CN109884303A - The Multiple immunizations histochemical analysis kit and its application method of a kind of liver cancer and application - Google Patents
The Multiple immunizations histochemical analysis kit and its application method of a kind of liver cancer and application Download PDFInfo
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Abstract
The present invention provides Multiple immunizations histochemical analysis kit and its application method and the application of a kind of liver cancer, it is related to Multiple immunizations group technical field, Multiple immunizations histochemical analysis kit of the present invention includes the first monoclonal antibody group (panel 1) and second monoclonal antibody group (panel 2) therein defining monoclonal antibody group；The first monoclonal antibody group includes PanCK monoclonal antibody, PDL1 monoclonal antibody, CD8 monoclonal antibody, CD66B monoclonal antibody and 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.Multiple immunizations histochemical analysis kit of the present invention is detected using Isolated human liver paraffin section as sample, and then predicts immunologic test point inhibitor to the action effectiveness of liver cancer.
Description
Technical field
The present invention relates to the Multiple immunizations histochemical analysis reagents of Multiple immunizations group technical field more particularly to a kind of liver cancer
Box and its application method and application.
Background technique
Liver cancer is the relevant cause of death of world's second largest cancer.It is estimated that the new hair liver cancer number of cases in the whole world in 2012 reaches
78.2 ten thousand, PLC mortality case 74.5 ten thousand, wherein about 50% occurs in China.Hepatocellular carcinoma (HCC) accounts for primary carcinoma of liver
90%, can by hepatitis type B virus (HBV) or Hepatitis C Virus (HCV) chronic infection, excessive drinking and with diabetes and fertilizer
Fat relevant metabolic syndrome causes.In the past ten years, the Molecular pathogenesis of HCC achieves great progress,
But current treatment method is still very limited.Only a small number of liver cancer patients are diagnosed in early days, treatment method at this time
Such as perform the operation excision, transplanting or local ablation is still effective to early liver cancer patient.Late in patient, survival rate uniquely can be improved
Systemic treatment be more tyrosine kinase inhibitor Sorafenibs (Sorafenib, a line) and Rui Gelafeini
(Regorafenib, two wires), nevertheless, the average life expectancy of patient is also less than 2 years.
In recent years, immunologic test point inhibitor discharges the immune response of body itself by the regulatory pathway of targeting T-cells
Tumour is attacked, shows significant curative effect in different entities tumour.Ipilimumab(anti-CTLA-4),Nivolumab
(anti-PD1), 8 kinds of immunologic test point inhibitor such as Pembrolizumab (anti-PD1) have obtained batch of regulatory agency
Standard changes the prospect for the treatment of of cancer.Liver cancer patient prognosis is generally poor, there is 5 years survival rate < 5% of symptomatic liver cancer patient.
In addition, these tumours have been demonstrated there is comparable drug resistance to radiation and chemotherapy, in hepatocellular carcinoma, immunization therapy is a kind of
Very attractive selection.It is studied based on CheckMate-040, FDA acceleration in 2017 has approved anti-programmed death receptor 1
(PD1) treatment of advanced stage HCC patient of the monoclonal antibody Nivolumab for previously once receiving Sorafenib treatment.Receive Nivolumab
The objective remission rate of the patient for the treatment of is 20%, and disease control rate 64%, the alleviation duration reaches 17 months, Progression free survival
It is 4 months, middle position is always survived and had not yet been reached, and it is 15 months that the middle position of dosage escalation group, which is always survived, while good security.New
Experimental data shows that T cell infiltration in tumor, interferon (IFN) signal, checkpoint molecule (PD1, PDL1 expression) or high tumour are prominent
The presence for becoming burden is advantageously possible for clinical remission.Amynologic characteristic unfortunately for hepatic carcinoma and how to utilize
These information improve the reactivity to immunotherapy to the maximum extent, we know little about it.So detection hepatic carcinoma microenvironment
Whether the case where middle immunocyte subgroup and immunologic test point molecule applies immunologic test point inhibitor medicaments for prediction patient
It is effectively very favorable.
Existing immunohistochemistry often uses diaminobenzidine (i.e. 3,3 ,-diaminobenzidine, DAB) to develop the color, former
Reason is to lose electronics because diaminobenzidine is the chromogenic substrate of peroxidase in the presence of hydrogen peroxide and show
Color change accumulation, forms sepia insoluble product.DAB is mainly combined with NH2 the or SH group of protein, is formed stable
Nn key or ns key, then the colour developing group in DAB can show color, be tagged on the protein being exposed through, thus aobvious
Show protein distribution and the type etc. of target cell.The prior art is sliced upper one molecule of generally labeling at one.Although at present
There is the immunohistochemical staining method of some multiple labellings, but these multiple histochemical staining methods can be to previous wheel shape in elution
At dyeing impact, generally require addition dyeing intensifier etc., testing result is inaccurate.
Summary of the invention
The purpose of the present invention is to provide the Multiple immunizations histochemical analysis kit and its application method of a kind of liver cancer and answer
With the kit can effectively predict the validity of immunologic test point inhibitor, good excellent with high sensitivity, specificity
Point.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of Multiple immunizations histochemical analysis kits of liver cancer, including monoclonal antibody group, antigen to repair
The junket ammonia salt that multiple liquid, the secondary antibody of horseradish peroxidase-labeled, hydrogen peroxide, nuclear staining agent and fluorophor mark, it is described glimmering
The quantity one of the number of species and monoclonal antibody type in monoclonal antibody group of fluorophor in the junket ammonia salt of light group label
It causes；
The monoclonal antibody group includes the first monoclonal antibody group and second monoclonal antibody group；First monoclonal
Antibody group includes PanCK monoclonal antibody, PDL1 monoclonal antibody, CD8 monoclonal antibody, CD66B monoclonal antibody and CD163
Monoclonal antibody；The second monoclonal antibody group includes that PanCK monoclonal antibody, PDL1 monoclonal antibody, CD8 monoclonal are anti-
Body, PD1 monoclonal antibody and CD68 monoclonal antibody.
Preferably, the kit further includes anti-quencher, detergent and confining liquid.
Preferably, the cleaning solution is TBST buffer.
Preferably, the nuclear staining agent is DAPI coloring agent.
Preferably, the fluorophor includes 520-FITC, 570-Cy3,620-Cy3.5,650-Cy5 and 690-Cy5.5.
The present invention also provides a kind of application method of the Multiple immunizations histochemical analysis kit of liver cancer described in above scheme,
The following steps are included:
(1) sample to be tested is mixed with any one monoclonal antibody in the monoclonal antibody group, is incubated for, washing,
Obtain antigen-primary antibody compound；
(2) antigen-primary antibody compound is mixed with the secondary antibody of horseradish peroxidase-labeled, is incubated for, washing is resisted
Former-one anti-secondary antibody compound；
(3) junket ammonia salt and hydrogen peroxide that the anti-secondary antibody compound of antigen-one, any one fluorophor mark are mixed
It closes and carries out fluorescent staining, be incubated for, washing obtains the first fluorescent marker compound；
(4) the first fluorescent marker compound is mixed, microwave treatment with antigen retrieval buffers, washs, obtains the second fluorescence mark
Remember compound；
The condition of the microwave treatment include: 750~850w handle 1~3min, then with 200~300w processing 12~
20min；
(5) step (1)~(4) are repeated using the second fluorescent marker compound as sample to be tested, are repeated to the monoclonal
Until each monoclonal antibody in antibody group is all excessively primary in conjunction with sample to be tested, multiple labelling compound is obtained；
Wherein, the monoclonal antibody for repeating to use for the first time and every time in step (1) is different, first in step (3)
Secondary and each junket ammonia salt for repeating the fluorophor used label is different；
(6) nuclear staining agent is added into the resulting multiple labelling compound of step (5), is incubated for, washing, mounting, with continuous
Light spectrum image-forming, detection.
Preferably, in step (1), when incubation monoclonal antibody be CD66B monoclonal antibody, PanCK monoclonal antibody,
When CD163 monoclonal antibody, CD8 monoclonal antibody, PD1 monoclonal antibody or CD68 monoclonal antibody, incubation temperature be 35~
42℃；When the monoclonal antibody of incubation is PDL1 monoclonal antibody, incubation temperature is 2~6 DEG C；First monoclonal antibody
In group antibody incubation sequence successively are as follows: CD66B monoclonal antibody, PanCK monoclonal antibody, PDL1 monoclonal antibody, CD163
Monoclonal antibody, CD8 monoclonal antibody；In the second monoclonal antibody group antibody incubation sequence successively are as follows: PD-1 Dan Ke
Grand antibody, PanCK monoclonal antibody, PDL1 monoclonal antibody, CD8 monoclonal antibody, CD68 monoclonal antibody.
Preferably, extension rate is 50~500 times when monoclonal antibody uses in the step (1)；The step (2)
In, the dosage of the secondary antibody of horseradish peroxidase-labeled is 50~200 μ L/ samples.
The present invention also provides the Multiple immunizations histochemical analysis kits of liver cancer described in above scheme in prediction immunologic test
Point inhibitor is to the application in liver cancer treatment validity, which is characterized in that the application includes:
S1, CD8 in the detection sample to be tested of kit described in above scheme is utilized+PDL1-, CD8+PD1-, CD68+PD1-,
CD68+PDL1-, CD68+And CD8+Quantity；
S2, CD68 is calculated according to the measurement result of S1+/CD8+Ratio；
S3, measurement and calculated result according to S1 and S2, as CD8 in sample to be tested+PDL1-> 2.14, CD8+PD1->
When 2.83, it is high to judge that immunologic test point inhibitor treats validity to the patient in the sample to be tested source；
Or as CD68 in sample to be tested+/CD8+< 5.49, CD68+PD1-< 8.15 and CD68+PDL1-When < 8.15, sentence
It is high that disconnected immunologic test point inhibitor treats validity to the patient in the sample to be tested source.
It is that the present invention obtains the utility model has the advantages that
The present invention provides a kind of Multiple immunizations histochemical analysis kits of liver cancer, and therein defining monoclonal antibody group packets
Include the first monoclonal antibody group and second monoclonal antibody group；The first monoclonal antibody group include PanCK monoclonal antibody,
PDL1 monoclonal antibody, CD8 monoclonal antibody, CD66B monoclonal antibody and CD163 monoclonal antibody；Second monoclonal
Antibody group includes PanCK monoclonal antibody, PDL1 monoclonal antibody, CD8 monoclonal antibody, PD1 monoclonal antibody and CD68 mono-
Clonal antibody.Multiple immunizations histochemical analysis kit provided by the invention with using Isolated human liver paraffin section as sample into
Row detection can mark simultaneously multiple immunocyte markers, tumour cell marker and immune phase in same tissue sample
Molecule is closed, CD8 in the detection sample to be tested of kit is utilized+PDL1-, CD8+PD1-, CD68+PD1-, CD68+PDL1-And CD68+, CD8+Quantity, and calculate CD68+/CD8+Ratio；It is higher or lower than threshold value according to each index to judge that immunologic test point presses down
The probability that preparation obtains good therapeutic effect to the patient in the sample to be tested source is higher.
The present invention also provides the application method of the Multiple immunizations histochemical analysis kit of liver cancer described in above-mentioned technical proposal,
First with one of labeling of monoclonal antibody sample to be tested in monoclonal antibody group, antigen-primary antibody compound is formed；Again with
Two anti-binding primary antibodies of horseradish peroxidase (HRP) label, form the anti-secondary antibody compound of antigen-one；Add hydrogen peroxide and
After the junket ammonia salt of one of fluorophor label, in the presence of hydrogen peroxide, the junket ammonia salt of fluorophor label exists
The lower enzymatic preparation (having fluorophor) formed containing covalent bonding site of HRP catalysis, with surrounding (including antigen, primary antibody
On secondary antibody) protein residues (tryptophan, histidine and junket ammonia salt residue etc.) combine, in this way in antigen-primary antibody binding site
Enzymatic preparation by aggregation largely containing fluorophor label, thus antigen (the immunologic test point that primary antibody is identified) quantity is got over
The enzymatic preparation of more then its combination is more, and the fluorophor having is also more, and then its detection signal is stronger.
The fluorescent marker that enzymatic reaction is obtained carries out microwave treatment and separates antigen and primary antibody (in monoclonal antibody group
Monoclonal antibody) combination, monoclonal antibody can be removed after elution；It is micro- since enzymatic preparation and antigen are Covalent bonding together
Influence of the wave processing to enzymatic preparation and antigen binding is smaller, will not influence the fluorescence signal intensity marked on antigen, thus
It realizes and completely removes last round of antibody in the case where ensuring that last round of fluorescent label signal is not lost, it will not be to next round list
Clonal antibody label interferes.Using the junket ammonia salt of different monoclonal antibody and different fluorophor labels, according to upper
The method of stating repeats, and can be realized and marks multiple molecules on same sample to be tested, without having to worry about cross reaction, testing result
Accurately, at most can simultaneously six kinds of label different immunological marker objects.Sample to be tested after multiple labelling is dyed, is sealed
Piece utilizes the immunological marker object concentration in multispectral imaging detection sample to be tested.
Multiple molecules are marked on same sample to be tested as it can be seen that can be realized using application method provided by the invention, and
And the interference of previous round polyclonal antibody is eliminated, the accuracy of detection is high, detection efficiency is high.
Detailed description of the invention
Fig. 1 is the case display diagram of 1 group of immunotherapeutic effects quality of panel in embodiment 1；Wherein Figure 1A is panel 1
The case spectrogram of group immunization therapy weak curative effect, Figure 1B are the eutherapeutic case spectrogram of 1 group of immunization therapy of panel；
Fig. 2 is the case display diagram of 2 groups of immunotherapeutic effects quality of panel in embodiment 1；Wherein Fig. 2A is panel 2
The case spectrogram of group immunization therapy weak curative effect, Fig. 2 B are the eutherapeutic case spectrogram of 2 groups of immunization therapies of panel；
Fig. 3 is A-Factorimportance forest map；
Fig. 4 is ROC curve figure.
Specific embodiment
The present invention provides a kind of Multiple immunizations histochemical analysis kits of liver cancer, including monoclonal antibody group, antigen to repair
The junket ammonia salt that multiple liquid, the secondary antibody of horseradish peroxidase-labeled, hydrogen peroxide, nuclear staining agent and fluorophor mark, it is described glimmering
The quantity one of the number of species and monoclonal antibody type in monoclonal antibody group of fluorophor in the junket ammonia salt of light group label
It causes；
The monoclonal antibody group includes the first monoclonal antibody group and second monoclonal antibody group；First monoclonal
Antibody group includes PanCK monoclonal antibody, PDL1 monoclonal antibody, CD8 monoclonal antibody, CD66B monoclonal antibody and CD163
Monoclonal antibody；The second monoclonal antibody group includes that PanCK monoclonal antibody, PDL1 monoclonal antibody, CD8 monoclonal are anti-
Body, PD1 monoclonal antibody and CD68 monoclonal antibody.
In the present invention, the kit further includes anti-quencher, detergent and confining liquid.
In the present invention, each monoclonal antibody in the monoclonal antibody group is to be detected immune for identifying respectively
Checkpoint.In the present invention, the Multiple immunizations histochemical analysis kit has the advantages that high specificity, high sensitivity.
In the present invention, the various monoclonal antibodies in the monoclonal antibody group are preferably source of mouse monoclonal antibody
And/or rabbit resource monoclonal antibody；In a specific embodiment of the present invention, the secondary antibody that PanCK, CD163, PD-1, CD68 are used is
The PV-6002 (enzyme mark goat anti-mouse IgG polymer) of Zhong Shan Golden Bridge production；The secondary antibody that CD66B, PDL1, CD8 are used is middle China fir
PV-6001 (the enzyme mark goat anti-rabbit igg polymer of Golden Bridge's production.The present invention is to the various Dan Ke in the monoclonal antibody group
The preparation method of grand antibody is not particularly limited, and is voluntarily prepared using commercial goods or conventional method.
In the present invention, the antigen retrieval buffers prevent to mark completely when immunohistochemical staining for repairing antigen.
The present invention is not particularly limited the source of the antigen retrieval buffers, using this field commercial goods or known formulations, this hair
Using the Opal7-colorManual IHC Kit of Perkin Elmer production as antigen retrieval in bright specific embodiment
Liquid.
In the present invention, the secondary antibody of horseradish peroxidase-labeled is used for anti-with the monoclonal in the monoclonal antibody group
Body combines, and horseradish peroxidase therein is in the presence of having hydrogen peroxide, the junket ammonia salt of catalytic fluorometry group label
Generate have covalent bond site enzymatic preparation, the enzymatic preparation can in conjunction with the protein residues in sample to be tested, thus
Make fluorophor label on sample to be tested.The present invention is to the secondary antibody source of the horseradish peroxidase-labeled without special limit
It is fixed, using commercial goods.
In the present invention, the hydrogen peroxide provides condition for the enzymatic reaction of horseradish peroxidase.The present invention is to institute
The source for stating hydrogen peroxide is not particularly limited.
In the present invention, the junket ammonia salt of the fluorophor label, to generate enzymatic reaction, is utilized as reaction substrate
Enzymatic preparation is in conjunction with the protein residues in antigen, and then the fluorophor that enzymatic preparation is had marks on sample to be tested,
And fluorophor marker number is directly proportional to the marker quantity that the primary antibody of use is identified, and then realizes to be identified anti-
Former quantitative detection.It is currently preferred, the fluorophor include 520-FITC, 570-Cy3,620-Cy3.5,650-Cy5 and
690-Cy5.5。
In the present invention, the nuclear staining agent is preferably DAPI coloring agent.In the present invention, the nuclear staining agent is used for
Mark nucleus.
In the present invention, the kit further includes anti-quencher, and the anti-quencher plays anti-fluorescence decay and prevents
The only effect of fluorescent quenching.The present invention is not particularly limited the type of the anti-quencher, using this field commercial goods.
In a specific embodiment of the present invention, the anti-quencher is preferably the anti-fluorescence decay mountant of Boble Ryder.
In the present invention, the confining liquid is used for blocking antigen, improves detection accuracy.The present invention is to the confining liquid
Type is not particularly limited, the Antibody Diluen/ generated in specific embodiments of the present invention using Perkin Elmer
Block is as confining liquid.
In the present invention, for the detergent use in washing step in the detection process, the present invention is excellent to the detergent
It is selected as TBST buffer.
The Multiple immunizations histochemical analysis kit of liver cancer provided by the invention inhibits situation for the immunologic test point of liver cancer
It is designed, provides corresponding monoclonal antibody group and identified, high specificity, high sensitivity.
The present invention also provides a kind of application method of the Multiple immunizations histochemical analysis kit of liver cancer described in above scheme,
The following steps are included:
(1) sample to be tested is mixed with any one monoclonal antibody in the monoclonal antibody group, is incubated for, washing,
Obtain antigen-primary antibody compound；
(2) antigen-primary antibody compound is mixed with the secondary antibody of horseradish peroxidase-labeled, is incubated for, washing is resisted
Former-one anti-secondary antibody compound；
(3) junket ammonia salt and hydrogen peroxide that the anti-secondary antibody compound of antigen-one, any one fluorophor mark are mixed
It closes and carries out fluorescent staining, be incubated for, washing obtains the first fluorescent marker compound；
(4) the first fluorescent marker compound is mixed, microwave treatment with antigen retrieval buffers, washs, obtains the second fluorescence mark
Remember compound；
The condition of the microwave treatment include: 750~850w handle 1~3min, then with 200~300w processing 12~
20min；
(5) step (1)~(4) are repeated using the second fluorescent marker compound as sample to be tested, are repeated to the monoclonal
Until each monoclonal antibody in antibody group is all excessively primary in conjunction with sample to be tested, multiple labelling compound is obtained；
Wherein, the monoclonal antibody for repeating to use for the first time and every time in step (1) is different, first in step (3)
Secondary and each junket ammonia salt for repeating the fluorophor used label is different；
(6) nuclear staining agent is added into the resulting multiple labelling compound of step (5), is incubated for, washing, mounting, with continuous
Light spectrum image-forming, detection.
In the present invention, the sample to be tested is preferably the histotomy of liver cancer, it is furthermore preferred that the histotomy is stone
Wax slice.In the present invention, when the sample to be tested is liver cancer tissue paraffin section, the present invention is in sample to be tested and monoclonal
Dewaxing and aquation are also carried out before antibody mixing, specifically includes the following steps:
A, liver cancer tissue paraffin section is toasted to 100~150min at 50~70 DEG C, obtains baking slice；
B, baking slice successively with xylene extraction twice, dehydrated alcohol is extracted twice, 95% ethanol solution of mass fraction
Extraction is primary, and the extraction of 90% ethanol solution of mass fraction is primary, and the extraction of 85% ethanol solution of mass fraction is primary, mass fraction
The extraction of 80% ethanol solution is primary, and the extraction of 75% ethanol solution of mass fraction is primary, and the liver cancer tissue after obtaining dewaxing aquation is made
For sample to be tested.
Currently preferred, each extraction time of dimethylbenzene is preferably 8~12min in the step B；Dehydrated alcohol
Each extraction time is preferably 3~8min；95% ethanol solution of mass fraction, 90% ethanol solution of mass fraction, mass fraction
Independently extraction time is preferably 3 for 85% ethanol solution, 80% ethanol solution of mass fraction and 75% ethanol solution of mass fraction
~8min.
It in the present invention, first will be to if Multiple immunizations histochemical analysis kit of the present invention further includes confining liquid
Sample is mixed with monoclonal antibody again after being closed with confining liquid, and the dosage of the confining liquid is preferably 50~150 μ L/ samples
Product, more preferably 100 μ L/ samples.In the present invention, the off-period of the confining liquid is preferably 8~15min, more preferably
10min。
The present invention mixes sample to be tested with any one monoclonal antibody in the monoclonal antibody group, is incubated for, washes
It washs, obtains antigen-primary antibody compound.It is currently preferred, in order to further increase the prediction accuracy of kit, institute of the present invention
State the incubation sequence of antibody in the first monoclonal antibody group in monoclonal antibody group successively are as follows: CD66B monoclonal antibody,
PanCK monoclonal antibody, PDL1 monoclonal antibody, CD163 monoclonal antibody, CD8 monoclonal antibody；Monoclonal of the present invention
The incubation sequence of antibody is successively in second monoclonal antibody group in antibody group are as follows: PD-1 monoclonal antibody, PanCK monoclonal are anti-
Body, PDL1 monoclonal antibody, CD8 monoclonal antibody, CD68 monoclonal antibody.
In the present invention, when the monoclonal antibody of incubation is CD66B monoclonal antibody, PanCK monoclonal antibody, CD163
When monoclonal antibody, CD8 monoclonal antibody, PD1 monoclonal antibody or CD68 monoclonal antibody, incubation temperature is preferably 35~42
DEG C, more preferably 37 DEG C.In the present invention, the incubation time of said monoclonal antibody is preferably 1h.
In the present invention, when the monoclonal antibody of incubation is PDL1 monoclonal antibody, incubation temperature is preferably 2~6 DEG C,
More it is selected as 3~5 DEG C.In the present invention, the incubation time of said monoclonal antibody is preferably 12~16h.
In the present invention, the monoclonal antibody is the commercially available monoclonal antibody stoste of purchase, by the Dan Ke when use
Grand antibody uses after diluting 50~500 times；In the present invention, it is preferred to use antibody dilution/confining liquid (opalTMKit) it is dilute
Release monoclonal antibody.In the present invention, the dosage of the monoclonal antibody is 100~300 μ L/ samples.
In the present invention, when in the multiple group of change immunoassay kits including cleaning solution, the washing is preferred
It is washed with cleaning solution；In the present invention, the washing is preferred repeats 2~3 times.In the present invention, the wash time is each
Preferably 3~10min, more preferably 5min.Washing step operation in following steps of the process is all the same, repeats no more.
After obtaining antigen-primary antibody compound, the present invention is by the two of antigen-primary antibody compound and horseradish peroxidase-labeled
Anti- mixing is incubated for, and washing obtains the anti-secondary antibody compound of antigen-one.
In the present invention, the secondary antibody of the horseradish peroxidase-labeled is preferably the goat anti-rabbit igg polymerization of HRP label
Object or enzyme mark goat anti-mouse IgG polymer.In the present invention, the secondary antibody of the horseradish peroxidase-labeled is the city of purchase
Sell commodity.In the present invention, the secondary antibody usage amount of the horseradish peroxidase-labeled is preferably 50~200 μ L/ samples, more excellent
It is selected as 100 μ L/ samples.
In the present invention, the incubation time of the secondary antibody of the horseradish peroxidase-labeled is preferably 8~15min, more excellent
It is selected as 10min.In the present invention, the temperature of the incubation is preferably 35~38 DEG C, and more preferably 37 DEG C.
After obtaining the anti-secondary antibody compound of antigen-one, the present invention is by the anti-secondary antibody compound of antigen-one, any one fluorescence
The junket ammonia salt of group label and hydrogen peroxide mixing carry out fluorescent staining, are incubated for, and it is compound to obtain the first fluorescent marker for washing
Object.
In the present invention, the hydrogen peroxide is preferably provided in the form of signal amplification liquid, and the signal amplifies the city Ye Wei
Commodity are sold, wherein being mixed with hydrogen peroxide.It is currently preferred to be used after 80~120 times of dilution of commercially available signal amplification liquid, more
Preferably dilute 100 times.In the present invention, the junket ammonia salt of the fluorophor label is preferably purchased from commercial goods, in this hair
Using the junket ammonia salt of the fluorophor label in commercially available OpalTM 7- color fluorescent dyeing reagent box in bright specific embodiment；
It is currently preferred, it is used after the junket ammonia salt that each fluorophor marks is diluted 50~150 times when use, it is preferred to dilute
100 times.It is currently preferred, 80~150 μ L/ sample of solution after the junket ammonia salt usage amount of the fluorophor label preferably dilutes
Product, more preferably 100 μ L/ samples.
In the present invention, the incubation time is preferably 8~15min, more preferably 10min.In the present invention, described to incubate
The temperature educated is preferably 35~38 DEG C, and more preferably 37 DEG C.
After obtaining the first fluorescent marker compound, the present invention mixes the first fluorescent marker compound with antigen retrieval buffers,
Microwave treatment, washing, obtains the second fluorescent marker compound；The condition of the microwave treatment include: 750~850w processing 1~
3min, then 12~20min is handled with 200~300w.In the present invention, the microwave treatment conditions preferably include: 80w preheating
5min, then 800w handle 2min, and last 240w handles 15min.
In the present invention, the antigen retrieval buffers come from commercial goods, use in a specific embodiment of the present invention
OpalTMAntigen retrieval buffers in 7- color fluorescent dyeing reagent box；The dosage of the antigen retrieval buffers is preferably 150~300mL/ sample
Product, more preferably 200mL/ sample.
In the present invention, monoclonal antibody of the first fluorescent marker compound under microwave treatment, with antigen binding
It is dissociated, the monoclonal antibody of disengaging and the secondary antibody of HRP label can be removed by washing, will not influence next round reaction
It carries out, prevents from interfering with each other when multiple labelling.
After obtaining the second fluorescent marker compound, the present invention is using obtained the second fluorescent marker compound as to test sample
Product repeat said monoclonal antibody and identify to the step of obtaining fluorescent marker compound, repeat into the monoclonal antibody group
All monoclonal antibodies it is excessively primary in conjunction with sample to be tested until, obtain multiple labelling compound.
In repetitive process of the invention, the monoclonal antibody that each round uses when repeating is used with an any other wheel
Monoclonal antibody type it is different；Correspondingly, the fluorescent base in the junket ammonia salt of the fluorophor label used when each round repeats
Group is also different from the type of an any other wheel.Mark different monoclonals anti-respectively using different fluorophors to realize
The purpose of body.
After obtaining multiple labelling compound, the present invention adds nuclear staining agent into multiple labelling compound and is incubated for, and washes
It washs, sealing, continuous spectrum imaging, image procossing and observation analysis.
In the present invention, the additive amount of the nuclear staining agent is preferably 80~150 μ L/ samples, more preferably 100 μ L/ samples
Product.
In the present invention, the incubation time is preferably 5~10min, more preferably 8min.
In the present invention, it when the multiple group of change immune reagent kit further includes anti-quencher, is incubated for, after washing described
Anti- quencher is added, then covers coverslip and carries out mounting.
In the present invention, the Multiple immunizations histochemical analysis kit is able to satisfy existing histology spectral imaging apparatus to more
A molecule is imaged simultaneously, i.e. multispectral imaging.
Multispectral imaging splits dependent on spectrum data gathering and spectrum and calculates two processes:
Spectrum data gathering: multispectral data acquisition technological means there are many kinds of, such as grating beam splitting, prismatic decomposition,
Liquid crystal tunable filter light splitting etc..With the Vectra system (liquid crystal tunable filter, LCTF) of PerkinElmer company
The spectral signal of filtering acquisition specific band.LCTF is made of liquid crystal material, changes light in crystalline substance by adjusting additional voltage
Light path in body, selectivity export the optical signal of specific wavelength, realize the purpose of light splitting.The continuous filtering of CCD exposure cooperation LCTF
Wave, so that it may the picture signal of accurate recording different wave length section.
Spectrum is split: each pixel signal of spectrum picture is the folded of different fluorescent dyes and sample spontaneous emissions
Add, using the spectral signature curve of every kind of dyestuff as standard, the signal being superimposed in spectrum picture is restored by mathematical method
Operation, so that the process for obtaining single channel image, which is known as spectrum, splits calculating.It is that entire light spectrum image-forming can not that spectrum, which splits and calculates,
The important link lacked directly affects the accuracy of data result.
Use " pure spectrum fractionation algorithm " that the color signal of up to 10 colors superposition can be split, by what is hidden in spectrum picture
" pure " dye signal accurately parses, and obtains the special distribution of every kind of dyestuff in the picture.And it can be by " true mesh
Mark signal " extracted from autofluorescence background, obtain the image of superelevation signal-to-noise ratio, allow the fluorescence signal of weak expression be able to from
It is displayed in background.
Analyze software can common location at least four molecule on the same coordinate position organizationally, check whether there is antigen
Molecule is expressed on a cell simultaneously.
The present invention also provides the Multiple immunizations histochemical analysis kits of liver cancer described in above scheme in prediction immunologic test
Point inhibitor is to the application in liver cancer treatment validity, which is characterized in that the application includes:
S1, CD8 in the detection sample to be tested of kit described in above scheme is utilized+PDL1-, CD8+PD1-, CD68+PD1-,
CD68+PDL1-, CD68+And CD8+Quantity；
S2, CD68 is calculated according to the measurement result of S1+/CD8+Ratio；
S3, measurement and calculated result according to S1 and S2, as CD8 in sample to be tested+PDL1-> 2.14 and CD8+PD1->
When 2.83, it is high to judge that immunologic test point inhibitor treats validity to the patient in the sample to be tested source；
Or as CD68 in sample to be tested+/CD8+< 5.49, CD68+PD1- < 8.15 and CD68+PDL1-When < 8.15, sentence
It is high that disconnected immunologic test point inhibitor treats validity to the patient in the sample to be tested source.
In the present invention, when the multiple group of change immunoassay kits is for predicting the effective of immunologic test point inhibitor
When property, using Isolated human liver paraffin section as sample to be tested.
Specifically, the present invention is using Isolated human liver paraffin section as sample, according to side shown in above-mentioned technical proposal
Method is detected with Multiple immunizations histochemical analysis kit described in preceding solution, and detection obtains contained in sample to be tested
PanCK (Pan-cytokeratin, wide spectrum cytokeratin), PDL1 (immunologic test point molecule), CD66B (neutrophil leucocyte mark
Will object), the content of CD163 (M2 type macrophage marker) and CD8 (T cell marker), or detection obtains sample to be tested
In contained PanCK, PDL1, PD-1 (immunologic test point molecule), CD68 (macrophage marker) and CD8 content.
CD68 is calculated according to said determination result+/CD8+Ratio；
According to said determination and calculated result, as CD8 in sample to be tested+PDL1-> 2.14 and CD8+PD1-When > 2.83,
It is high to judge that immunologic test point inhibitor treats validity to the patient in the sample to be tested source；
Or as CD68 in sample to be tested+/CD8+< 5.49, CD68+PD1-< 8.15 and CD68+PDL1-When < 8.15, sentence
It is high that disconnected immunologic test point inhibitor treats validity to the patient in the sample to be tested source.Not up to above-mentioned condition when, then judge
It is low that immunologic test point inhibitor treats validity to the patient in the sample to be tested source.
The spy that immunologic test point inhibitor validity is predicted using Multiple immunizations histochemical analysis kit of the present invention
Anisotropic strong, high sensitivity, to realize that accurate medication provides condition.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
Embodiment 1
Choose 20 liver cancer patient tumor tissues immunization therapy curative effects it is poor (overall survival phase (Overall Survival,
OS) shorter (number 1~10)) and immunization therapy curative effect preferably each 10 of (OS longer (number 11~20)), specific life cycle
It is shown in Table 1.
1 20 liver cancer patient life cycles of table
The Multiple immunizations histochemical analysis of each albumen in tumor microenvironment is carried out as follows.Each patient 2 opens slice
(panel 1 and panel 2), 5 molecules of each biopsy marker.Its dyeing sequence are as follows: Panel1:CD66B, PanCK, PDL1,
CD163,CD8；Panel 2:PD-1,PanCK,PDL1,CD8,CD68.Primary antibody, secondary antibody, fluorescent dye information in experimentation
Such as table 2:
2 primary antibody of table, secondary antibody and fluorescent dye information
Embodiment operating procedure:
1. will be placed in 60 DEG C of insulating boxs containing 20 paraffin sections and toast 120min；
2. dewaxing and aquation: dimethylbenzene (10min) → dimethylbenzene (10min) → dehydrated alcohol (5min × 2 time) → 95%
Ethyl alcohol (5min × 2) → 90% ethyl alcohol (5min) → 85% ethyl alcohol (5min) → 80% ethyl alcohol (5min) → 75% ethyl alcohol
(5min)；
3. distilled water flushing 2 times, 5min/ times；
4. antigen retrieval: with OpalTM 7- color fluorescent dyeing reagent box (Opal 7-colorManual IHC Kit,
PerkinElmer) endoantigen repairs liquid Microwave method, and 80w preheats 5min, low fire 15min (beauty in 800w high fire 2min, 240w
Micro-wave oven M1-231A)；
5. room temperature natural cooling 15min；
6. washing: TBST buffer solution is washed 3 times, 5min/ times；
7. closing: using confining liquid (producer Perkin Elmer；Trade name Antibody Diluent/Block), room temperature envelope
Close 10min；
8. primary antibody be incubated for: be added dropwise primary antibody (100~300 μ L of primary antibody working solution), CD66B, PanCK, CD163, CD8, CD68,
The incubation conditions of PD1 are 37 DEG C of incubation 1h；The incubation conditions of PD-L1 are 4 DEG C of overnight incubations；The dyeing of primary antibody is divided into two
Panel, dyeing sequence are as follows: Panel 1:CD66B, PanCK, PDL1, CD163, CD8；Panel 2:PD-1,PanCK,PDL1,
CD8,CD68；
9. washing: TBST buffer solution is washed 3 times, 5min/ times；
10. secondary antibody is incubated for: secondary antibody in kit, 37 DEG C of incubation 10min are added dropwise；PanCK,CD163,PD-1,CD68；It uses
Secondary antibody be Zhong Shan Golden Bridge production PV-6002 (enzyme mark goat anti-mouse IgG polymer)；CD66B, PDL1, CD8 use two
Resist the PV-6001 (enzyme mark goat anti-rabbit igg polymer) for the production of Zhong Shan Golden Bridge；
11. washing: TBST buffer solution is washed 3 times, 5min/ times；
12. fluorescence developing: TSA is added dropwise and dilutes the opal fluorescent staining after 100 times, room temperature 10min；
13. washing: TBST buffer solution is washed 3 times, 5min/ times；
14. antibody successively dyes: first antibody dyeing terminates, and subsequent each antibody is both needed to repetition step 4 and arrives step 13,
Successively mark all antibody；Antibody dyeing sequence are as follows: panel1:CD66B, PanCK, PDL1, CD163, CD8；panel2:PD-
1,PanCK,PDL1,CD8,CD68；
15. microwave treatment: repeating step 4) and arrive step 6)；
16.DAPI dyeing, 5~10min of room temperature；
17. washing: TBST is washed 3 times, 5min/ times；
18. sealing: anti-quencher mounting (the anti-fluorescence decay mountant of Boble Ryder)；
19. continuous spectrum imaging, image procossing and observation analysis
According to the above operating procedure, the immunocyte marker and immune correlation to 20 hepatic carcinoma histotomies are completed
The multiple labelling of molecule.With PerkinElmer company Vectra system carry out continuous spectrum acquisition, and do image procossing and
Observation analysis.Immunization therapy weak curative effect and the eutherapeutic case of immunization therapy respectively show 1, and every each displaying 1 is schemed, such as Fig. 1
(A/B) and shown in Fig. 2 (A/B), different colours represent different markers in Fig. 1, specific as shown in Table 3 and Table 4.
The 1 marker color of panel shown in FIG. 1 of table 3
| Marker | PanCK | CD8 | PDL1 | CD66B | CD163 |
| Color | Green | Aubergine | It is orange | Bright pink color | Blue-green |
The 2 marker color of panel shown in Fig. 2 of table 4
| Marker | PanCK | CD8 | PDL1 | CD68 | PD1 |
| Color | Green | Aubergine | It is orange | Blue-green | Carmetta |
To each molecule of tumor region statistical analysis, the immunocyte of 20 patients and immunologic test point Molecular Detection
Percent positive statistical result is as shown in 5~table of table 8:
The positive expression percentage statistical result of each molecule in the patient tumors region of 5 panel of table, 1 therapeutic effect difference
(%)
The positive expression percentage statistical result of each molecule in the good patient tumors region of 6 panel of table, 1 therapeutic effect
(%)
| Number | 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 |
The positive expression percentage statistical result of each molecule in the poor patient tumors region of table 7 panel, 2 therapeutic effects
(%)
The positive expression percentage statistical result of each molecule in the good patient tumors region of 8 panel of table, 2 therapeutic effect
(%)
| Number | 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 |
Multifactor point of progress is preferably organized to immunization therapy curative effect poorer group and immunization therapy curative effect using random forests algorithm
Analysis.To each decision tree, selects the outer data of corresponding bag to calculate the outer data error of bag, be denoted as errOOB1.At random to number outside bag
Noise jamming is added according to the feature X of all samples, calculates the outer data error of bag again, is denoted as errOOB2.The importance of feature X
=∑ (errOOB2-errOOB1)/k.The determination method of important factor are as follows: calculate the importance of each feature and descending sort.
Important factor is characterized several top 20%.
The drafting of ROC curve: to every one tree, the true positive rate and false positive rate of sample are measured.It is (sensitive with true positive rate
Spend %) it is ordinate, false positive rate (1- specificity %) is that abscissa draws ROC curve.Immunization therapy curative effect poorer group and exempt from
The multiplicity result that epidemic disease treatment curative effect is preferably organized: Factor importance forest map is as shown in Figure 3, ROC curve is such as schemed
Shown in 4.
From the point of view of multiplicity result, distinguish immunization therapy curative effect poorer group and immunization therapy curative effect preferably organize it is important
The factor has 5, respectively CD8 in tumor region+PDL1-, CD8+PD1-, CD68+PD1-, CD68+PDL1-And CD68+/CD8+Ratio.
In ROC curve, orange point indicates maximum (the Youden index=sensitivity+specificity-of the Youden index of the point
1), i.e. sensitivity and specificity is best.AUC=0.89, P=0.002 indicate there is statistical significance.When a subject examines
The index of above 2 panel is surveyed, and index of correlation and its ratio meet or when better than corresponding threshold value, this subject will
The probability for carrying out the clinical Benefit from immunization therapy will be much higher than other incongruent subjects.
It can be seen that using Multiple immunizations groupization multiple immunocyte markers in tagged tissue paraffin section, swollen simultaneously
Oncocyte marker and immunologic test point molecule, for predicting whether multiple patients apply immunologic test point inhibitor medicaments simultaneously
It is effectively very favorable.
Immunization therapy curative effect poorer group and immunization therapy curative effect are preferably organized using Mann-Whitney test and carry out Dan Yin
Element analysis, obtains the threshold value of characterization factor.The threshold value of 5 indexs is as follows, is shown in Table 9:
The threshold value of 95 indexs of table
As CD8 in sample to be tested+PDL1-> 2.14, CD8+PD1-When > 2.83, the clinical Benefit from immunization therapy
Probability is higher；As subject CD68+/CD8+< 5.49, CD68+PD1-< 8.15 and CD68+PDL1-When < 8.15, from immune
The probability for treating middle clinical Benefit is higher.
Embodiment 2
A kind of Multiple immunizations histochemical analysis kit of liver cancer, including monoclonal antibody group, antigen retrieval buffers, horseradish peroxide
The junket ammonia salt and nuclear staining agent that secondary antibody, hydrogen peroxide, the fluorophor of compound enzyme label mark；
The monoclonal antibody group are as follows: PanCK monoclonal antibody, PDL1 monoclonal antibody, CD8 monoclonal antibody, CD66B
Monoclonal antibody and CD163 monoclonal antibody；
The fluorophor is 520-FITC, 570-Cy3,620-Cy3.5,650-Cy5 and 690-Cy5.5；
The kit further includes anti-quencher, detergent and confining liquid.
Embodiment 3
A kind of Multiple immunizations histochemical analysis kit of liver cancer, including monoclonal antibody group, antigen retrieval buffers, horseradish peroxide
The junket ammonia salt and nuclear staining agent that secondary antibody, hydrogen peroxide, the fluorophor of compound enzyme label mark；
The monoclonal antibody group are as follows: PanCK monoclonal antibody, PDL1 monoclonal antibody, CD8 monoclonal antibody, PD1 are mono-
Clonal antibody and CD68 monoclonal antibody；
The fluorophor is 520-FITC, 570-Cy3,620-Cy3.5,650-Cy5 and 690-Cy5.5；
The kit further includes anti-quencher, detergent and confining liquid.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of Multiple immunizations histochemical analysis kit of liver cancer, including monoclonal antibody group, antigen retrieval buffers, horseradish peroxidating
The junket ammonia salt that secondary antibody, hydrogen peroxide, nuclear staining agent and the fluorophor of object enzyme label mark, the junket ammonia of the fluorophor label
The number of species of fluorophor are consistent with the quantity of monoclonal antibody type in monoclonal antibody group in salt；
The monoclonal antibody group includes the first monoclonal antibody group and second monoclonal antibody group；First monoclonal antibody
Group includes PanCK monoclonal antibody, PDL1 monoclonal antibody, CD8 monoclonal antibody, CD66B monoclonal antibody and CD163 Dan Ke
Grand antibody；The second monoclonal antibody group include PanCK monoclonal antibody, PDL1 monoclonal antibody, CD8 monoclonal antibody,
PD1 monoclonal antibody and CD68 monoclonal antibody.
2. kit according to claim 1, which is characterized in that the kit further include anti-quencher, detergent and
Confining liquid.
3. kit according to claim 2, which is characterized in that the cleaning solution is TBST buffer.
4. kit according to claim 1, which is characterized in that the nuclear staining agent is DAPI coloring agent.
5. kit according to claim 1, which is characterized in that the fluorophor include 520-FITC, 570-Cy3,
620-Cy3.5,650-Cy5 and 690-Cy5.5.
6. a kind of application method of the Multiple immunizations histochemical analysis kit of liver cancer described in Claims 1 to 5 any one, including
Following steps:
(1) sample to be tested is mixed with any one monoclonal antibody in the monoclonal antibody group, is incubated for, washing obtains
Antigen-primary antibody compound；
(2) antigen-primary antibody compound is mixed with the secondary antibody of horseradish peroxidase-labeled, is incubated for, washing obtains antigen-one
Anti- secondary antibody compound；
(3) junket ammonia salt and hydrogen peroxide that the anti-secondary antibody compound of antigen-one, any one fluorophor mark are mixed into
Row fluorescent staining is incubated for, and washing obtains the first fluorescent marker compound；
(4) the first fluorescent marker compound is mixed, microwave treatment with antigen retrieval buffers, is washed, it is multiple to obtain the second fluorescent marker
Close object；
The condition of the microwave treatment includes: that 750~850w handles 1~3min, then handles 12~20min with 200~300w；
(5) step (1)~(4) are repeated using the second fluorescent marker compound as sample to be tested, are repeated to the monoclonal antibody
Until each monoclonal antibody in group is all excessively primary in conjunction with sample to be tested, multiple labelling compound is obtained；
Wherein, the monoclonal antibody for repeating to use for the first time and every time in step (1) is different, in step (3) for the first time and
The junket ammonia salt for repeating the fluorophor label used every time is different；
(6) nuclear staining agent is added into the resulting multiple labelling compound of step (5), is incubated for, washing, mounting, with continuous spectrum
Imaging, detection.
7. application method according to claim 6, which is characterized in that in step (1), when the monoclonal antibody of incubation is
CD66B monoclonal antibody, PanCK monoclonal antibody, CD163 monoclonal antibody, CD8 monoclonal antibody, PD1 monoclonal antibody or
When CD68 monoclonal antibody, incubation temperature is 35~42 DEG C；When the monoclonal antibody of incubation is PDL1 monoclonal antibody, it is incubated for
Temperature is 2~6 DEG C；In the first monoclonal antibody group antibody incubation sequence successively are as follows: CD66B monoclonal antibody, PanCK
Monoclonal antibody, PDL1 monoclonal antibody, CD163 monoclonal antibody, CD8 monoclonal antibody；The second monoclonal antibody group
The incubation sequence of middle antibody is successively are as follows: PD-1 monoclonal antibody, PanCK monoclonal antibody, PDL1 monoclonal antibody, CD8 Dan Ke
Grand antibody, CD68 monoclonal antibody.
8. application method according to claim 6, which is characterized in that dilute when monoclonal antibody uses in the step (1)
Releasing multiple is 50~500 times；In the step (2), the dosage of the secondary antibody of horseradish peroxidase-labeled is 50~200 μ L/ samples
Product.
9. the Multiple immunizations histochemical analysis kit of liver cancer described in Claims 1 to 5 any one is in prediction immunologic test point suppression
Preparation is to the application in liver cancer treatment validity, which is characterized in that the application includes:
S1, CD8 in the detection sample to be tested of kit described in claim 1~6 any one is utilized+PDL1-, CD8+PD1-, CD68+
PD1-, CD68+PDL1-, CD68+And CD8+Quantity；
S2, CD68 is calculated according to the measurement result of S1+/CD8+Ratio；
S3, measurement and calculated result according to S1 and S2, as CD8 in sample to be tested+PDL1- > 2.14, CD8+PD1-> 2.83
When, it is high to judge that immunologic test point inhibitor treats validity to the patient in the sample to be tested source；
Or as CD68 in sample to be tested+/CD8+< 5.49, CD68+PD1-< 8.15 and CD68+PDL1-When < 8.15, judgement is exempted from
It is high that epidemic disease checkpoint inhibitor treats validity to the patient in the sample to be tested source.
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