Kits for multiplex immunoassays and their applications

Abstract

The present invention belongs to the technical field of biochemical detection, and specifically relates to a kit and application for multiple immunoassays. It can be used for multi-target detection of circulating tumor cells in different body fluids such as blood, or for projects involving multi-target detection in tissue sections, cell slides and other detection processes. It is particularly suitable for multi-target staining experiments of antibodies of the same species but different subtypes. By digesting the anti-mouse IgG antibodies obtained by goat immunization with papain, purifying the Fab fragments and labeling them with dyes, a GM Fab secondary antibody with subtype recognition ability is prepared, which is used for specific blocking operations after multiple rounds of primary antibody staining, significantly reducing cross-interference between different primary antibodies. After multiple staining verification in tumor tissues and circulating tumor cells after blood enrichment, the results show that the signal distribution of each target is clear, the positioning is accurate, and the background interference is low, which is suitable for tumor immune microenvironment analysis and immunotherapy evaluation.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemical detection, and particularly relates to a kit for multiple immunoassay and its application. Background Art

[0002] Multiplex immunoassay is a technology that simultaneously detects multiple target proteins in the same tissue or cell sample. It is widely used in the fields of circulating tumor cell detection, tumor microenvironment analysis, immune cell phenotyping, and disease heterogeneity research.

[0003] Currently, multiplex immunostaining techniques primarily rely on combinations of primary antibodies from different sources (species) in order to perform multiple rounds of staining with corresponding species-specific secondary antibodies, thereby avoiding the problem of cross-recognition of secondary antibodies. However, the majority of primary antibodies available in real-world applications are mouse-derived monoclonal antibodies, with a limited number of other primary antibodies, such as rabbit, rat, and human (recombinant), being the most widely used in clinical pathology and basic scientific research. They offer advantages such as high target specificity and good batch stability, but they still cannot meet the requirements of a variety of immunofluorescence assays. Therefore, in scenarios where multiple primary antibodies are used simultaneously, traditional species differentiation strategies are difficult to apply, and can easily lead to cross-reactions between secondary antibodies in different rounds, thereby affecting detection specificity and image interpretation accuracy. This difficulty has resulted in current circulating tumor cell testing being limited to detecting a limited number of markers. Furthermore, in the detection of tissue sections, multiple sections of the same tissue are required for the detection of different markers. Tumor heterogeneity also affects the accuracy of test results.

[0004] To address signal interference from the same primary antibody during multiple rounds of staining, a series of blocking or signal elimination techniques have been developed. These include antigen shedding, heat repair to remove antibodies, and chemical methods to destroy the previous round of labeling. However, these methods are often associated with issues such as antigen damage, decreased signal intensity, or increased background staining, compromising detection sensitivity and reliability.

[0005] Therefore, how to achieve sequential immunodetection of multiple primary antibodies of the same species in tissue samples while retaining the convenience and signal quality of conventional primary antibodies and effectively avoiding nonspecific staining caused by cross-reactions is a technical challenge that needs to be urgently solved in the current field of multiple immunostaining technology. Summary of the Invention

[0006] To address the cross-reaction problem of primary antibodies of the same species in existing multiple immunoassay technologies, particularly the signal interference problem caused by traditional secondary antibodies when multiple monoclonal antibodies need to be sequentially applied to the same sample for sequential staining, the present invention provides a kit and application method for multiple immunoassays, which can achieve high-specificity, low-background co-staining of multiple targets in tissue or cell samples without changing the species and structure of conventional primary antibodies, significantly improving staining accuracy and spatial resolution.

[0007] Specifically, the present invention proposes: a kit for multiple immunoassay, which contains a goat anti-mouse secondary antibody (GM Fab secondary antibody) that has been purified by papain hydrolysis and retains only the Fab segment and lacks the Fc segment, wherein the amino acid sequence of the light chain variable region of the GMFab secondary antibody is SEQ ID NO: 1, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 2, and are encoded by SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The addition of the GM Fab secondary antibody after the first round of immune reaction can form a specific binding with the primary antibody bound to the sample and block its antigen binding site, thereby blocking subsequent cross-reactions with the same species primary antibody.

[0008] In a preferred technical solution, it also includes a detection reagent that is compatible with the GM Fab secondary antibody. The detection reagent is selected from one or more of a fluorescent dye, horseradish peroxidase, alkaline phosphatase or a biotin labeling system, and can be coupled to the GM Fab secondary antibody through a flexible linker peptide segment SEQ ID NO: 5.

[0009] In a preferred technical solution, the working concentration of the GM Fab secondary antibody in the final reaction system is 0.1-10 μg / mL.

[0010] In a preferred technical solution, it also comprises a diluent, a washing solution and a blocking solution suitable for immunohistochemistry or immunofluorescence staining.

[0011] The present invention also provides the use of the GM Fab secondary antibody in the preparation of a blocking agent for sequential detection of primary antibodies of the same species in multiple immunoassays. By adding the GM Fab secondary antibody after each round of immune reaction to block the bound primary antibody, cross-reaction of subsequent primary antibodies is avoided and signal specificity is ensured.

[0012] In a preferred technical solution, the sample for the multiple immunoassay is at least one of a tissue section, a cell slide or a smear.

[0013] The present invention also provides a multiplex immunoassay method for sequentially detecting multiple primary antibodies of the same species using the kit, comprising the following steps:

[0014] S1. Perform the first round of immune reaction on the sample to be tested and add the primary antibody that can be used to prepare monoclonal antibodies, including rat, rabbit, and human monoclonal antibodies.

[0015] S2. After incubation, the GM Fab secondary antibody is added and incubated to block the primary antibody bound in step S1;

[0016] S3, repeating steps S1-S2 at least once, using a different primary antibody each time and adding the GM Fab secondary antibody for blocking after each primary antibody incubation;

[0017] S4. Perform color development or fluorescence detection on the final signal to obtain spatial expression information of multiple targets.

[0018] In a preferred technical solution, the number of the multiple targets is 2-8, and the spectral overlap between the detection signals is less than 10%.

[0019] In a preferred technical solution, the multiplex immunoassay is used for co-staining and spatial heterogeneity analysis of immune-related markers such as PD-L1, CD8, and Ki-67 in tumor tissues.

[0020] In another aspect, the present invention provides an expression vector comprising a nucleic acid molecule as described herein.

[0021] In another aspect, the present invention provides a host cell comprising the expression vector as described herein.

[0022] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain an origin of replication.

[0023] Beneficial effects

[0024] The present invention provides a kit for multiple immunoassays and its application, which has the following significant technical effects and advantages:

[0025] 1. This invention effectively overcomes the problem of cross-reactivity with the same-species primary antibody during multiple rounds of immunostaining by introducing a papain-purified goat anti-mouse secondary antibody (GM Fab secondary antibody) that retains only the Fab fragment and lacks the Fc fragment. Because this Fab structure lacks the Fc fragment, it is not recognized by subsequent conventional anti-mouse secondary antibodies and does not nonspecifically bind to Fc receptors in tissues, significantly reducing background interference and improving the specificity and accuracy of the staining signal.

[0026] 2. The GM Fab secondary antibody possesses a single antigen-binding site, enabling selective binding to the primary antibody already bound to the sample surface after the first round of immune response, blocking its antigen-binding region and thereby creating an "antibody barrier" that blocks the nonspecific binding pathways of subsequent primary antibodies, enabling sequential immunoassay detection of multiple antibodies in the same sample. This strategy eliminates the need to change the primary antibody species, simplifies the protocol, and significantly enhances the method's versatility and clinical adaptability.

[0027] 3. The GM Fab secondary antibody in the kit of this invention can be flexibly coupled to fluorescent dyes and connected to functional tags via a flexible linker peptide, maintaining the antigen recognition ability and conformational stability of the Fab structure, making it compatible with a variety of colorimetric or fluorescent detection systems and suitable for various staining platforms such as immunohistochemistry and immunofluorescence.

[0028] 4. This invention is suitable for spatial expression co-staining of 2 to 8 different targets, which can achieve high-resolution, multi-target spatial information acquisition, and is conducive to revealing complex biological phenomena such as immune cell distribution and signal partitioning in the tumor microenvironment.

[0029] In summary, the kit and application scheme for multiple immunoassay provided by the present invention not only improve the specificity and stability of detection, but also significantly expand the application scope of antibodies of the same species in multiple rounds of immunostaining. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic diagram of the method flow of embodiment 1 of the present invention;

[0031] Figure 2 The multiple fluorescence images of the first embodiment of the present invention;

[0032] Figure 3 This is the multiple fluorescence image of Example 2 of the present invention;

[0033] Figure 4 This is the multiple fluorescence image of Example 3 of the present invention;

[0034] Figure 5This is a multiple fluorescence image according to the fourth embodiment of the present invention. DETAILED DESCRIPTION

[0035] The following examples are used to further illustrate the present invention, but are not intended to limit the scope of protection of the present invention. Unless otherwise specified, all reagents used in the examples were commercially available analytical grade reagents or prepared according to conventional techniques.

[0036] Example 1: Preparation and application of GM Fa secondary antibody in multiplex immunoassay

[0037] The method flow is as follows Figure 1 As shown:

[0038] Preparation of S1 and GM Fab secondary antibodies

[0039] Healthy goats were used as immunization hosts and injected with mouse IgG antigens via a standard immunization protocol to induce the production of high-titer anti-mouse IgG antibodies. After the immunization cycle, goat serum was collected and total IgG was purified using affinity chromatography (Protein G column).

[0040] Place the purified goat IgG in a papain digestion system to prepare the following digestion solution:

[0041] Final IgG concentration: 5 mg / mL;

[0042] Papain concentration: 0.05 mg / mL;

[0043] Digestion buffer: 20 mM phosphate buffer (pH 7.0), 10 mM EDTA, 20 mM cysteine;

[0044] The mixture was incubated at 37°C in a constant temperature shaker for 6 hours. After the reaction was completed, iodoacetamide was added to terminate the digestion.

[0045] The reaction solution was purified using a Protein A column to remove the Fc fragment and unreacted whole antibody. The unbound fraction was collected as the target GM Fab secondary antibody. The fraction was further concentrated by ultrafiltration and buffer exchanged (PBS) to a final concentration of 1 mg / mL. The fraction was sterilized with a 0.22 μm filter and stored in aliquots at -20°C.

[0046] Confirmation and expression of S2 and GM Fab secondary antibody sequences

[0047] Amino acid sequencing of the GM Fab secondary antibody revealed its light chain variable region (VL) and heavy chain variable region (VH) sequences as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Based on these sequences, expression vectors were constructed by optimizing codon usage to obtain the light chain and heavy chain coding sequences (SEQ ID NO: 3 and SEQ ID NO: 4), respectively. These sequences were then transfected into the HEK293 expression system. Following affinity purification and functional verification, the resulting recombinant GM Fab secondary antibody demonstrated comparable performance to the native Fab fragment and could be used as an alternative.

[0048] S3. Multiple Immunostaining Applications

[0049] Paraffin-embedded human breast cancer tissue sections were selected and, after dewaxing, hydration, antigen retrieval, and endogenous enzyme blocking, the following procedures were performed in sequence:

[0050] First round of immune response: add mouse-derived anti-PD-L1 monoclonal antibody (IgG1) and incubate overnight at 4°C;

[0051] First blocking: After washing at room temperature, add GM Fab secondary antibody (prepared in this example, 1 μg / mL) and incubate at 37°C for 30 minutes to block the bound primary antibody;

[0052] Second round of immune response: Add mouse-derived anti-CD8 antibody (IgG2a) and incubate at 4°C;

[0053] Second blocking: Repeat GM Fab secondary antibody blocking as above;

[0054] The third round of immune response: mouse anti-Ki-67 antibody (IgG2b) was added and incubated at 4°C;

[0055] Final detection: incubate with secondary antibodies labeled with anti-mouse IgG1, IgG2a, and IgG2b subtype-specific dyes (labeled with Cy3, FITC, and DAPI), counterstain nucleic acids with DAPI, and wash and seal with PBS.

[0056] S4. Image analysis and effect evaluation

[0057] The stained samples were imaged using a multi-channel confocal scanning microscope, and the results showed:

[0058] The signals of each target (PD-L1, CD8, Ki-67) are clearly distributed without obvious cross-interference, such as Figure 2 As shown, PD-L1 is marked in yellow, CD8 is marked in green, and Ki-67 is marked in blue. The three marker colors are clearly distinguished, and there is no overlap of counterstaining.

[0059] The background signal was significantly reduced, especially in the highly expressed areas, and no nonspecific enrichment was observed;

[0060] Spatial colocalization analysis of three primary antibodies of the same mouse species in a single tissue sample was achieved.

[0061] This example effectively solves the cross-reaction problem existing in traditional multiple staining of antibodies of the same species by using the GM Fab secondary antibody provided by the present invention, and realizes high-specificity and high-throughput tissue multi-marker detection. It is applicable to various pathology platforms such as immunohistochemistry (IHC) and immunofluorescence (IF), and is particularly suitable for multi-target co-staining experiments in tumor immune microenvironment analysis and immunotherapy response evaluation.

[0062] Example 2: Application of GM Fab secondary antibody based on biotin labeling system in multiple immunostaining

[0063] S1. Preparation of biotinylated GM Fab secondary antibody

[0064] The GM Fab secondary antibody solution (1 mg / mL) purified in Example 1 was covalently labeled with NHS-biotin (N-Hydroxysuccinimide-Biotin). The reaction conditions were as follows:

[0065] GM Fab secondary antibody final concentration: 1 mg / mL;

[0066] NHS-biotin addition amount: molar ratio 1:20;

[0067] Reaction buffer: 0.1 M NaHCO3, pH 8.5;

[0068] Reaction temperature: room temperature;

[0069] Reaction time: 2 hours;

[0070] After the reaction, use a desalting column or ultrafiltration centrifuge tube (10 kDa) to remove free NHS-biotin, and replace the buffer with PBS to obtain biotin-labeled GM Fab secondary antibody (Biotin-GM Fab), which is stored at -20°C until use.

[0071] S2. Tissue sample preparation and staining process

[0072] Human colon cancer paraffin-embedded (FFPE) tissue sections were selected and, after dewaxing, hydration, antigen retrieval, and blocking, three rounds of primary antibody staining with the same species were performed according to the following protocol:

[0073] First round of dyeing

[0074] Add mouse anti-CD3 antibody (IgG1) and incubate overnight at 4°C;

[0075] After washing, Biotin-GM Fab secondary antibody (1 μg / mL) was added and incubated at 37°C for 30 min;

[0076] Then add HRP-labeled avidin (streptavidin-HRP) and incubate at room temperature for 20 minutes;

[0077] Color development was performed using AEC substrate (red signal) for 5 minutes, followed by termination of the reaction and washing.

[0078] Second round of dyeing

[0079] Mouse anti-CD68 antibody (IgG1) was added and incubated at 4°C;

[0080] Repeat the Biotin-GM Fab secondary antibody blocking step;

[0081] Streptavidin-AP (alkaline phosphatase) was added again for incubation, and NBT / BCIP was used for color development (blue signal);

[0082] Washing treatment.

[0083] Third round of dyeing

[0084] Mouse anti-Ki-67 antibody (IgG1) was added and incubated at 4°C;

[0085] Biotin-GM Fab secondary antibody was also used for blocking;

[0086] Then, fluorescently labeled avidin (streptavidin-Alexa Fluor 488) was added;

[0087] The sections were mounted after DAPI counterstaining.

[0088] S3. Image Acquisition and Analysis

[0089] A compound microscope was used for observation and image acquisition under bright field and fluorescence channels respectively. Figure 3 The display shown:

[0090] CD3-positive T cells appear red and are mainly distributed around the tumor;

[0091] CD68-positive macrophages are blue and distributed in the interstitial area;

[0092] Ki-67-positive proliferating cells showed green fluorescence inside the tumor nests;

[0093] DAPI staining is clear and has no background contamination;

[0094] The signal distribution among the three targets was clear and the positioning was accurate, with no cross-staining observed;

[0095] The tissue structure was intact, and no nonspecific background staining or signal diffusion was observed.

[0096] This example fully demonstrates the advantages of combining a GM Fab secondary antibody with a biotin-avidin system. By constructing a sequential blocking strategy, we successfully performed sequential detection of three primary antibodies from the same species in the same sample, compatible with multiple detection methods (enzyme-linked, fluorescent, etc.). This method offers high throughput, specificity, and sensitivity, and is widely applicable to multi-target detection scenarios in pathology analysis, immune monitoring, and drug development.

[0097] Example 3: Sequential blocking of GM Fab secondary antibodies in triple immunofluorescence staining of CD3 (green), Ki-67 (red), and CD68 (yellow)

[0098] Preparation and fluorescent labeling of S1 GM Fab secondary antibody

[0099] Purified GM Fab secondary antibodies (1 mg / mL) were obtained according to the methods in Example 1, S1-S2. To adapt to three-channel detection, the Fab fragment was covalently conjugated with FITC, TRITC, and Alexa 546 NHS-ester reagents at a molar ratio of 1:15 in 0.1 M NaHCO3 (pH 8.3) buffer for 2 h. Free dye was removed by dialysis to obtain three single-color labeled GM Fab secondary antibodies:

[0100] FITC-Fab (green; used for the first round of blocking);

[0101] TRITC-Fab (red; used for the second round of blocking);

[0102] Alexa 546-Fab (yellow; used for the third round of blocking);

[0103] S2 Tissue samples and pretreatment

[0104] Paraffin sections (4 µm) of human non-small cell lung cancer were obtained. After deparaffinization with xylene and hydration with graded ethanol, sections were microwave-fixed in 10 mM citrate buffer (pH 6.0) for 15 minutes. After cooling, sections were incubated with 3% hydrogen peroxide for 10 minutes to remove endogenous peroxidases and then blocked with 5% goat serum for 15 minutes at room temperature.

[0105] S3 three rounds of sequential immunostaining

[0106] First round: add mouse monoclonal antibody anti-CD3 (1 μg / mL) dropwise and incubate at 37 °C for 45 min; wash three times with PBS; add FITC-Fab (1 μg / mL) and incubate at 37 °C for 20 min to block the bound primary antibody; wash with PBS and inactivate the residual activity with 0.02N HCl for 1 min.

[0107] Second round: Add mouse monoclonal antibody anti-Ki-67 (2 μg / mL) and incubate at 37 °C for 45 min; wash with PBS; add TRITC-Fab (1 μg / mL) and incubate at 37 °C for 20 min; wash with PBS and inactivate.

[0108] Round 3: Add mouse monoclonal antibody anti-CD68 (1 µg / mL) and incubate in the same manner; wash with PBS; add Alexa 546-Fab (1 µg / mL) and incubate at 37 °C for 20 min; wash with PBS, counterstain cell nuclei with DAPI (1 µg / mL) for 5 min, wash with PBS, and then mount the slides with fluorescent mounting solution.

[0109] S4 Image Acquisition and Effect Evaluation

[0110] The slices were imaged using a confocal laser scanning microscope equipped with three laser channels. Green, red, yellow, and blue images were collected in the FITC, TRITC, Alexa 546, and DAPI channels, respectively. Figure 4 As shown:

[0111] CD3 signals were clearly distributed in the tumor edge area and appeared green;

[0112] Ki-67 positive cells are concentrated in the nuclear region and appear red;

[0113] CD68-positive signals mainly appeared in macrophages in the tumor interstitial area and appeared yellow;

[0114] Background DNA appears blue;

[0115] No cross-contamination or signal crosstalk between channels was observed.

[0116] This example uses a fluorescent dye-labeled GM Fab secondary antibody for specific blocking after each round of immunostaining, effectively preventing cross-binding between primary antibodies of different subtypes within the same species. Through sequential staining and Fab segment blocking, multiple immunostaining with three mouse homologous antibodies for CD3, Ki-67, and CD68 was successfully achieved in the same tissue section. This method is simple to use, offers clear staining signals, low background, and high reproducibility, making it suitable for studying the tumor immune microenvironment and analyzing the spatial localization of multiple targets.

[0117] Example 4: Application of GM Fab Secondary Antibody in Multiple Immunofluorescence Detection of CTC Multi-markers

[0118] S1. Sample preparation and pretreatment

[0119] A 5 mL peripheral blood sample from a liver cancer patient was collected and enriched for peripheral blood mononuclear cells (PBMCs) using density gradient centrifugation. The cells were then transferred to a multichannel microfluidic chip or polylysine-coated slide. The cells were fixed with 4% paraformaldehyde for 10 minutes, rinsed with PBS, and permeabilized with 0.1% Triton X-100 for 5 minutes. Nonspecific sites were then blocked with 3% BSA and incubated at room temperature for 30 minutes.

[0120] S2 and GM Fab secondary antibody preparation and sequence characteristics

[0121] The GM Fab secondary antibody is derived from IgG antigens from healthy goats immunized against mice. It is obtained by papain digestion, retaining only the Fab fragment and removing the Fc fragment. This Fab fragment is purified by affinity chromatography, concentrated, and buffered with PBS to a final concentration of 1 mg / mL.

[0122] The amino acid sequence of its light chain variable region is SEQ ID NO: 1, the amino acid sequence of its heavy chain variable region is SEQ ID NO: 2, and the corresponding nucleotide sequences are SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Both can be obtained by HEK293 cell expression and recombination, and the performance is consistent with that of natural Fab.

[0123] S3. Multiple immunofluorescence staining steps

[0124] After the sample is permeabilized and blocked, the following immune reactions and GM Fab secondary antibody blocking procedures are performed in sequence:

[0125] First round of immune response: CD45 detection (leukocyte marker); mouse anti-human CD45 antibody (IgG1, 1 µg / mL) was added and incubated overnight at 4°C; the next day, after washing, GM Fab secondary antibody (1 µg / mL) was added and incubated at 37°C for 30 minutes; fluorescence was developed using an anti-IgG1 secondary antibody labeled with Alexa Fluor™ 555 (orange).

[0126] Second round of immunoreaction: pan-CK detection (epithelial marker): Mouse anti-human pan-CK antibody (IgG2a, 1 µg / mL) was added, incubated, and blocked; anti-IgG2a secondary antibody labeled with Alexa Fluor™ 488 (green) was used for color development.

[0127] Third round of immunoreaction: EpCAM detection (cell adhesion marker): Mouse anti-human EpCAM antibody (IgG2b, 1µg / mL) was added, incubated, and blocked; anti-IgG2b secondary antibody labeled with Alexa Fluor™ 532 (yellow) was used for color development.

[0128] Fourth round of immunoreaction: Vimentin detection (mesenchymal marker) was performed by adding mouse anti-human vimentin antibody (IgG1) and blocking again; Alexa Fluor™ 647 (purple)-labeled secondary antibody was used for color development.

[0129] Fifth round of immune response: PD-L1 detection (immunosuppression marker): Mouse anti-human PD-L1 antibody (IgG2a) was added for blocking; Alexa Fluor™ 594 (red)-labeled secondary antibody was used for color development.

[0130] Nuclear staining: Use DAPI (blue) to stain cell nuclei (1 µg / mL), incubate at room temperature for 5 minutes, and then rinse.

[0131] S4. Image acquisition and signal analysis:

[0132] Use a confocal laser scanning microscope to collect six channel images (DAPI, orange, green, yellow, purple, and red) and use image overlay software (such as ImageJ or HALO) to perform spatial analysis of cell marker expression. The judgment criteria are as follows:

[0133] DAPI + Indicates nucleated cells; CD45 - Exclude leukocyte background; CK + / EpCAM + / Vimentin + Characterize different types of CTC phenotypes (epithelial, mesenchymal, or mixed); PD-L1 + Determination of the immunosuppressive phenotype of CTCs.

[0134] S5. Result evaluation:

[0135] like Figure 5 As shown, blue is DAPI, orange is CD45, green is pan-CK, yellow is EpCAM, purple is Vimentin, and red is PD-L1. Multiple phenotypic characteristics of different CTC types can be observed in the image, which effectively supports heterogeneity analysis.

[0136] This example successfully achieved sequential staining and signal separation of six primary antibodies from the same species of mouse by introducing the GM Fab secondary antibody provided by this invention into a multiplex immunofluorescence assay for CTCs. This strategy is particularly suitable for spatial expression analysis of multiple CTC markers in tumor liquid biopsies. This strategy significantly improves staining specificity and throughput, making it suitable for clinical immunophenotyping, tumor metastasis mechanism research, and immunotherapy prognosis assessment.

[0137] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A kit for multiple immunoassay, characterized in that: The kit contains a goat anti-mouse secondary antibody, namely a GM Fab secondary antibody, which has been purified by papain hydrolysis and retains only the Fab segment and lacks the Fc segment; The amino acid sequence of the light chain variable region of the GM Fab secondary antibody is SEQ ID NO: 1, encoded by SEQ ID NO: 3; the amino acid sequence of the heavy chain variable region is SEQ ID NO: 2, encoded by SEQ ID NO: 4; the addition of the GM Fab secondary antibody after the first round of immune response can form specific binding with the primary antibody bound to the sample and block its antigen binding site, thereby blocking subsequent cross-reactions with the primary antibody of the same species.

2. The kit according to claim 1, wherein It also contains a detection reagent that matches the GM Fab secondary antibody. The detection reagent is selected from one or more of fluorescent dyes, horseradish peroxidase, alkaline phosphatase or biotin labeling systems, and can be coupled to the GM Fab secondary antibody through a flexible linker peptide segment SEQ ID NO:

5.

3. The kit according to claim 1 or 2, characterized in that The working concentration of the GM Fab secondary antibody in the final reaction system is 0.1-10 μg / mL.

4. The kit according to any one of claims 1 to 3, characterized in that Also included are diluents, wash solutions, and blocking solutions suitable for immunohistochemistry or immunofluorescence staining.

5. A multiplex immunoassay method for sequentially detecting multiple primary antibodies of the same species using the kit according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. Perform the first round of immune reaction on the sample to be tested and add the first primary antibody that can be used to prepare monoclonal antibodies, including rat, rabbit or human monoclonal antibodies; S2. After incubation, the GM Fab secondary antibody is added and incubated to block the primary antibody bound in step S1; S3, repeating steps S1-S2 at least once, each time using a different primary antibody capable of preparing monoclonal antibodies and adding the GM Fab secondary antibody for blocking after each primary antibody incubation; S4. Perform color development or fluorescence detection on the final signal to obtain spatial expression information of multiple targets.

6. The method according to claim 5, characterized in that The number of the multiple targets is 2-8, and the spectral overlap between the detection signals is less than 10%.

Images