Composition for immunohistochemical staining of PAUF protein comprising Anti-PAUF monoclonal antibody and use thereof

Abstract

The present invention relates to: a composition and kit for immunohistochemistry (IHC) staining, comprising an anti-PAUF monoclonal antibody that specifically binds to a PAUF protein; and an immunohistochemical staining method using same. The present invention exhibits significantly improved staining sensitivity and clarity over conventional PAUF staining methods, thereby providing the advantage of obtaining more accurate and reliable results regarding the expression of the PAUF protein. In addition, the present invention enables accurate staining of a large amount of clinical tissues in a short time by using specialized staining equipment together, thereby providing the advantage of enhancing the reliability of staining results. Accordingly, it is possible to specifically, rapidly, and accurately identify only PAUF within cancer tissues of cancer patients, thereby enabling rapid and efficient cancer diagnosis, and by using this, early cancer diagnosis is possible, thereby enabling early treatment for cancer patients in need of targeted therapy.

Description

Composition for immunohistochemical staining of PAUF protein containing anti-PAUF monoclonal antibody and use thereof

[0001] The present invention relates to a composition for immunohistochemical staining of PAUF proteins comprising an anti-PAUF monoclonal antibody, and to a kit and an immunohistochemical staining method using the same.

[0002] Targeted therapy is a treatment method that acts specifically on cancer cells. Generally, it selectively attacks only cancer cells by targeting specific proteins or genetic mutations. Unlike conventional chemotherapy, which affects normal cells as well as cancer cells, targeted therapy enables more precise treatment based on the specific physiological characteristics of cancer cells. Cytokines, monoclonal antibodies, recombinant antibodies, and nucleoside therapeutics are utilized in targeted therapy. Recently, such targeted therapies are being actively researched as methods to restore immune function or inhibit cancer growth by suppressing specific proteins.

[0003] For the successful application of targeted therapy, accurate diagnosis of cancer cells is an essential prerequisite. Specifically, it is necessary to first confirm whether a specific target protein is expressed in the cancer cells; this allows for determining whether targeted therapy is feasible for the cancer patient and suggesting a more appropriate treatment method. However, existing diagnostic methods for targeted therapy have been criticized for lacking sensitivity and accuracy, which limits their ability to clearly determine the expression of target proteins. In particular, regarding the PAUF (Pancreatic adenocarcinoma upregulated factor) protein, which is known to be overexpressed in cancer cells of various cancer types—such as pancreatic, ovarian, and cervical cancers—and involved in tumor cell development and mutation, a precise and efficient method capable of specifically staining PAUF has not yet been developed, despite the fact that diagnosing its expression could enable targeted therapy for various cancer types.

[0004] Consequently, the low reliability of diagnosing PAUF expression has made it difficult to accurately and rapidly identify patients requiring targeted therapy, acting as a limiting factor in the effectiveness of early cancer diagnosis and treatment. Therefore, there is a need to develop accurate and rapid diagnostic methods for PAUF protein expression.

[0005] The main objective of the present invention is to provide an immunohistochemistry (IHC) staining method with excellently improved sensitivity and clarity for PAUF (Pancreatic adenocarcinoma upregulated factor) protein in order to solve the aforementioned conventional problems.

[0006] Specifically, one objective of the present invention is to provide a composition for immunohistochemical staining of a PAUF protein comprising an anti-PAUF monoclonal antibody comprising, as a heavy chain variable region, a heavy chain CDR1 of SEQ ID NO. 1; a heavy chain CDR2 of SEQ ID NO. 2 or 39; and a heavy chain CDR3 of SEQ ID NO. 3, 4 or 30, and as a light chain variable region, a light chain CDR1 of SEQ ID NO. 5; a light chain CDR2 of SEQ ID NO. 6; and a light chain CDR3 of SEQ ID NO. 7 or 31.

[0007] In addition, another objective of the present invention is to provide a method for immunohistochemical staining of PAUF proteins using the anti-PAUF monoclonal antibody.

[0008] In addition, another objective of the present invention is to provide a kit for immunohistochemical staining of PAUF proteins comprising the above-mentioned anti-PAUF monoclonal antibody.

[0009] The purpose of the present invention is not limited to the description above and is provided for all cases in which appropriate effects can be obtained by utilizing the present invention.

[0010] The inventors conducted research to develop a technology capable of specifically, accurately, and relatively simply identifying PAUF (Pancreatic adenocarcinoma upregulated factor) protein. As a result, they discovered that when immunohistochemistry (IHC) staining is performed using the anti-PAUF monoclonal antibody of the present invention, which specifically binds to PAUF protein, sensitivity and clarity are significantly improved compared to conventional technology, thereby enabling more accurate diagnosis of PAUF protein expression, and thus completed the present invention.

[0011] Specifically, the present invention provides a composition for immunohistochemical staining of a PAUF protein, comprising an anti-PAUF monoclonal antibody comprising, as a heavy chain variable region, a heavy chain CDR1 of SEQ ID NO. 1; a heavy chain CDR2 of SEQ ID NO. 2 or 39; and a heavy chain CDR3 of SEQ ID NO. 3, 4 or 30, and as a light chain variable region, a light chain CDR1 of SEQ ID NO. 5; a light chain CDR2 of SEQ ID NO. 6; and a light chain CDR3 of SEQ ID NO. 7 or 31.

[0012] In addition, the present invention provides a composition for immunohistochemical staining of a PAUF protein, wherein the heavy chain variable region of the anti-PAUF monoclonal antibody comprises heavy chain FR1 of SEQ ID NO. 8, 19 or 32; heavy chain FR2 of SEQ ID NO. 9 or 20; heavy chain FR3 of SEQ ID NO. 10, 21 or 33; and heavy chain FR4 of SEQ ID NO. 11, 12, 22, 23 or 34, and the light chain variable region of the anti-PAUF monoclonal antibody comprises light chain FR1 of SEQ ID NO. 13, 24 or 35; light chain FR2 of SEQ ID NO. 14 or 25; light chain FR3 of SEQ ID NO. 15 or 26; and light chain FR4 of SEQ ID NO. 16, 27 or 36.

[0013] In addition, the present invention provides a composition for immunohistochemical staining of a PAUF protein, wherein, in the above composition, the heavy chain variable region of the anti-PAUF monoclonal antibody is composed of the amino acid sequence of SEQ ID NO. 17, 28, or 37; and the light chain variable region of the anti-PAUF monoclonal antibody is composed of the amino acid sequence of SEQ ID NO. 18, 29, or 38.

[0014] In addition, the present invention provides a composition for immunohistochemical staining of a PAUF protein, wherein, in the above composition, the heavy chain variable region of the anti-PAUF monoclonal antibody is composed of the amino acid sequence of SEQ ID NO. 37; and the light chain variable region of the anti-PAUF monoclonal antibody is composed of the amino acid sequence of SEQ ID NO. 38.

[0015] The present invention also provides a method for immunohistochemical staining of a PAUF protein, comprising the step of treating a biological sample isolated from an individual with a composition for immunohistochemical staining of a PAUF protein comprising the anti-PAUF monoclonal antibody.

[0016] In addition, the present invention provides an immunohistochemical staining method for PAUF proteins, wherein, in the above immunohistochemical staining method, after the step of treating the composition, the sample is further treated with an antibody that specifically recognizes the anti-PAUF monoclonal antibody as a secondary antibody.

[0017] In addition, the present invention provides an immunohistochemical staining method for PAUF protein, wherein the secondary antibody is an HRP-labeled goat-derived anti-human IgG Fc cross-adsorbed secondary antibody (Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody HRP).

[0018] In addition, the present invention provides a kit for immunohistochemical staining of a PAUF protein, comprising a composition for immunohistochemical staining of a PAUF protein comprising the anti-PAUF monoclonal antibody.

[0019] In addition, the present invention provides a kit for immunohistochemical staining of PAUF protein, wherein the kit further comprises, as a secondary antibody, an antibody that specifically recognizes the anti-PAUF monoclonal antibody.

[0020] In addition, the present invention provides a kit for immunohistochemical staining of PAUF protein, wherein the secondary antibody in the kit is an HRP-labeled goat-derived anti-human IgG Fc cross-adsorbed secondary antibody (Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody HRP).

[0021]

[0022] The present invention will be described in more detail below.

[0023]

[0024] The present invention, in a specific embodiment,

[0025] As a heavy chain variable region, it comprises heavy chain CDR1 of SEQ ID NO. 1; heavy chain CDR2 of SEQ ID NO. 2 or 39; and heavy chain CDR3 of SEQ ID NO. 3, 4 or 30, and

[0026] A composition for immunohistochemical staining of a PAUF protein is provided, comprising an anti-PAUF monoclonal antibody comprising light chain CDR1 of SEQ ID NO. 5; light chain CDR2 of SEQ ID NO. 6; and light chain CDR3 of SEQ ID NO. 7 or 31 as light chain variable regions.

[0027] In the present invention, the PAUF (Pancreatic adenocarcinoma upregulated factor) protein is known to be a protein involved in various physiological processes related to the growth, development, invasion, and metastasis of cancer cells. The PAUF protein is known to be overexpressed in cancer cells such as pancreatic cancer, ovarian cancer, colorectal cancer, and cervical cancer, and has been reported to be a target for the diagnosis and treatment of cancer.

[0028] In the present invention, the anti-PAUF monoclonal antibody refers to an antibody capable of specifically binding to the PAUF protein and inhibiting the activity of the PAUF protein.

[0029] In the present invention, the antibody generally has a heavy chain and a light chain, and each heavy chain and light chain includes a constant region and a variable region. The variable regions of the light chain and heavy chain include three complementarity-determining regions (hereinafter referred to as "CDRs") and four framework regions (hereinafter referred to as "FRs"). The CDRs of each chain primarily serve to bind to the epitopes of the antigen and are typically named sequentially starting from the N-terminus as CDR1, CDR2, and CDR3. Additionally, the FRs of each chain may be named sequentially starting from the N-terminus as FR1, FR2, FR3, and FR4.

[0030] In the present invention, the variable region of the heavy chain is 'V H ', the variable region of the light chain is 'V L It can be named ', and the CDR of the heavy chain is each 'V H -CDR1', 'V H -CDR2', 'V H -CDR3', where the CDR of the light chain is 'V L -CDR1', 'V L -CDR2', 'V L As '-CDR3', the FR of the heavy chain is 'V' respectively H -FR1', 'V H -FR2', 'V H -FR3', 'V H As in '-FR4', the FR of the light chain is 'V L -FR1', 'V L -FR2', 'V L -FR3', 'V L It can be named '-FR4'.

[0031] In the present invention, the anti-PAUF monoclonal antibody comprises, as a heavy chain variable region, a heavy chain CDR1 of SEQ ID NO. 1; a heavy chain CDR2 of SEQ ID NO. 2 or 39; and a heavy chain CDR3 of SEQ ID NO. 3, 4 or 30, and as a light chain variable region, a light chain CDR1 of SEQ ID NO. 5; a light chain CDR2 of SEQ ID NO. 6; and a light chain CDR3 of SEQ ID NO. 7 or 31.

[0032] In the present invention, the heavy chain variable region of the anti-PAUF monoclonal antibody may more specifically comprise heavy chain FR1 of SEQ ID NO. 8, 19, or 32; heavy chain FR2 of SEQ ID NO. 9 or 20; heavy chain FR3 of SEQ ID NO. 10, 21, or 33; and heavy chain FR4 of SEQ ID NO. 11, 12, 22, 23, or 34, and the light chain variable region of the anti-PAUF monoclonal antibody may more specifically comprise light chain FR1 of SEQ ID NO. 13, 24, or 35; light chain FR2 of SEQ ID NO. 14 or 25; light chain FR3 of SEQ ID NO. 15 or 26; and light chain FR4 of SEQ ID NO. 16, 27, or 36. In this case, the affinity and specificity for the PAUF protein may be further enhanced.

[0033] In the present invention, the heavy chain variable region of the anti-PAUF monoclonal antibody may more specifically be composed of the amino acid sequence of SEQ ID NO. 17, 28, or 37, and the light chain variable region of the anti-PAUF monoclonal antibody may more specifically be composed of the amino acid sequence of SEQ ID NO. 18, 29, or 38. In this case, the affinity and specificity for the PAUF protein may be further improved.

[0034] In the present invention, the heavy chain variable region of the anti-PAUF monoclonal antibody may more specifically be composed of the amino acid sequence of SEQ ID NO. 37, and the light chain variable region of the anti-PAUF monoclonal antibody may more specifically be composed of the amino acid sequence of SEQ ID NO. 38. In this case, the affinity and specificity for the PAUF protein may be further improved.

[0035] In the anti-PAUF monoclonal antibody of the present invention, the antibody in which the heavy chain variable region is composed of the amino acid sequence of SEQ ID NO. 37 and the light chain variable region is composed of the amino acid sequence of SEQ ID NO. 38 was named the "20C5" antibody.

[0036] The amino acid sequence list for the anti-PAUF monoclonal antibody of the present invention is as shown in [Table 1] below.

[0037] Sequence Number Classification Sequence 1V H -CDR1GFNIKDYY2V H -CDR2IDPENGNT3V H -CDR3ARRAITTATAWFA4V H -CDR3ARRAITTATAWFAY5V L -CDR1QSLLNSRTRKNY6V L -CDR2WAS7V L -CDR3KQSYNLY8V H -FR1QVQLKQSGAELVRPGALVKLSCKAS9V H -FR2MHWVKQRPEQGLEWIGW10V H -FR3IYDPKFQGKASITADTSSNTAYLQLSSLTSEDTAVYYC11V H -FR4YWGQGTLVTVSA12V H -FR4WGQGTLVTVSA13V L -FR1DIVMTQSPSSLAVSAGEKVTMSCKSS14V L -FR2LAWYQQKPGQSPKLLIY15V L -FR3TRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC16V L-FR4TFGAGTKLELK17중쇄가변영역(V H )QVQLKQSGAELVRPGALVKLSCKASGFNIKDYYMHWVKQRPEQGLEWIGWIDPENGNTIYDPKFQGKASITADTSSNTAYLQLSSLTSEDTAVYYCARRAITTATAWFAYWGQGTLVTVSA18경쇄가변영역(V L )DIVMTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLYTFGAGTKLELK19V H -FR1QVQLVQSGAEVKKPGASVKVSCKAS20V H -FR2MHWVRQAPGQGLEWMGW21V H -FR3NYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYC22V H -FR4YWGQGTLVTVSS23V H -FR4WGQGTLVTVSS24V L -FR1DIVMTQSPDSLAVSLGERATINCKSS25V L -FR2LAWYQQKPGQPPKLLIY26V L -FR3TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC27V L -FR4TFGQGTKVEIK28중쇄가변영역(V H )QVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQGLEWMGWIDPENGNTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARRAITTATAWFAYWGQGTLVTVSS29경쇄가변영역(V L )DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNLYTFGQGTKVEIK30V H -CDR3ARRGWLPAWFAY31V L-CDR3KQSYNLYT32V H -FR1QVQLKESGAELVRPGALVKLSCKAS33V H -FR3IYDPKFQGKASLTADTSSNTAYLQLSSLTSEDTAVYYC34V H -FR4WGQGTLVTVSA35V L -FR1DIVMTQSPSSLAVSAGEKVTLSCKSS36V L -FR4FGGGTKLEIK37Multi-chain variable region(V H )QVQLKESGAELVRPGALVKLSCKASGFNIKDYYMHWVKQRPEQGLEWIGWIDPEHGNTIYDPKFQGKASLTADTSSNTAYLQLSSLTSEDTAVYYCARRGWLPAWFAYWGQGTLVTVSA38 light chain variable region (V L )DIVMTQSPSSLAVSAGEKVTLSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLYTFGGGTKLEIK39V H -CDR2IDPEHGNT

[0038]

[0039] Among the anti-PAUF monoclonal antibodies of the present invention, the amino acid sequence list for the 20C5 antibody is as shown in [Table 2] below.

[0040] Sequence Number Classification Sequence 37 Heavy Chain Variable Region (V H )QVQLKESGAELVRPGALVKLSCKASGFNIKDYYMHWVKQRPEQGLEWIGWIDPEHGNTIYDPKFQGKASLTADTSSNTAYLQLSSLTSEDTAVYYCARRGWLPAWFAYWGQGTLVTVSA1V H -CDR1GFNIKDYY39V H -CDR2IDPEHGNT30V H -CDR3ARRGWLPAWFAY32V H -FR1QVQLKESGAELVRPGALVKLSCKAS9V H -FR2MHWVKQRPEQGLEWIGW33VH -FR3IYDPKFQGKASLTADTSSNTAYLQLSSLTSEDTAVYYC34V H -FR4WGQGTLVTVSA38 light chain variable region(V L )DIVMTQSPSSLAVSAGEKVTLSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLYTFGGGTKLEIK5V L -CDR1QSLLNSRTRKNY6V L -CDR2WAS31V L -CDR3KQSYNLYT35V L -FR1DIVMTQSPSSLAVSAGEKVTLSCKSS14V L -FR2LAWYQQKPGQSPKLLIY15V L -FR3TRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC36V L -FR4FGGGTKLEIK

[0041]

[0042] The anti-PAUF monoclonal antibody of the present invention may additionally include the content regarding the anti-PAUF antibody disclosed in Korean Registered Patent No. KR10-1856904 (registered on May 3, 2018).

[0043]

[0044] In another specific embodiment, the present invention provides a method for immunohistochemical staining of a PAUF protein, comprising the step of treating a biological sample isolated from an individual with a composition for immunohistochemical staining of a PAUF protein comprising the anti-PAUF monoclonal antibody of the present invention.

[0045] In the present invention, the immunohistochemistry (IHC) staining generally refers to a technique that visualizes the presence and distribution of specific proteins or antigens to identify them within a tissue, and involves a method of detecting target proteins in specific parts of a tissue using antibodies. Since immunohistochemistry staining allows for the visual confirmation of the expression and distribution of specific proteins in biological tissues, it can be utilized in various ways for the diagnosis of diseases or research.

[0046] In the present invention, the immunohistochemical staining can be performed according to a generally known standard procedure. The immunohistochemical staining can generally be performed by preparing a tissue sample, performing a blocking treatment, then treating it with a primary antibody which is the main antibody, and then treating it with a secondary antibody, and the staining results can be obtained by observing and analyzing them through a microscope.

[0047] More specifically, the tissue sample preparation described above involves preparing a tissue sample to observe the location and expression level of a specific protein within the tissue. This can be performed by fixing the tissue sample isolated from an individual to prevent deformation of the cell structure, then cutting it into an appropriate size and attaching it to a slide glass. The blocking treatment of the tissue sample may be performed by treating the tissue slide with a blocking solution to prevent non-specific binding and ensure high specificity for the target protein. The primary antibody treatment involves treating the tissue slide with a primary antibody capable of recognizing and specifically binding to the target protein. This can be performed by treating the tissue slide with the primary antibody, storing it for a certain period of time, and then washing it out. The secondary antibody treatment involves treating the tissue slide with an antibody capable of specifically binding to the primary antibody to amplify the signal and aid in visualization. Generally, the secondary antibody may be an antibody labeled with an enzyme (e.g., HRP (Horseradish Peroxidase), AP (Alkaline Phosphatase), etc.) that reacts with a specific substrate to induce a color change or fluorescence. The above secondary antibody treatment can be performed by treating a tissue slide with a secondary antibody, storing it for a certain period of time, washing it, and then treating it with a substrate for the enzyme to induce color development. The stained result can generally be observed using an optical microscope or a fluorescence microscope.

[0048] In the present invention, the anti-PAUF monoclonal antibody may be used as a primary antibody in immunohistochemical staining.

[0049] In the present invention, when the anti-PAUF monoclonal antibody is used as a primary antibody in immunohistochemical staining, it can be treated by diluting it to 1:350 to 1:650, specifically 1:400 to 1:600, more specifically 1:450 to 1:550, and even more specifically about 1:500.

[0050] In the present invention, the immunohistochemical staining method may further include, after the step of treating a composition containing the anti-PAUF monoclonal antibody, the step of treating the sample with an antibody that specifically recognizes the anti-PAUF monoclonal antibody as a secondary antibody.

[0051] Non-limiting examples of the secondary antibody in the immunohistochemical staining method of the present invention may include anti-human IgG. In order to achieve superior staining sensitivity, specificity, and clarity by using the anti-PAUF monoclonal antibody of the present invention as the primary antibody, an HRP-labeled antibody may be used as the secondary antibody, and more specifically, when using an HRP-labeled goat-derived anti-human IgG Fc cross-adsorbed secondary antibody (Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody HRP), an excellent synergistic effect with the anti-PAUF monoclonal antibody of the present invention is exhibited, which has the advantage of obtaining more reliable results with improved speed and accuracy of the staining results.

[0052] In the immunohistochemical staining method of the present invention, the secondary antibody may be treated by dilution to 1:50 to 1:350, specifically 1:100 to 1:300, more specifically 1:150 to 1:250, and even more specifically about 1:200.

[0053] According to a specific embodiment of the present invention, immunohistochemical staining for PAUF protein can be performed in the following manner. Specifically, each tissue slide is incubated in a drying oven at a temperature of 45 to 75°C, more specifically 50 to 70°C, more specifically 55 to 65°C, and more specifically about 60°C for 45 to 75 minutes, more specifically 50 to 70 minutes, more specifically 55 to 65 minutes, and more specifically about 60 minutes, to allow the tissue to adhere firmly to the slide.

[0054] Next, each tissue slide is treated with xylene for 1 to 25 minutes, more specifically 3 to 20 minutes, more specifically 5 to 15 minutes, and more specifically about 10 minutes to remove paraffin, and this process is repeated once or 2 to 4 times.

[0055] Next, in 100% ethanol for 1 to 15 minutes, specifically 1 to 10 minutes, more specifically 1 to 5 minutes, and even more specifically about 3 minutes; in 95% ethanol for 0.1 to 6 minutes, specifically 0.3 to 4 minutes, more specifically 0.5 to 2 minutes, and even more specifically about 1 minute; in 80% ethanol for 0.1 to 6 minutes, specifically 0.3 to 4 minutes, more specifically 0.5 to 2 minutes, and even more specifically about 1 minute; and in 70% ethanol for 0.1 to 6 minutes, specifically 0.3 to 4 minutes, more specifically 0.5 to 2 minutes, and even more specifically about 1 minute; After rehydrating by treatment for a period of time, wash in PBS for 1 to 15 minutes, specifically 1 to 10 minutes, more specifically 1 to 5 minutes, and even more specifically about 3 minutes, repeating once or 2 to 4 times.

[0056] Next, to restore antigenicity, each tissue slide is placed in citrate buffer (10 mM sodium citrate buffer pH 6.0) and boiled for 15 to 45 minutes, more specifically 20 to 40 minutes, more specifically 25 to 35 minutes, and more specifically about 30 minutes. Then, to remove endogenous peroxidase activity, the slide is placed in a hydrogen peroxide blocking solution (30% H2O2 in methanol) for 1 to 25 minutes, more specifically 5 to 20 minutes, more specifically 5 to 15 minutes, and more specifically about 10 minutes. Afterward, the slide is washed with PBS for 1 to 15 minutes, more specifically 1 to 10 minutes, more specifically 1 to 5 minutes, and more specifically about 3 minutes, repeated once or 2 to 4 times.

[0057] Next, treat with a blocking solution (1.5% horse serum in PBS) for 15 to 45 minutes, specifically 20 to 40 minutes, more specifically 25 to 35 minutes, and more specifically about 30 minutes; then, as a primary antibody, treat with an anti-PAUF monoclonal antibody (5 to 25 μg / ml, more specifically 10 to 20 μg / ml, more specifically about 15 μg / ml) diluted to 1:350 to 1:650, specifically 1:400 to 1:600, more specifically 1:450 to 1:550, and more specifically about 1:500; and then at 1 to 15°C, specifically 1 to 10°C, more specifically 2 to 6°C, and more specifically about 4°C for 5 to 35 hours, In detail, after storing for 10 to 30 hours, more specifically 15 to 25 hours, and even more specifically about 20 hours, wash with PBS for 1 to 15 minutes, in detail 1 to 10 minutes, more specifically 1 to 5 minutes, and even more specifically about 3 minutes, repeating once or 2 to 4 times.

[0058] Next, the HRP-labeled secondary antibody is treated at a dilution ratio of 1:50 to 1:350, more specifically 1:100 to 1:300, more specifically 1:150 to 1:250, and more specifically about 1:200, and stored at room temperature (20 to 30°C, more specifically 23 to 29°C, more specifically 25 to 27°C) for 45 to 75 minutes, more specifically 50 to 70 minutes, more specifically 55 to 65 minutes, and more specifically about 60 minutes, and then washed with PBS for 1 to 15 minutes, more specifically 1 to 10 minutes, more specifically 1 to 5 minutes, and more specifically about 3 minutes, once or 2 to 4 times.

[0059] Next, DAB (diaminobenzidine) is applied to check for a color reaction for 15 to 45 seconds, more specifically 20 to 40 seconds, more specifically 25 to 35 seconds, and even more specifically about 30 seconds.

[0060] Next, counterstain with Mayer's hematoxylin is performed, and after treatment with triple-distilled water for 0.1 to 6 minutes, specifically 0.3 to 4 minutes, more specifically 0.5 to 2 minutes, and even more specifically about 1 minute, the sample is washed with PBS for 1 to 15 minutes, specifically 1 to 10 minutes, more specifically 1 to 5 minutes, and even more specifically about 3 minutes, repeated once or 2 to 4 times. Then, the sample is treated with distilled water in 70% ethanol for 0.1 to 6 minutes, specifically 0.3 to 4 minutes, more specifically 0.5 to 2 minutes, and even more specifically about 1 minute; Dehydration is performed by treating with distilled water in 80% ethanol for 0.1 to 6 minutes, specifically 0.3 to 4 minutes, more specifically 0.5 to 2 minutes, and more specifically about 1 minute; with distilled water in 95% ethanol for 0.1 to 6 minutes, specifically 0.3 to 4 minutes, more specifically 0.5 to 2 minutes, and more specifically about 1 minute; and with 100% ethanol for 1 to 15 minutes, specifically 1 to 10 minutes, more specifically 1 to 5 minutes, and more specifically about 3 minutes. Then, the process of removing paraffin is repeated once or 2 to 4 times by treating with xylene for 1 to 25 minutes, specifically 3 to 20 minutes, more specifically 5 to 15 minutes, and more specifically about 10 minutes.

[0061] Next, each tissue slide that has completed immunohistochemical staining is fixed using a mounting medium, and PAUF expression is read using an optical microscope.

[0062]

[0063] In another specific embodiment, the present invention provides a kit for immunohistochemical staining of a PAUF protein, comprising a composition for immunohistochemical staining of a PAUF protein comprising the anti-PAUF monoclonal antibody.

[0064] In the immunohistochemical staining kit of the present invention, the anti-PAUF monoclonal antibody may be used as a primary antibody.

[0065] In the present invention, the immunohistochemical staining kit may further include an antibody that specifically recognizes the anti-PAUF monoclonal antibody as a secondary antibody.

[0066] The description of the secondary antibody in the immunohistochemical staining kit of the present invention is replaced by the previously described content.

[0067] The immunohistochemical staining kit of the present invention may further include preparations or reagents generally required when performing immunohistochemical staining, in addition to the primary antibody and secondary antibody. For example, a blocking reagent (e.g., albumin, serum, gelatin, etc.) to prevent non-specific binding of antibodies and increase the accuracy of the staining result; a staining reagent (chromogenic or fluorescent substrate) to react with a secondary antibody conjugated with an enzyme such as HRP or AP to induce a color change or fluorescence (e.g., DAB, BCIP / NBT, etc.); and a washing buffer (e.g., PBS, TBS, etc.) to remove non-specific binding or unconjugated reagents. A staining reagent for distinguishing nuclear or other cellular structures to contrast and / or stained tissues may include a counterstaining reagent for microscopic examination (e.g., hematoxylin, etc.) and may further include other auxiliary reagents (e.g., pH adjusting reagent, reaction enhancer, etc.).

[0068] In the present invention, immunohistochemical staining using the anti-PAUF monoclonal antibody can also be performed using an automated immunohistochemical staining (automated IHC) method using automated equipment, and can be performed based on a suitable protocol provided according to the automated equipment used.

[0069]

[0070] Terms not otherwise defined in the present invention shall be interpreted as having the meanings commonly used in the relevant technical field. Additionally, the expression "or" as used in the present invention may be interpreted as a concept including "and" unless otherwise noted.

[0071] The scope of the present invention is not limited by the specific descriptions disclosed herein, and each description and embodiment disclosed herein may be applied to each other description and embodiment. That is, all possible combinations of the various elements disclosed herein are to be interpreted as falling within the scope of the present invention. Furthermore, a person skilled in the art may recognize or identify a number of equivalents to specific embodiments of the present invention through ordinary experimentation, and such equivalents are to be interpreted as falling within the scope of the present invention.

[0072] The present invention relates to a composition for immunohistochemistry (IHC) staining comprising an anti-PAUF monoclonal antibody that specifically binds to a PAUF protein, a kit, and a method for immunohistochemistry staining using the same.

[0073] Compared to conventionally presented PAUF staining methods, the present invention has the advantage of significantly improving staining sensitivity and clarity, thereby enabling the derivation of more accurate and reliable results regarding the expression of PAUF proteins.

[0074] Furthermore, the present invention has the advantage of improving the reliability of staining results by utilizing specialized staining equipment to accurately stain a large volume of clinical tissue in a short period of time. Through this, only PAUF can be specifically, quickly, and accurately identified in the cancerous tissue of cancer patients, enabling rapid and efficient cancer diagnosis. Additionally, by utilizing this, early diagnosis of cancer is possible, which has the advantage of allowing for early treatment in cancer patients requiring targeted therapy.

[0075] Figure 1 shows the results of performing immunohistochemical staining (IHC) and hematoxylin-eosin staining (H&E) on PAUF protein in normal pancreatic tissue and pancreatic cancer tissue using the 20C5 antibody of the present invention in one embodiment of the present invention.

[0076] Figure 2 shows a comparison of the results of immunohistochemical staining (IHC) for PAUF protein in normal pancreatic tissue and pancreatic cancer tissue using the 20C5 antibody of the present invention in one embodiment of the present invention, and the results of the results of the test using the ZG16B antibody (control group).

[0077] Figure 3 shows the results of automated immunohistochemical staining (IHC staining) for PAUF protein in normal pancreatic tissue and pancreatic cancer tissue using the 20C5 antibody of the present invention in one embodiment of the present invention.

[0078] The present invention will be described in more detail below through specific embodiments. However, these embodiments are merely examples for explaining the present invention and should not be interpreted as limiting the scope of the present invention in any way.

[0079]

[0080] [Example 1] Confirmation of Immunohistochemical Staining in Normal Pancreas and Pancreatic Cancer Tissues Using 20C5 Antibody

[0081] Immunohistochemical staining was performed on normal pancreatic tissue and pancreatic cancer tissue using the 20C5 antibody of the present invention. As the tissues to be stained, paraffin-infiltrated tissue slides (US Biomax, Inc) consisting of normal pancreatic tissue (n=11) and pancreatic cancer tissue (n=21), respectively, were used.

[0082] Specifically, each tissue slide was incubated in a dry oven at 60°C for 60 minutes to ensure the tissue adhered firmly to the slide. Next, each tissue slide was treated with xylene for 10 minutes to remove paraffin, a process repeated twice. Subsequently, the slides were rehydrated by treatment in 100% ethanol for 3 minutes, 95% ethanol for 1 minute, 80% ethanol for 1 minute, and 70% ethanol for 1 minute, followed by washing in PBS for 3 minutes, repeated twice. Finally, to restore antigenicity, each tissue slide was placed in citric acid buffer (10 mM sodium citrate buffer pH 6.0) and boiled for 30 minutes. Next, to remove endogenous peroxidase activity, the sample was left in a hydrogen peroxide blocking solution (30% H2O2 in methanol) for 10 minutes, followed by washing with PBS for 3 minutes, repeated three times. Then, it was treated with a blocking solution (1.5% horse serum in PBS) for 30 minutes. Next, as the primary antibody, a 20C5 antibody (15 μg / ml) diluted 1:500 was applied, stored at 4°C for 20 hours, and then washed with PBS for 3 minutes, repeated three times. Next, as a secondary antibody against the 20C5 antibody, an HRP-labeled goat-derived anti-human IgG Fc cross-adsorbed secondary antibody (Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody HRP (Invitrogen, 31413)) was treated at a dilution ratio of 1:200 and stored at room temperature (25–27°C) for 60 minutes, after which it was washed three times with PBS for 3 minutes each. Then, DAB (diaminobenzidine, Abcam, ab64238) was treated, and the color reaction was observed for 30 seconds.Next, counterstaining was performed with Mayer's hematoxylin. Then, the samples were washed with triple-distilled water for 1 minute, followed by washing with PBS twice for 3 minutes each. Next, the samples were dehydrated by treatment with 70% ethanol for 1 minute, 80% ethanol for 1 minute, 95% ethanol for 1 minute, and 100% ethanol for 3 minutes. Subsequently, the samples were treated twice with xylene for 10 minutes each. Afterward, each tissue slide that had undergone immunohistochemical staining was fixed using a mounting medium, and PAUF expression was analyzed using an optical microscope.

[0083] In the experiment, Human PAUF / ZG16B antibody (R&D systems, MAB7777) diluted 1:500 was used as the primary antibody for the control group, and HRP-labeled goat-derived anti-mouse IgG Fc secondary antibody (Goat anti-Mouse IgG Fc Secondary Antibody HRP (Invitrogen, A16084)) diluted 1:200 was used as the secondary antibody.

[0084] In addition, hematoxylin-eosin staining (H&E) was performed on normal pancreatic tissue and pancreatic cancer tissue to confirm the pathological morphology of the cancerous tissue. Paraffin-infiltrated tissue slides (US Biomax, Inc.) consisting of normal pancreatic tissue (n=11) and pancreatic cancer tissue (n=21), respectively, were used as the tissues to be stained. H&E tissue staining allows for the differentiation of whether the tissue is cancerous, thereby enabling the distinction between cancer cells and normal cells. Accordingly, by comparing the results of the immunohistochemical staining using the 20C5 antibody with the results of the H&E tissue staining, it is possible to confirm whether the immunohistochemical staining using the 20C5 antibody was reliably and properly performed.

[0085] Specifically, for H&E staining, each tissue slide was incubated in a drying oven at 60°C for 60 minutes to ensure the tissue adhered firmly to the slide. Next, each tissue slide was treated with xylene for 10 minutes to remove paraffin, a process repeated twice. Subsequently, the slides were rehydrated by treatment with 100% ethanol for 3 minutes, 95% ethanol for 1 minute, 80% ethanol for 1 minute, and 70% ethanol for 1 minute, followed by washing with triple-distilled water for 3 minutes. Next, nucleus staining was performed with Mayer's hematoxylin for 10 minutes, followed by washing with triple-distilled water for 1 minute. Finally, the slides were immersed in 1% HCl 1-2 times to remove over-stained areas, and then washed with triple-distilled water for 3 minutes. Next, counterstaining was performed with eosin (Eosin Y solution, alcohol) for 1 minute. Then, the samples were washed with triple-distilled water for 3 minutes. Next, the samples were dehydrated by treatment with 70% ethanol for 1 minute, 80% ethanol for 1 minute, 95% ethanol for 1 minute, and 100% ethanol for 3 minutes. Then, the samples were treated twice with xylene for 10 minutes each. After the immunohistochemical staining was completed, each tissue slide was fixed using a mounting medium, and the cells and tissue structures were examined under an optical microscope.

[0086] As shown in Figure 1, when immunohistochemical staining was performed using the 20C5 antibody of the present invention, the expression of PAUF protein in pancreatic cancer tissue was clearly confirmed compared to normal pancreatic tissue. The results of the immunohistochemical staining using the 20C5 antibody were confirmed to be highly reliable through comparison with H&E staining results.

[0087] In addition, as shown in Figure 2, in the case of the control group in immunohistochemical staining, the staining results between normal pancreatic tissue and pancreatic cancer tissue were not clear in the microscopic images, and an error occurred in which the proportion of the area stained positively for PAUF protein was higher in normal pancreatic tissue than in pancreatic cancer tissue, confirming that the sensitivity and accuracy were significantly reduced. In contrast, when the 20C5 antibody of the present invention was used, a clear difference in staining between normal pancreatic tissue and pancreatic cancer tissue was confirmed even in the microscopic images. When the area stained positively with PAUF protein was quantified and compared, the average value was approximately 24.82 in normal pancreatic tissue and approximately 60.07 in pancreatic cancer tissue, indicating that the value was measured to be about 2.42 times higher in pancreatic cancer tissue, confirming that the sensitivity and accuracy are excellent.

[0088]

[0089] [Example 2] Confirmation of Autologous Immunohistochemical Staining in Normal Pancreas and Pancreatic Cancer Tissues Using 20C5 Antibody

[0090] Automated immunohistochemical staining (IHC) was performed on normal pancreatic tissue and pancreatic cancer tissue using the 20C5 antibody of the present invention. Paraffin-infiltrated tissue slides consisting of normal pancreatic tissue (n=5) and pancreatic cancer tissue (n=5), respectively, were used as the tissues to be stained, and an automated immunohistochemical staining machine (Leica, BOND RxM) was used.

[0091] Specifically, 1% BSA, 20C5 (5 μg / ml) as the primary antibody, and Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, HRP (Invitrogen, 31413, 1:200) as the secondary antibody were pre-diluted in PBS to their respective concentrations and loaded into the reagent tray. Next, the power to the equipment and the PC connected to the equipment was turned on, and the 'BOND' program was accessed. Then, the reagent settings icon was selected to add 1% BSA, the primary antibody, and the secondary antibody to the list. At this time, the type was selected as 'Auxiliary'. Next, the open containers containing each reagent were registered, the protocol settings icon was selected, and the step-by-step protocol (steps 1–25) was set and saved as shown in [Table 3] below. The items listed in [Table 3] below correspond to the items displayed on the screen of the automated immunohistochemistry staining system (Leica, BOND RxM), and any omitted step numbers in [Table 3] below represent the washing steps.

[0092] Step No. Reagent Supplier Incubation Time (min) 1 Peroxide Block Leica Microsystems 10:00 5 1% BSA 30:00 9 Primary Antibody 30:00 1 3 Secondary Antibody 8:00 1 7 Mixed DAB Refine Leica Microsystems 0:00 1 8 Mixed DAB Refine Leica Microsystems 10:00 2 2 Hematoxylin Leica Microsystems 5:00

[0093]

[0094] Next, the slide settings icon was selected, followed by "Add Study." The information for the study to be conducted was entered and saved. At this time, the dosage was set to 150 μl and the preparation protocol to "Dewax." Then, "Add Slide" was selected, and a description of the slide to be stained was added. The marker was set to the primary antibody container registered during the open container registration process, the HIER (Heat-Induced Epitope Retrieval) to "HIER 20 min with ER2," and the staining was set to the protocol according to Table 3. Next, a label was printed and attached to the slide, the slide was placed on the slide tray, covered with a covertile, and the tray was mounted on the instrument. Then, the lids of all markers and the detection system were opened, and the reagent tray was mounted on the instrument. When the green light on the instrument turned on, the start icon was clicked to execute the corresponding protocol. After the immunohistochemical staining was completed, the slide tissue was washed under running water for 5 minutes, and then the slide was dehydrated twice in 100% ethanol for 1 minute each. Next, the slide was cleared in xylene for 1 minute, fixed using an mounting agent, and then PAUF expression was read using an optical microscope.

[0095] As shown in Figure 3, when automated immunohistochemical staining was performed using the 20C5 antibody of the present invention, a clear difference in staining between normal pancreatic tissue and pancreatic cancer tissue was confirmed in the microscopic images. When the regions where the PAUF protein was expressed and stained positive were quantified and compared, the average value was approximately 4.39 in normal pancreatic tissue and approximately 52.77 in pancreatic cancer tissue, indicating that the value was measured to be about 12.02 times higher in pancreatic cancer tissue, confirming that the sensitivity and accuracy are significantly excellent.

[0096]

[0097] This specification omits detailed descriptions of matters that can be sufficiently recognized and inferred by those skilled in the art, and various modifications are possible within the scope of not altering the technical concept or essential configurations of the invention, in addition to the specific examples described herein. Accordingly, the invention may be implemented in a manner different from that specifically described and exemplified in this specification, and this is a matter that can be understood by those skilled in the art.

[0098] The sequence listings mentioned in this specification are submitted as separate electronic sequence listing (XML) files in accordance with PCT Rule 13ter and WIPO Standard ST.26, and constitute part of this specification.

Claims

1. As a heavy chain variable region, it comprises heavy chain CDR1 of SEQ ID NO. 1; heavy chain CDR2 of SEQ ID NO. 2 or 39; and heavy chain CDR3 of SEQ ID NO. 3, 4 or 30, and A composition for immunohistochemical staining of PAUF protein, comprising an anti-PAUF monoclonal antibody comprising light chain CDR1 of SEQ ID NO. 5; light chain CDR2 of SEQ ID NO. 6; and light chain CDR3 of SEQ ID NO. 7 or 31 as light chain variable regions.

2. In claim 1, the heavy chain variable region of the anti-PAUF monoclonal antibody comprises heavy chain FR1 of SEQ ID NO. 8, 19, or 32; heavy chain FR2 of SEQ ID NO. 9 or 20; heavy chain FR3 of SEQ ID NO. 10, 21, or 33; and heavy chain FR4 of SEQ ID NO. 11, 12, 22, 23, or 34, and A composition for immunohistochemical staining of PAUF protein, wherein the light chain variable region of the above anti-PAUF monoclonal antibody comprises light chain FR1 of SEQ ID NO. 13, 24, or 35; light chain FR2 of SEQ ID NO. 14 or 25; light chain FR3 of SEQ ID NO. 15 or 26; and light chain FR4 of SEQ ID NO. 16, 27, or 36.

3. In claim 1, the heavy chain variable region of the anti-PAUF monoclonal antibody is composed of the amino acid sequence of SEQ ID NO. 17, 28, or 37; A composition for immunohistochemical staining of PAUF protein, wherein the light chain variable region of the above anti-PAUF monoclonal antibody consists of the amino acid sequence of SEQ ID NO. 18, 29, or 38.

4. In claim 1, the heavy chain variable region of the anti-PAUF monoclonal antibody is composed of the amino acid sequence of SEQ ID NO. 37; A composition for immunohistochemical staining of PAUF protein, wherein the light chain variable region of the above anti-PAUF monoclonal antibody consists of the amino acid sequence of SEQ ID NO.

38.

5. A method for immunohistochemical staining of a PAUF protein, comprising the step of treating a biological sample separated from an individual with a composition for immunohistochemical staining of a PAUF protein comprising the anti-PAUF monoclonal antibody according to any one of claims 1 to 4.

6. A method for immunohistochemical staining of PAUF protein according to claim 5, further comprising, after the step of treating the composition, the step of treating the sample with an antibody that specifically recognizes the anti-PAUF monoclonal antibody as a secondary antibody.

7. A method for immunohistochemical staining of PAUF protein according to claim 6, wherein the secondary antibody is an HRP-labeled goat-derived anti-human IgG Fc cross-adsorbed secondary antibody (Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody HRP).

8. A kit for immunohistochemical staining of a PAUF protein, comprising a composition for immunohistochemical staining of a PAUF protein comprising the anti-PAUF monoclonal antibody according to any one of claims 1 to 4.

9. A kit for immunohistochemical staining of PAUF protein according to claim 8, wherein the kit further comprises an antibody that specifically recognizes the anti-PAUF monoclonal antibody as a secondary antibody.

10. A kit for immunohistochemical staining of PAUF protein, wherein, in claim 9, the secondary antibody is an HRP-labeled goat-derived anti-human IgG Fc cross-adsorbed secondary antibody (Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody HRP).

Images