs to multiple patients is effective or not.
And performing single-factor analysis on the group with poor immunotherapy curative effect and the group with good immunotherapy curative effect by adopting a Mann-Whitney test to obtain the threshold value of the characteristic factor. The thresholds for the 5 indices are as follows, see table 9:
table 95 threshold values
When CD8 is in the sample to be tested+PDL1-＞2.14、CD8+PD1-Greater than 2.83, the probability of clinical benefit from immunotherapy is high; when subject is CD68+/CD8+＜5.49、CD68+PD1-< 8.15 and CD68+PDL1-Below 8.15, there is a high probability of clinical benefit from immunotherapy.
Example 2
A multiple immunohistochemical analysis kit for liver cancer comprises a monoclonal antibody group, an antigen repairing solution, a horseradish peroxidase-labeled secondary antibody, hydrogen peroxide, a fluorescent group-labeled tyrosine salt and a nuclear staining agent;
the monoclonal antibody group is as follows: PanCK monoclonal antibody, PDL1 monoclonal antibody, CD8 monoclonal antibody, CD66B monoclonal antibody, and CD163 monoclonal antibody;
the fluorescent groups are 520-FITC, 570-Cy3, 620-Cy3.5, 650-Cy5 and 690-Cy5.5;
the kit also comprises an anti-quenching agent, a detergent and a confining liquid.
Example 3
A multiple immunohistochemical analysis kit for liver cancer comprises a monoclonal antibody group, an antigen repairing solution, a horseradish peroxidase-labeled secondary antibody, hydrogen peroxide, a fluorescent group-labeled tyrosine salt and a nuclear staining agent;
the monoclonal antibody group is as follows: PanCK monoclonal antibody, PDL1 monoclonal antibody, CD8 monoclonal antibody, PD1 monoclonal antibody, and CD68 monoclonal antibody;
the fluorescent groups are 520-FITC, 570-Cy3, 620-Cy3.5, 650-Cy5 and 690-Cy5.5;
the kit also comprises an anti-quenching agent, a detergent and a confining liquid.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (3)
1. A multiple immunohistochemical analysis kit for liver cancer comprises a monoclonal antibody group, an antigen retrieval solution, a horseradish peroxidase-labeled secondary antibody, hydrogen peroxide, a nuclear staining agent and a fluorescent group-labeled tyrosine salt, wherein the number of types of fluorescent groups in the fluorescent group-labeled tyrosine salt is consistent with the number of types of monoclonal antibodies in the monoclonal antibody group;
the monoclonal antibody groups include a first monoclonal antibody group and a second monoclonal antibody group; the first monoclonal antibody group comprises a PanCK monoclonal antibody, a PDL1 monoclonal antibody, a CD8 monoclonal antibody, a CD66B monoclonal antibody and a CD163 monoclonal antibody; the second monoclonal antibody group includes PanCK monoclonal antibody, PDL1 monoclonal antibody, CD8 monoclonal antibody, PD1 monoclonal antibody and CD68 monoclonal antibody;
the nuclear stain is a DAPI stain;
the fluorescent groups comprise 520-FITC, 570-Cy3, 620-Cy3.5, 650-Cy5 and 690-Cy5.5.
2. The kit of claim 1, further comprising an anti-quenching agent, a detergent, and a blocking solution.
3. The kit of claim 2, wherein the detergent is a TBST buffer.
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