Multiplex fluorescence immunohistochemical detection kit for rare gastrointestinal tumors and application of multiplex fluorescence immunohistochemical detection kit

Abstract

The invention provides a multiplex fluorescence immunohistochemical detection kit for rare gastrointestinal tumors and application of the multiplex fluorescence immunohistochemical detection kit, and belongs to the technical field of multiplex immunohistochemistry. The invention provides a multiplex fluorescence immunohistochemical detection reagent combination, which comprises a monoclonal antibody designed for protein markers and a fluorescent dye, and can be applied to multiplex fluorescence immunohistochemical detection for detecting the protein markers such as PD-L1, PD-1, CTLA-4, LAG-3, TIM-3 and TIGIT. The invention further provides a kit capable of being used for multiplex fluorescence immunohistochemical detection, staining detection of six protein markers on a single section can be achieved, and multiplex fluorescence staining of disease samples, especially complex-structure gastrointestinal tract rare tumor samples is achieved by matching six protein targets according to a specific staining sequence and fluorescence channels. And false positive caused by staining crosstalk of each channel is prevented.

Description

Technical Field

[0001] This invention belongs to the field of multiplex immunohistochemistry technology, specifically relating to a multiplex fluorescent immunohistochemistry detection kit for rare gastrointestinal tumors and its application. Background Technology

[0002] Gastrointestinal tumors, including gastric cancer, colon cancer, and rectal cancer, have seen a rising incidence rate in recent years, especially among people aged 40-60. The early diagnosis rate of gastrointestinal tumors is less than 30%, and approximately 70% of early-stage patients are asymptomatic and not screened in time. Most are diagnosed at an advanced stage, often with multiple metastases, limited surgical opportunities, and poor prognosis, with a 5-year survival rate of less than 30%. Furthermore, due to the heterogeneity and complexity of advanced gastrointestinal tumors, especially rare types, surgical intervention alone is often insufficient for a biological cure; therefore, multidisciplinary treatment is necessary to improve outcomes. Multidisciplinary comprehensive treatment strategies encompass preoperative treatment, surgical resection, postoperative adjuvant chemotherapy, radiotherapy, targeted drug therapy, and immunotherapy, aiming to improve treatment effectiveness and patient prognosis. The application of targeted drugs and immunotherapy has significantly improved the prognosis of advanced-stage patients. For example, imatinib has enabled 20% of patients with metastatic gastrointestinal stromal tumors to survive for more than 10 years.

[0003] Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the digestive tract. These tumors originate from undifferentiated stromal cells in the gastrointestinal tract, with 60%–70% occurring in the stomach and 20%–30% in the small intestine. They are considered rare diseases. The age of onset is primarily between 40 and 65 years, with an average age of onset of approximately 60 years. There is no significant difference in the male-to-female ratio. Early-stage GISTs can be resected via endoscopic minimally invasive surgery or laparoscopic resection. Advanced or metastatic patients can have their survival significantly prolonged with comprehensive targeted therapy (such as avatinib or retinotinib). Similarly, gastrointestinal neuroendocrine tumors (GI-NETs) are also rare diseases, with approximately 60% occurring in the gastrointestinal tract. The age of onset is wide, but they are most common between 40 and 60 years of age, with no significant difference in the male-to-female ratio.

[0004] Over the past decade, the treatment of gastrointestinal tumors has evolved from single surgery or chemotherapy to a precision medicine era combining surgery, targeted therapy, immunotherapy, and radiotherapy. Minimally invasive techniques improve surgical safety, targeted drugs target specific gene mutations, immunotherapy overcomes the limitations of traditional chemotherapy, and multidisciplinary collaboration and comprehensive management further optimize efficacy. Among these, the application of PD-1 / PD-L1 inhibitors and the combination therapy of PD-1 / CTLA-4 bispecific antibodies have significantly improved patient survival. However, due to the slow clinical progress of immunotherapy drugs and the lack of attention paid to rare types of gastrointestinal tumors, they are easily confused with conventional gastrointestinal tumors. Therefore, immune protein target detection methods (such as PD-1 / PD-L1 / CTLA-4) applicable to gastrointestinal tumors, especially rare types, are particularly important.

[0005] Currently, the primary method for detecting the expression levels of various drug-guided target proteins in formalin-fixed paraffin sections of tumor tissues is immunohistochemistry. This method utilizes the fundamental principle of immunology—the antigen-antibody reaction—where the binding between antibodies and antigens is highly specific. Specific antibodies detect similar antigenic substances in tissues or cells. However, since antigen-antibody complexes are colorless, histochemical methods (fluorescein, enzymes, metal ions, isotopes) are necessary to visualize the antigen-antibody binding sites for qualitative, localization, or quantitative analysis of antigens in tissues. However, conventional immunohistochemistry can only detect the expression of PD-L1, PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT proteins in gastrointestinal tumor samples, requiring multiple sections to detect multiple targets. This wastes scarce and valuable clinical samples. Furthermore, conventional immunohistochemistry, by only showing the expression level of a single target per section, cannot reveal the spatial interactions between different target expression sites, significantly limiting the in-depth analysis of immunohistochemical results. Summary of the Invention

[0006] This invention provides a multiplex fluorescent immunohistochemical detection kit for rare gastrointestinal tumors and its application, combining the advantages of "multi-channel + drug-related targets" to provide a reliable basis for immunotargeted therapy of various diseases, especially gastrointestinal tumors.

[0007] This invention provides a combination of multiplex fluorescent immunohistochemical detection reagents, including monoclonal antibodies and fluorescent dyes designed for protein biomarkers;

[0008] The protein biomarkers include PD-L1, PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT;

[0009] The fluorescent dye is specifically linked to the conjugates of monoclonal antibodies and secondary antibodies designed for each of the protein markers.

[0010] In a preferred embodiment of the present invention, the monoclonal antibody includes PD-L1 monoclonal antibody, PD-1 monoclonal antibody, CTLA-4 monoclonal antibody, LAG-3 monoclonal antibody, TIM-3 monoclonal antibody and TIGIT monoclonal antibody;

[0011] The secondary antibody includes an HPR-labeled anti-mouse / rabbit IgG polymeric secondary antibody.

[0012] In a preferred embodiment of the present invention, the fluorescent dye comprises 480 fluorescent dye, 520 fluorescent dye, 570 fluorescent dye, 620 fluorescent dye, 670 fluorescent dye, 780 fluorescent dye and biotin.

[0013] In a preferred embodiment of the present invention, the correspondence between the fluorescent dye and the protein marker includes: PD-L1 correspondence. 620 fluorescent dye, CTLA-4 corresponding 570 fluorescent dye, corresponding to LAG-3 670 fluorescent dye, corresponding to TIGIT 520 fluorescent dye, TIM-3 corresponding 480 fluorescent dye, PD-1 corresponds to biotin and 780 fluorescent dye.

[0014] This invention also provides the application of the above-mentioned combination of multiplex fluorescent immunohistochemical detection reagents in the preparation of multiplex fluorescent immunohistochemical detection kits.

[0015] In a preferred embodiment of the present invention, the detection biomarkers of the multiplex fluorescent immunohistochemical detection kit include PD-L1, PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT.

[0016] In a preferred embodiment of the present invention, the multiplex fluorescent immunohistochemical detection kit is used to detect tumor samples.

[0017] The present invention also provides a multiplex fluorescent immunohistochemistry detection kit, comprising the above-mentioned multiplex fluorescent immunohistochemistry detection reagent combination, and further comprising buffer, elution buffer, blocking buffer, DAPI nuclear staining reagent, antigen retrieval solution and signal amplification solution.

[0018] The present invention also provides a method for using the above-mentioned multiplex fluorescence immunohistochemistry detection kit, including the following steps: (1) placing the detection sample in On the X30 fully automated staining instrument, after baking, dewaxing, antigen retrieval, buffer washing and blocking, pretreated sections are obtained;

[0019] (2) Pre-treated sections were incubated with PD-L1 monoclonal antibody and then labeled after binding with secondary antibody. The first fluorescently stained section was obtained by eluting and blocking the 620 fluorescent dye.

[0020] (3) The first fluorescently stained sections were incubated with CTLA-4 monoclonal antibody and then labeled with secondary antibody. The 570 fluorescent dye was eluted and then blocked to obtain the second fluorescently stained section;

[0021] (4) The slides were incubated with LAG-3 monoclonal antibody using a second fluorescent staining method, and then labeled after binding with the secondary antibody. The 670 fluorescent dye was eluted and then blocked to obtain the third fluorescently stained section;

[0022] (5) The third fluorescently stained sections were incubated with TIGIT monoclonal antibody and then labeled after binding with secondary antibody. The 520 fluorescent dye was eluted and then blocked to obtain the fourth fluorescently stained section;

[0023] (6) The TIM-3 monoclonal antibody was incubated with the fourth fluorescently stained section and then labeled after binding with the secondary antibody. 480 fluorescent dye was used to elute and block the sample to obtain the fifth fluorescently stained section.

[0024] (7) The fifth fluorescently stained sections were incubated with PD1 monoclonal antibody, and biotin was labeled after binding with secondary antibody. After elution, biotin was labeled again. 780 fluorescent dye and DAPI nuclear dye.

[0025] The present invention also provides the application of the above-mentioned combination of multiplex fluorescent immunohistochemical detection reagents or the above-mentioned multiplex fluorescent immunohistochemical detection kit in the preparation of the following products, the application including at least one of the following: (1) products for predicting the effectiveness of disease treatment;

[0026] (2) Screening products for targeted drugs;

[0027] (3) Products that predict the expression levels of disease protein biomarkers.

[0028] Beneficial Effects: This invention provides a multiplex fluorescence immunohistochemistry (MHI) assay kit, comprising monoclonal antibodies and fluorescent dyes designed for protein biomarkers. It can be applied to the multiplex fluorescence immunohistochemistry detection of protein biomarkers PD-L1, PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT. This invention also provides a kit for multiplex fluorescence immunohistochemistry detection, enabling staining detection of six protein biomarkers on a single slide. This saves sample quantity while increasing the spatial location information of each protein target. It overcomes the problem of similar staining positions of PD-L1, PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT, and achieves multiplex fluorescence staining of disease samples, especially rare gastrointestinal tumors with complex structures, by combining the six protein targets in a specific staining order and fluorescence channels. This prevents false positives caused by crosstalk between channels. The high sensitivity and strong specificity of the biotin-streptavidin system compensate for the weakness of the conventional 780 nm near-infrared spectral channel, which is prone to false negatives.

[0029] This invention targets the aforementioned six targets, all of which have associated marketed or clinically-stage drugs, such as PD-L1 and PD-1 related drugs like Pembrolizumab, nivolumab, atezolizumab, and durvalumab; CTLA-4 related drugs like ipilimumab combined with nivolumab dual immunotherapy; and TIGIT antibody drugs and LAG-3 inhibitors like Relatlimab, which are currently in clinical trials. The multiplex detection method used in this invention can simultaneously detect all six drug-related targets, and the results reflect the expression levels of these targets in the sample, serving as a reference for clinical medication. Rather than being limited to detecting a single immune microenvironment or a single drug-related target using existing technologies, this increases the actual utilization rate of panel channels, providing the possibility of more tangible benefits for a wider range of patients. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of a fluorescence channel without staining crosstalk. In the diagram, A: TIM-3- 480 / CTLA-4- 570 fluorescence channels, B: TIM-3- 480+ DAPI fluorescence channels, C: CTLA-4- 570+ DAPI fluorescence channels;

[0031] Figure 2 This is a schematic diagram of fluorescence channel staining crosstalk. In the diagram, A: TIM-3- 520 / CTLA-4- 570 fluorescence channels, B: TIM-3- 520+DAPI fluorescence channels, C:CTLA-4- 570+ DAPI fluorescence channels;

[0032] Figure 3 This diagram illustrates the combination of 6 target sites and nuclear staining targets in a colon cancer sample. Figure A shows the staining pattern of PD-L1 / PD-1 / CTLA-4 / LAG-3 / TIM-3 / TIGIT, and Figure B shows the staining pattern of TIM-3. 480)+DAPI staining map, C:TIGIT( 520)+DAPI staining map, D:CTLA-4( 570)+DAPI staining map, E:PD-L1( 620)+DAPI staining pattern, F: LAG-3 ( 690)+DAPI staining map, G:PD-1( 780)+DAPI staining map;

[0033] Figure 4 This diagram illustrates the combination of 6 target sites and nuclear staining targets in a gastric stromal tumor sample. Figure A shows PD-L1 / PD-1 / CTLA-4 / LAG-3 / TIM-3 / TIGIT staining, and Figure B shows TIM-3 (…). 480)+DAPI staining map, C:TIGIT( 520)+DAPI staining map, D:CTLA-4( 570)+DAPI staining map, E:PD-L1( 620)+DAPI staining pattern, F: LAG-3 ( 690)+DAPI staining map, G:PD-1( 780)+DAPI staining map;

[0034] Figure 5 This diagram illustrates the combination of six target sites and nuclear staining targets for small intestinal neuroendocrine tumors. Figure A shows PD-L1 / PD-1 / CTLA-4 / LAG-3 / TIM-3 / TIGIT staining, and Figure B shows TIM-3 (…). 480)+DAPI staining map, C:TIGIT( 520)+DAPI staining map, D:CTLA-4( 570)+DAPI staining map, E:PD-L1( 620)+DAPI staining pattern, F: LAG-3 ( 690)+DAPI staining map, G:PD-1( 780)+DAPI staining diagram. Detailed Implementation

[0035] This invention provides a combination of multiplex fluorescent immunohistochemical detection reagents, including monoclonal antibodies and fluorescent dyes designed for protein biomarkers;

[0036] The protein biomarkers include PD-L1, PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT;

[0037] The fluorescent dye is specifically linked to the conjugates of monoclonal antibodies and secondary antibodies designed for each of the protein markers.

[0038] In this invention, there are English abbreviations. The correspondence between each abbreviation and its Chinese name and English full name is shown in Table 1.

[0039] Table 1. Correspondence between the Chinese and English names of this invention

[0040]

[0041] The protein biomarkers PD-L1, PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT described in this invention can serve as diagnostic, therapeutic, and prognostic markers for various diseases. They can be used for the detection of gastrointestinal diseases such as gastrointestinal tumors, especially rare gastrointestinal tumors. Using these as intermediate data, combined with other examinations, they can be used to make disease diagnoses, pre-diagnoses, treatments, prognoses, drug screenings, and evaluations of treatment efficacy. The gastrointestinal tumors described in this invention can be gastric adenocarcinoma or colon cancer, and rare gastrointestinal tumors can be GIST, GI-NETs, ​​gastrointestinal stromal cell sarcoma, or leiomyosarcoma.

[0042] The monoclonal antibodies described in this invention include PD-L1 monoclonal antibody, PD-1 monoclonal antibody, CTLA-4 monoclonal antibody, LAG-3 monoclonal antibody, TIM-3 monoclonal antibody, and TIGIT monoclonal antibody; the secondary antibody includes HPR-labeled anti-mouse / rabbit IgG polymer secondary antibody. This invention does not specifically limit the source of the monoclonal antibodies and secondary antibodies; conventional commercially available products in the art can be used.

[0043] This invention does not specifically limit the type of fluorescent dye, as long as it can be linked to a single protein marker-monoclonal antibody-secondary antibody complex. For example, in one embodiment of this invention, a specific fluorescent dye was selected. 480 fluorescent dye, 520 fluorescent dye, 570 fluorescent dye, 620 fluorescent dye, 670 fluorescent dye, 780 fluorescent dyes and biotin, and each fluorescent dye also has a correspondence with a protein marker: PD-L1 corresponds to 620 fluorescent dye, CTLA-4 corresponding 570 fluorescent dye, corresponding to LAG-3 670 fluorescent dye, corresponding to TIGIT 520 fluorescent dye, TIM-3 corresponding 480 fluorescent dye, PD-1 corresponds to biotin and 780 fluorescent dyes. In one embodiment of the present invention, the above-mentioned seven fluorescent dyes are selected from Alpha. 7-color fluorescent staining kit - AlphaX Bio (Catalog#AXT37100041).

[0044] This invention also provides the application of the above-mentioned combination of multiplex fluorescent immunohistochemical detection reagents in the preparation of multiplex fluorescent immunohistochemical detection kits.

[0045] The multiplex immunohistochemical assay kit of this invention detects biomarkers including PD-L1, PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT. The multiplex immunohistochemical assay kit of this invention can be used to detect tumor samples, especially gastrointestinal tumor samples.

[0046] The present invention also provides a multiplex fluorescent immunohistochemistry detection kit, comprising the above-mentioned multiplex fluorescent immunohistochemistry detection reagent combination, and further comprising buffer, elution buffer, blocking buffer, DAPI nuclear staining reagent, antigen retrieval solution and signal amplification solution.

[0047] In a preferred embodiment of the present invention, the buffer solution is a mixed solution composed of tris(hydroxymethyl)aminomethane, sodium chloride, Tween-20 and ProClin300;

[0048] The sealing solution is a mixed solution composed of BSA, TBS and ProClin300;

[0049] The signal amplification solution is a mixed solution composed of magnesium sulfate, 4-bromophenylboronic acid and Tris-HCl.

[0050] The antigen retrieval solution is a solution composed of disodium ethylenediaminetetraacetate and tris(hydroxymethyl)aminomethane.

[0051] The elution buffer is a solution composed of disodium ethylenediaminetetraacetate and tris(hydroxymethyl)aminomethane, with the same composition as the antigen retrieval buffer. It is used to elute the primary and secondary antibody complexes after one round of fluorescent staining.

[0052] The specifications of the multiplex immunohistochemistry kit constructed in this invention are shown in Table 2:

[0053] Table 2. One specification of the multiplex immunohistochemistry reagent kit of the present invention.

[0054]

[0055] The present invention also provides a method for using the above-mentioned multiplex fluorescence immunohistochemistry detection kit, including the following steps: (1) placing the detection sample in On the X30 fully automated staining instrument, after baking, dewaxing, antigen retrieval, buffer washing and blocking, pretreated sections are obtained;

[0056] (2) Pre-treated sections were incubated with PD-L1 monoclonal antibody and then labeled after binding with secondary antibody. The first fluorescently stained section was obtained by eluting and blocking the 620 fluorescent dye.

[0057] (3) The first fluorescently stained sections were incubated with CTLA-4 monoclonal antibody and then labeled with secondary antibody. The 570 fluorescent dye was eluted and then blocked to obtain the second fluorescently stained section;

[0058] (4) The slides were incubated with LAG-3 monoclonal antibody using a second fluorescent staining method, and then labeled after binding with the secondary antibody. The 670 fluorescent dye was eluted and then blocked to obtain the third fluorescently stained section;

[0059] (5) The third fluorescently stained sections were incubated with TIGIT monoclonal antibody and then labeled after binding with secondary antibody. The 520 fluorescent dye was eluted and then blocked to obtain the fourth fluorescently stained section;

[0060] (6) The TIM-3 monoclonal antibody was incubated with the fourth fluorescently stained section and then labeled after binding with the secondary antibody. 480 fluorescent dye was used to elute and block the sample to obtain the fifth fluorescently stained section.

[0061] (7) The fifth fluorescently stained sections were incubated with PD1 monoclonal antibody, and biotin was labeled after binding with secondary antibody. After elution, biotin was labeled again. 780 fluorescent dye and DAPI nuclear dye.

[0062] In this invention, the fluorescent dye is used 480 520 570 620 670 and When staining 780, a fluorescent dye working solution is required. The fluorescent dye working solution uses a signal amplification solution as a solvent, and is prepared by mixing the fluorescent dye stock solution and the signal amplification solution in the kit at a volume ratio of 1:150.

[0063] The detection platform of the multiplex immunohistochemistry kit of the present invention includes: The X30 multi-target pathological staining system (Alpha X Bio) and the ZEISS AXIOSCAN 7 whole-slice imaging system (ZEISS) are used in conjunction with the X30 multi-target pathological staining system. The detection process includes dewaxing, antigen retrieval, cycling labeling with six antibodies and their combinations (primary antibody incubation, secondary antibody binding, and fluorescein labeling), and nuclear counterstaining. The entire staining process is carried out within [the specified timeframe]. The staining process was performed using the X30 fully automated staining system, with strict control over the time and temperature of each reagent incubation step. Dewaxing and antigen retrieval exposed the antigens in the tissue sections. Antibodies against PD-L1, CTLA-4, LAG-3, TIGIT, TIM-3, and PD1 were used to specifically bind to their corresponding proteins in the tissue. The secondary antibody from the multiplex immunohistochemical staining kit was bound to the primary antibody, and then fluorescein was used to conjugate the secondary antibody. After six cycles of labeling, the staining was completed by DAPI nuclear counterstaining. After mounting with anti-fluorescence attenuation mounting media, the expression of PD-L1, CTLA-4, LAG-3, TIGIT, TIM-3, and PD1 proteins in the samples could be observed using a fluorescence scanning imaging system and software.

[0064] The staining procedure for the target protein described in this invention is shown in Table 3. The room temperature referred to in this invention is 15-30°C.

[0065] Table 3. Staining procedure for the target protein of this invention.

[0066]

[0067]

[0068]

[0069] The antibody staining sequence and fluorescein pairings for each protein target in the detection method of this invention are shown in Table 4. Multiplex fluorescence staining of the samples is performed on the six protein targets using the specific staining sequence and fluorescence channel pairings shown in the table below, demonstrating no staining crosstalk at their respective specific locations. In this embodiment of the invention, the target of the DAPI used is the cell nucleus, and the color is blue.

[0070] Table 4. Staining order and fluorescein pairings

[0071]

[0072] The present invention also provides the application of the above-mentioned combination of multiplex fluorescent immunohistochemical detection reagents or the above-mentioned multiplex fluorescent immunohistochemical detection kit in the preparation of the following products, the application including at least one of the following: (1) products for predicting the effectiveness of disease treatment;

[0073] (2) Screening products for targeted drugs;

[0074] (3) Products that predict the expression levels of disease protein biomarkers.

[0075] The detection range of the detection reagent combination and detection kit described in this invention includes various subtypes of gastrointestinal tumors, such as gastric adenocarcinoma, colon cancer, GIST, GI-NETs, ​​gastrointestinal stromal cell sarcoma, or leiomyosarcoma.

[0076] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a multiplex fluorescent immunohistochemical detection kit for rare gastrointestinal tumors provided by the present invention and its application, should not be construed as limiting the scope of protection of the present invention.

[0077] Unless otherwise specified, all materials used in the embodiments of this invention are conventional commercially available products in the art. For example, the specific sources of the monoclonal antibodies used in the embodiments are as follows:

[0078] (1)PD-L1 Recombinant Rabbit Monoclonal Antibody[PD01-02]-HUABIO(Catalog#HA721176);

[0079] (2)PD1 Recombinant Rabbit Monoclonal Antibody[SJ01-91]-HUABIO(Catalog#ET1606-41);

[0080] (3)LAG-3Recombinant Rabbit Monoclonal Antibody[PS01-33]-HUABIO(Catalog#HA721358);

[0081] (4)TIM 3Mouse Monoclonal Antibody[1E3]-HUABIO(Catalog#EM1701-19);

[0082] (5)CTLA-4Recombinant Rabbit Monoclonal Antibody[PS01-34]-HUABIO(Catalog#HA721269);

[0083] (6) TIGIT(E5Y1W) RabbitmAb-CST (Catalog #99567).

[0084] The detection platform used in this embodiment of the invention is X30 Multi-Target Pathological Staining Machine - Alpha X Bio and ZEISS AXIOSCAN 7 Whole Slice Imaging System - ZEISS.

[0085] Example 1

[0086] The detection process includes dewaxing, antigen retrieval, cycling labeling with six antibodies and their combinations (primary antibody incubation, secondary antibody binding, and fluorescein labeling), and nuclear counterstaining. The entire staining process takes place within... The staining process was completed on a fully automated staining instrument, with the time and temperature of each reagent incubation step strictly controlled by the instrument. Dewaxing and antigen retrieval exposed the antigens in the tissue sections. PD-L1, PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT antibodies were used to specifically bind to the corresponding proteins in the tissue. The secondary antibody from the multiplex immunohistochemical staining kit was then bound to the primary antibody, and fluorescein was used to ligate and label the secondary antibody. The staining process was completed after six cycles of labeling and DAPI nuclear counterstaining. The specific procedure is shown in Table 3, using the optimized parameters listed in Table 3.

[0087] After the stained sections are mounted with anti-fluorescence attenuation mounting medium, the expression of PD-L1, PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT proteins in the samples can be observed using a fluorescence scanning imaging system and software.

[0088] Six protein targets were used for multiplex fluorescence staining of the samples in the specific staining order and fluorescence channel combination shown in Table 3, such as... Figure 1 As shown, TIM-3 positive cells (cyan) and CTLA-4 positive cells (orange) are located at their respective specific positions, indicating no staining crosstalk in the fluorescence channels.

[0089] Comparative Example 1

[0090] Except for the staining procedure shown in Table 5, all other operations were the same as in Example 1, and the results were as follows: Figure 2 As shown, TIM-3 positive cells (green) and CTLA-4 positive cells (orange) overlap in most positions (yellow), indicating staining crosstalk between the two fluorescence channels, which causes false positives in one channel.

[0091] Example 2: Colon Cancer

[0092] After undergoing the same staining and imaging procedures as in Example 1, the following schematic diagrams were obtained: 6 target points combined with nuclear staining targets, and individual target points combined with nuclear staining targets. Figure 3 It can assess the expression levels of each target. The expression levels of TIM-3, TIGIT, CTLA-4, PD-L1, LAG-3, and PD-1 were approximately 1%, 10%, 5%, 1%, 10%, and 1%, respectively.

[0093] Example 3: Gastrointestinal stromal tumor

[0094] After undergoing the same staining and imaging procedures as in Example 1, the following schematic diagrams were obtained: 6 target points combined with nuclear staining targets, and individual target points combined with nuclear staining targets. Figure 4 It can assess the expression levels of each target. The expression levels of TIM-3, TIGIT, CTLA-4, PD-L1, LAG-3, and PD-1 were approximately 5%, 1%, 5%, 0%, 1%, and 1%, respectively.

[0095] Example 4: Small intestinal neuroendocrine tumor

[0096] After undergoing the same staining and imaging procedures as in Example 1, the following schematic diagrams were obtained of the combinations of the six target points with nuclear staining targets and the combinations of each target point alone with nuclear staining targets. Figure 5 It can assess the expression levels of each target. The expression levels of TIM-3, TIGIT, CTLA-4, PD-L1, LAG-3, and PD-1 were approximately 1%, 1%, 5%, 5%, 0%, and 1%, respectively.

[0097] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A multiplex fluorescent immunohistochemical detection reagent combination, characterized in that, This includes monoclonal antibodies and fluorescent dyes designed for protein biomarkers; The protein biomarkers include PD-L1, PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT; The fluorescent dye is specifically linked to the conjugates of monoclonal antibodies and secondary antibodies designed for each of the protein markers.

2. The multiplex fluorescent immunohistochemical detection reagent combination according to claim 1, characterized in that, The monoclonal antibodies include PD-L1 monoclonal antibody, PD-1 monoclonal antibody, CTLA-4 monoclonal antibody, LAG-3 monoclonal antibody, TIM-3 monoclonal antibody and TIGIT monoclonal antibody; The secondary antibody includes an HPR-labeled anti-mouse / rabbit IgG polymeric secondary antibody.

3. The multiplex fluorescent immunohistochemical detection reagent combination according to claim 1 or 2, characterized in that, The fluorescent dye includes 480 fluorescent dye, 520 fluorescent dye, 570 fluorescent dye, 620 fluorescent dye, 670 fluorescent dye, 780 fluorescent dye and biotin.

4. The multiplex fluorescent immunohistochemical detection reagent combination according to claim 3, characterized in that, The correspondence between the fluorescent dye and the protein marker includes: PD-L1 correspondence 620 fluorescent dye, CTLA-4 corresponding 570 fluorescent dye, corresponding to LAG-3 670 fluorescent dye, corresponding to TIGIT 520 fluorescent dye, TIM-3 corresponding 480 fluorescent dye, PD-1 corresponds to biotin and 780 fluorescent dye.

5. The use of the combination of multiplex fluorescent immunohistochemical detection reagents according to any one of claims 1 to 4 in the preparation of multiplex fluorescent immunohistochemical detection kits.

6. The application according to claim 5, characterized in that, The biomarkers detected by the multiplex fluorescent immunohistochemistry assay kit include PD-L1, PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT.

7. The application according to claim 5, characterized in that, The multiplex fluorescent immunohistochemistry detection kit can be used to detect tumor samples.

8. A multiplex fluorescent immunohistochemical detection kit, characterized in that, The reagent combination includes the multiplex fluorescent immunohistochemical detection reagent as described in any one of claims 1 to 4, and further includes buffer, elution buffer, blocking buffer, DAPI nuclear staining reagent, antigen retrieval solution and signal amplification solution.

9. The method of using the multiplex fluorescence immunohistochemistry detection kit according to claim 8, characterized in that, The steps include: (1) placing the test sample in On the X30 fully automated staining instrument, after baking, dewaxing, antigen retrieval, buffer washing and blocking, pretreated sections are obtained; (2) Pre-treated sections were incubated with PD-L1 monoclonal antibody and then labeled with secondary antibody. The first fluorescently stained section was obtained by eluting and blocking the 620 fluorescent dye. (3) The first fluorescently stained sections were incubated with CTLA-4 monoclonal antibody and then labeled with secondary antibody. The 570 fluorescent dye was eluted and then blocked to obtain the second fluorescently stained section; (4) The slides were incubated with LAG-3 monoclonal antibody using a second fluorescent staining method, and then labeled after binding with the secondary antibody. The 670 fluorescent dye was eluted and then blocked to obtain the third fluorescently stained section; (5) The third fluorescently stained sections were incubated with TIGIT monoclonal antibody and then labeled after binding with secondary antibody. 520 fluorescent dye was used to elute and then block the sample to obtain the fourth fluorescently stained section. (6) The TIM-3 monoclonal antibody was incubated with the fourth fluorescently stained section and then labeled after binding with the secondary antibody. 480 fluorescent dye was used, and after elution and blocking, the fifth fluorescently stained section was obtained. (7) The fifth fluorescently stained sections were incubated with PD1 monoclonal antibody, and biotin was labeled after binding with secondary antibody. After elution, biotin was labeled again. 780 fluorescent dye and DAPI nuclear dye.

10. The use of the combination of multiplex fluorescent immunohistochemical detection reagents according to any one of claims 1 to 4 or the multiplex fluorescent immunohistochemical detection kit according to claim 8 in the preparation of the following products, characterized in that, The application includes at least one of the following: (1) products for predicting the effectiveness of disease treatment; (2) Screening products for targeted drugs; (3) Products that predict the expression levels of disease protein biomarkers.

Images