CN1928563A - Immunodiagnosis reagent kit for breast cancer and testing sheet - Google Patents

Immunodiagnosis reagent kit for breast cancer and testing sheet Download PDF

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CN1928563A
CN1928563A CN 200510029534 CN200510029534A CN1928563A CN 1928563 A CN1928563 A CN 1928563A CN 200510029534 CN200510029534 CN 200510029534 CN 200510029534 A CN200510029534 A CN 200510029534A CN 1928563 A CN1928563 A CN 1928563A
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her2
expression
tissue
staining
tumor
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CN 200510029534
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Chinese (zh)
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CN1928563B (en )
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郭亚军
王皓
侯盛
钱卫珠
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上海张江生物技术有限公司
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Abstract

The disclosed testing block for Her2 express in mammary tissue includes: the Her2 high-express area, the express contrast area, the no-express contrast area, and the testing area for target tissue. This invention can provide accurate evaluation standard for clinic diagnosis on breast cancer.

Description

乳腺癌的免疫组化诊断试剂盒和测试片 Breast immunohistochemical diagnostic kits and test piece

技术领域 FIELD

本发明属于免疫学领域,涉及一种肿瘤诊断检测用品,进一步地,本发明涉及检测乳腺组织中Her2表达的测试片以及含该测试片的试剂盒。 The present invention is in the field of immunology, a tumor relates to diagnostic test kits, further, the present invention relates to a strip detection Her2 expression in breast tissue and a kit containing the test sheet.

背景技术 Background technique

乳腺癌是妇女常见的恶性肿瘤,在欧美国家的发病率占女性恶性肿瘤的首位。 Breast cancer is a common malignancy in women, accounting for the first female cancer incidence in Europe and the United States. 我国虽然属乳腺癌相对低发地区,但发病率正逐年上升。 Although China is a relatively low breast cancer-prone areas, but the incidence is rising year by year. 多数乳腺癌患者早期手术的预后较好,但约25-30%的患者的癌组织中有Her2受体的过度表达,这些患者的预后明显差于Her2低表达的患者,体现为手术后复发率高、病情发展迅速、化疗缓解期短,以及对化疗药物易产生耐药,无瘤生存率和总生存率均低于Her2阴性的乳腺癌。 Early surgical prognosis most preferably breast cancer, but about 25-30% of cancer patients Her2 overexpression of the receptor, which significantly worse prognosis in patients with low expression of Her2, embodied as the recurrence rate after surgery high, rapid progression of the disease, chemotherapy, remission is short and easy to produce drug chemotherapy drugs, disease-free survival and overall survival rates were lower than Her2-negative breast cancer. Her2在体内广泛分布,编码该蛋白的基因erbB-2,又称为neu基因,是人类肿瘤中发生改变频率最高的基因之一。 Her2 is widely distributed in the body, the gene encoding the protein erbB-2, also known as neu gene is altered in human tumors one of the highest frequency of the gene. 美国肿瘤学会2000年曾推荐“Her2/neu”作为乳腺癌标记物,无论对诊断还是复发的检测都具有重要价值。 American Cancer Society in 2000 had recommended "Her2 / neu" as a marker for breast cancer, regardless of diagnosis or detection of recurrence are of great value.

Her2是肿瘤靶向治疗的理想靶点之一,国内外制备了多种抗Her2单克隆抗体,发现它们可以显著抑制Her2阳性肿瘤细胞的体外、体内生长。 Her2 is the ideal target for cancer targeted therapy, more anti-Her2 monoclonal antibodies were found to be significantly inhibits Her2 positive tumor cells grown in vivo prepared at home and abroad. 由于鼠源抗体注射至人体后会产生严重的异种抗原反应,人们又对鼠源抗体进行了人源化改造,如1998年美国FDA批准了新型抗Her2人源化抗体用于治疗Her2阳性的转移性乳腺癌,该抗体即Herceptin,在临床试验和后续的应用中取得了十分确切的疗效。 Since the human body can cause serious reactions heterogeneous antigen murine antibody injection to, people of a murine antibody humanization reform, such as the US FDA in 1998 approved the transfer of a new anti-Her2 positive Her2 humanized antibody for the treatment of breast cancer, the antibody that is Herceptin, made a very definite effect in clinical trials and subsequent applications. 国内专利01132225.X也公开了一种抗Her2抗体,前期的临床前研究已经证实其可在体外与Her2受体阳性的乳腺癌细胞特异、可逆、高亲和力地结合,可抑制Her2阳性表达的肿瘤细胞的体外增殖,还可抑制Her2阳性表达的肿瘤细胞裸鼠异种移植模型的肿瘤生长。 Domestic Patent No. 01132225.X also discloses an anti-Her2 antibody, preclinical studies have demonstrated that may be pre-receptor positive breast cancer cells in vitro and Her2-specific, reversible, binding with high affinity, inhibit tumor expression of Her2 in vitro cell proliferation, inhibition of tumor cells may further expression of Her2 nude mouse xenograft model of tumor growth. 荷瘤小鼠注射该抗体后24~60小时检测发现,抗体集中分布于肿瘤组织中。 After the tumor-bearing mice were injected with antibody testing found 24 to 60 hours, concentrated antibody in the tumor tissues.

由于抗Her2单克隆抗体药物的适应症为Her2阳性的乳腺癌患者,因此在进行临床研究乃至今后临床应用时,需要确定备选患者肿瘤组织Her2抗原的表达情况。 Due to indications of anti-Her2 monoclonal antibody drug for Her2 positive breast cancer patients, and therefore the conduct of clinical research as well as clinical applications in the future, we need to determine the expression of tumor tissue in patients with Her2 antigen candidate. 但是,目前已有的乳腺癌诊断试剂普遍存在操作程序烦琐,非特异性高的问题,不能简便、迅速、准确地判断患者肿瘤组织Her2抗原的表达情况。 However, there are widespread breast cancer diagnostic reagents cumbersome procedures, high non-specific problems, not simply, quickly and accurately determine the expression of tumor tissue in patients with Her2 antigen. 因此,目前医学上迫切需要开发一种能够快速、便捷且准确地进行乳腺癌体外诊断的用品。 Therefore, it is medically urgent need to develop a fast, easy and accurate diagnosis of breast cancer in vitro supplies.

发明内容 SUMMARY

本发明的目的是提供一种在进行临床研究和诊断时,能够准确测定受试者乳腺组织的Her2抗原的表达情况的测试片。 Object of the present invention is to provide one kind when conducting clinical research and diagnosis, can be accurately measured the expression of the antigen Her2 breast tissue subject test piece.

本发明的另一目的是提供一种乳腺癌的免疫组织化学诊断试剂盒。 Another object of the present invention is to provide a breast cancer diagnostic kit immunohistochemistry.

本发明的另一目的是提供一种检测受试者肿瘤组织中Her2抗原表达水平的方法。 Another object of the present invention is to provide a method of detecting the expression level of Her2 antigen tumor tissue subject method.

在本发明的一个方面,提供了一种检测乳腺组织中Her2表达的测试片,所述的测试片的一个主表面上具有以下区域:(a)Her2高表达对照区,所述的高表达对照区设有Her2高表达的肿瘤组织切片;(b)Her2中表达对照区,所述的中表达对照区设有Her2中表达的肿瘤组织切片;(c)Her2无表达对照区,所述的无表达对照区设有Her2无表达的组织切片;(d)Her2检测区,所述的检测区用于放置待检测的乳腺组织。 In one aspect of the present invention, there is provided a test piece for detecting Her2 expression in breast tissue, the region having a major surface of the test piece: (a) Her2 expression control region, the expression control region tumor tissue sections with high expression of Her2; expression control zone (B) Her2, the expression control region is provided in the tissue sections in Her2 expressing tumors; (C) no Her2 expression control region, the free expression of tissue sections with no control area expressed Her2; (d) Her2 detection zone, the detection zone for the detection of breast tissue to be placed.

作为本发明的一个优选例,在所述的检测乳腺组织中Her2表达的测试片中,所述Her2高表达的肿瘤组织切片选自:D2F2/E2、HTB-20或SK-BR-3肿瘤组织切片。 As a preferred embodiment of the present invention, test pieces for detecting the expression of Her2 breast tissue, high expression of the Her2 tumor tissue sections were selected: D2F2 / E2, HTB-20, or SK-BR-3 tumor slice.

作为本发明的一个优选例,在所述的检测乳腺组织中Her2表达的测试片中,所述Her2中表达的肿瘤组织切片选自:KYC或LYX肿瘤组织切片。 As a preferred embodiment of the tumor tissue of the present invention, Her2 expression in the test pieces for detecting expression in breast tissue Her2, the slice is selected from: KYC LYX or tumor tissue sections.

作为本发明的一个优选例,在所述的检测乳腺组织中Her2表达的测试片中,所述Her2无表达的组织切片选自:A431、QYC、MCF-7、RENCA肿瘤组织切片或Her2无表达的正常组织切片。 As a preferred embodiment of the present invention, the detection of test pieces Her2 expression in breast tissue, no expression of the Her2 selected tissue sections: A431, QYC, MCF-7, RENCA tumor tissue sections or no expression of Her2 normal tissue sections.

作为本发明的一个优选例,在所述的检测乳腺组织中Her2表达的测试片中,所述的组织切片为石蜡包埋的切片。 As a preferred embodiment of the present invention, the expression in said detection test pieces Her2 breast tissue, the tissue sections of paraffin-embedded sections.

在本发明的第二方面,提供了一种乳腺癌的Her2免疫组化诊断试剂盒,其包含所述的检测乳腺组织中Her2表达的测试片。 In a second aspect of the present invention, there is provided a Her2 immunohistochemistry breast cancer diagnostic kit, comprising a detection test strip of Her2 expression in breast tissue.

作为本发明的一个优选例,在所述的乳腺癌的Her2免疫组化诊断试剂盒中,所述试剂盒中还包含免疫组化染色试剂,所述免疫组化染色试剂包含:非特异性结合位点的阻断剂,与组织抗原连接的初始抗体,与初始抗体连接的第二抗体,酶,底物,显色剂。 As a preferred embodiment of the present invention, in the breast Her2 immunohistochemical diagnostic kits, the kit further comprises immunohistochemical staining agent, the immunohistochemical staining agent comprising: non-specific binding blockers point, the initial tissue antigens linked antibody, a second antibody linked to the initial antibody, enzymes, substrates, color-developing agent.

作为本发明的一个优选例,在所述的乳腺癌的Her2免疫组化诊断试剂盒中,所述的初始抗体为抗Her2人源化单克隆抗体或鼠源抗Her2单克隆抗体,所述的第二抗体为生物素标记的抗人IgG或抗鼠IgG。 As a preferred embodiment of the present invention, the Her2 immunohistochemical diagnostic kit of breast cancer, the initial anti-Her2 antibody is a humanized monoclonal antibody or a murine anti-Her2 monoclonal antibody, said the second antibody is biotin-labeled anti-human IgG or anti-mouse IgG.

作为本发明的一个优选例,在所述的乳腺癌的Her2免疫组化诊断试剂盒中,所述的非特异性结合位点的阻断剂选自:动物血清,白蛋白;所述的酶为辣根过氧化物酶;所述的底物为H2O2;所述的显色剂选自:DAB-H2O2,AEC-H2O2,CN-H2O2。 As a preferred embodiment of the present invention, in the breast Her2 immunohistochemical diagnostic kits, the non-specific binding sites blocker is selected from: animal serum albumin; the enzyme is horseradish peroxidase; said substrate is by H2O2; the color developing agent is selected from: DAB-H2O2, AEC-H2O2, CN-H2O2.

在本发明的第三方面,提供了一种体外检测样品中Her2抗原表达水平的检测方法,所述的样品是肿瘤组织,该方法包括步骤:将肿瘤组织制成切片;将所述切片置于所述测试片中的Her2检测区,进行免疫组化染色检测,将样品的染色情况与测试片上Her2高表达、中表达、无表达的组织切片的染色情况进行比对,结果判定如下:染色情况≤Her2低表达区,判为阴性(-);染色情况介于Her2低表达区和Her2中表达区之间,判为弱阳性(+);染色情况介于Her2中表达区和Her2高表达区之间,判为中强阳性(++);染色情况≥Her2高表达区,判为强阳性(+++)。 In a third aspect of the present invention, there is provided a method for detecting the level of expression of Her2 in an in vitro test sample antigen, said tissue sample is a tumor, the method comprising the steps of: tumor tissue sections were prepared; the sections were placed Her2 detection zone of the test sheet, immunohistochemical staining, the staining staining Her2 expression on the test piece of the sample, expressed, no expression tissue sections were aligned, the result is determined as follows: staining ≤Her2 low expression region, judged as negative (-); staining region between Her2 expression and low Her2 expression region, judged weak positive (+); staining region between Her2 expression and high expression of Her2 region between, judged as strongly positive (++); staining ≥Her2 expression area, judged as strongly positive (+++).

更特别的,在所述的检测方法中,所述Her2高表达的肿瘤组织切片选自D2F2/E2、HTB-20或SK-BR-3肿瘤组织切片;所述Her2中表达的肿瘤组织切片选自KYC或LYX肿瘤组织切片;所述Her2无表达的组织切片选自A431、QYC、MCF-7、RENCA或其它Her2无表达的组织切片。 More particularly, in the detection method, the expression of the tumor tissue slice selected Her2 D2F2 / E2, HTB-20, or SK-BR-3 tumor tissue sections; Her2 expressed in tumor tissue is selected from the slice KYC LYX or from tumor tissue sections; tissue expression of the Her2 free slice selected A431, QYC, MCF-7, RENCA, or no expression of Her2 other tissue sections.

附图说明 BRIEF DESCRIPTION

图1显示了本发明的试剂盒中,组织切片在玻片上的分布示意图,其中,1、2和3分别表示Her2高表达、中表达、低表达对照区,4表示检测区。 Figure 1 shows the kit of the present invention, a schematic diagram of the distribution of the tissue sections on the slide, wherein the 1, 2 and 3 show Her2 expression, the expression, the expression of the low control zone, 4 indicates the detection zone.

图2显示了几种细胞呈现不同表达强度荧光的流式细胞术检测结果,其中,A表示LYX细胞的结果,其Her2无表达;B表示QYC细胞的结果,其Her2无表达;C表示MCF-7细胞的结果,其Her2无表达;D表示RENCA细胞的结果,其Her2无表达;E表示KYC的结果,其Her2中度表达;F表示LYX细胞的结果,其Her2中度表达;G表示SK-BR-3细胞的结果,其Her2高度表达;H表示HTB-20细胞的结果,其Her2高度表达;I表示D2F2/E2细胞的结果,其Her2高度表达。 Figure 2 shows the results of flow cytometry showed different expression of several cell fluorescence intensity, wherein, A shows the results LYX cells, which expressed no Her2; QYC B shows the results of cells which expressed Her2 no; C represents MCF- results 7 cells, which expressed no Her2; D shows the result of RENCA cells, no expression of Her2; KYC E shows the results, which Her2 moderate expression; F. LYX shows the results of cells, medium expression of Her2; G represents SK results -BR-3 cells, which expressed Her2 height; H shows the results of HTB-20 cells, which expressed Her2 height; I means results D2F2 / E2 cells, which highly expressed Her2.

图3显示了强阳、弱阳和阴性的切片组织的一些照片,其中,A为MCF-7(Her2无表达);B为LYX(Her2中度表达);C为D2F2/E2(Her2高度表达);D为样品3(+);E为样品5(++);F为样品7(+++)。 Figure 3 shows the strong positive, weak positive and some pictures of the negative section of tissue, wherein, A is MCF-7 (Her2 no expression); B is a LYX (Her2 moderate expression); C was D2F2 / E2 Her2 highly expressed ( ); D sample 3 (+); E is a sample 5 (++); F is sample 7 (+++).

具体实施方式 Detailed ways

由于目前已有的乳腺癌诊断试剂操作程序烦琐,非特异性高,不能迅速、准确的判断患者肿瘤组织Her2抗原的表达情况。 Because there are breast cancer diagnostic reagents cumbersome procedures, high non-specific and can not quickly and accurately determine the expression of tumor tissue in patients with Her2 antigen. 因此,本发明提供了一种可简便地测试患者Her2抗原表达情况,进而可准确诊断病情。 Accordingly, the present invention provides an easy test antigen expression in patients with Her2, in turn, can accurately diagnose the disease. 本发明人经过长期的研究,选取出Her2抗原表达水平呈高度、中度及无表达的多种不同的细胞,制备成具有良好均质性的组织,做成组织切片后置于一定的载体上。 The present inventors have long studied to select a variety of different cell antigen Her2 expression levels were high, moderate, and no expression, prepared with good homogeneity tissue, tissue sections were made after a certain carrier . 在检测时,将受试者的组织切片与对照切片同时进行染色,然后进行对比,即可判断出受试者的Her2表达情况。 Upon detection, the tissue sections were incubated with control subjects, while sections were stained and compared, you can determine the condition of the subject Her2 expression.

本发明人选取Her2抗原表达水平呈高度、中度及无表达的多种不同的细胞,建立Balb/c裸鼠的异种移植模型,并使成瘤率大大提高。 The present invention is selected antigen Her2 expression levels take a wide variety of cells high, moderate, and no expression is established xenograft model Balb / c nude mice, and tumor formation rate was greatly improved. 因为采用细胞系体外扩增及特异性免疫功能缺陷的裸鼠体内接种获得的肿瘤组织,实为单克隆的肿瘤细胞增殖的结果,因此,肿瘤组织具有良好的均质性,即肿瘤的不同部位,抗原表达水平一致。 Because the use of tumor cell lines in vitro and specific immune function deficient nude mice inoculated obtained, in fact the result of a monoclonal tumor cell proliferation, and therefore, the tumor tissue with good homogeneity, i.e., different parts of the tumor , consistent with antigen expression level. 用这样的组织作为判定标准更为可靠,也使判定依据具有更好的稳定性。 With such criteria as the tissue more reliable, but also makes the determination based on better stability.

应理解,现有技术中存在许多种Her2无表达的正常组织,这些Her2无表达的正常组织也可用于本发明。 It should be understood that there are many normal tissues without expression of Her2 the prior art, no expression of Her2 these normal tissues may also be used in the present invention.

本发明选取固定、石蜡包埋的切片,而非冰冻切片,在保证实验结果可靠的前提下,降低了对实验设备及方法难度的要求,易于普及。 The present invention is selected fixed, paraffin-embedded sections, rather than frozen sections, under the premise of ensure reliable results, the requirements for reducing the difficulty of the test apparatus and method, easy to spread.

在本发明中,手术切除的组织及活检获得的组织都可用于检测,降低了对受检组织的要求,扩大了可接受检测患者的范围。 In the present invention, and surgical removal of tissue biopsy obtained can be used in the detection, reducing the requirements for the subject tissue and expand the range of acceptable patient is detected. 并且,受试者的肿瘤组织与Her2抗原呈不同表达水平的对照组织贴附于同一测试片,同时进行IHC染色,最大程度消除了操作过程微小变化对染色结果带来的误差。 And, subject to the tumor antigen was Her2 expression levels of different control tissue attached to the same test piece, simultaneously IHC staining, the maximum error is eliminated during the operation of small changes caused by staining.

本发明采用Her2表达水平呈高度、中度及无表达的明确肿瘤组织作为对照,可对IHC染色过程进行有效的监控,对IHC染色结果进行准确的评价,提高了检测结果的准确性,使假阳性和假阴性的结果都得到有效的控制。 The present invention employs Her2 expression levels were of the expression of high, moderate, and no expression as a control, can effectively monitor the IHC staining procedure, the results of IHC staining accurate evaluation, improves the accuracy of test results, false positives and false negative results have been effectively controlled.

下面结合具体实施例,进一步阐述本发明。 The following embodiments with reference to specific embodiments, further illustrate the present invention. 应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。 It should be understood that these embodiments are illustrative only and the present invention is not intended to limit the scope of the invention. 下列实施例中未注明具体条件的实验方法,通常按照常规条件,未详细说明的技术为常规技术。 Experimental methods without specific conditions in the examples below, are performed under routine conditions, described in detail techniques have not a conventional technique.

实施例1 Her2抗原呈不同表达水平的肿瘤细胞的选择采用以下步骤进行肿瘤细胞的选择:1)备选肿瘤细胞包括:人肝癌细胞QYC,人肝癌细胞KYC,人肝癌细胞LYX,小鼠肾腺癌细胞RENCA,D2F2/E2(前述五种细胞获自第二军医大学国际合作肿瘤研究所),人表皮癌细胞A431(购自上海午立生物技术有限公司)及MCF-7(获自美国国立癌症研究所),SK-BR-3(购自上海午立生物技术有限公司),HTB-20(购自广州维尔曼公司)。 Example 1-Her2 antigen showed varying expression levels of selected tumor cells using the steps of selecting tumor cells: 1) Alternative tumor cell comprising: a human hepatoma cell QYC, human hepatoma cells KYC, human hepatoma cells LYX, mouse adrenal cancer RENCA, D2F2 / E2 (obtained from the previous five cell tumor second Military Medical University, Institute of international cooperation), human epidermoid carcinoma A431 (purchased from Shanghai biological technology Co., Ltd. Kenneth Li) and MCF-7 (obtained from the American National cancer Institute), SK-BR-3 (purchased from Shanghai Kenneth Li biotechnology Co., Ltd.), HTB-20 (purchased from Guangzhou Wellman company). 体外培养至对数生长期,分别取各种细胞进行荧光染色;2)细胞计数,将待测肿瘤细胞加入流式细胞仪专用检测管中,2×105/管,200g×5分钟离心,弃上清;3)用含1%小牛血清的PBS将rhHer2-mAb(获自第二军医大学国际合作肿瘤研究所)稀释至10μg/mL,加入上述流式管中,100μL/管,冰浴,避光孵育45分钟;4)含1%小牛血清的PBS洗涤细胞2次,每次均200g×5分钟离心,弃上清;5)用含1%小牛血清的PBS将FITC-羊抗人IgG(购自上海生物制品研究所)稀释至1μg/mL,加入上述流式管中,100μL/管,冰浴,避光孵育45分钟;6)含1%小牛血清的PBS洗涤细胞2次,每次均200g×5分钟离心,弃上清;7)将细胞沉淀用300μL含1%小牛血清的PBS重悬,用流式细胞仪进行检测,Her2表达越强的细胞结合的荧光标记抗体越多。 To logarithmic phase cultured in vitro, cells were collected from a variety of fluorescent staining; 2) cells, the tumor cells to be tested was added FACS special test tube, 2 × 105 / tube, 200g × 5 minutes centrifugation, discard supernatant; 3) with calf serum in PBS containing 1% the rhHer2-mAb (available from cancer Institute international cooperation second Military Medical University) were diluted to 10μg / mL, was added to the flow tube, 100μL / tube, the ice bath was , in the dark for 45 min; 4) were washed with PBS containing 1% calf serum 2 times centrifugation was 200g × 5 minutes, and the supernatant was discarded; 5) with calf serum in PBS containing 1% sheep will FITC- anti-human IgG (purchased from Shanghai Institute of biological products) was diluted to 1μg / mL, was added to the flow tube, 100μL / tube on ice, in the dark for 45 min; 6) cells were washed with 1% PBS containing fetal calf serum 2, each time centrifuged at 200g × 5 minutes, and the supernatant was discarded; 7) with 300μL cell pellet containing 1% calf serum and resuspended in PBS, were detected by flow cytometry, Her2 expressing cells stronger binding more fluorescently labeled antibody.

通过对照荧光强度,获得Her2抗原呈高度表达的D2F2/E2、SK-BR-3、HTB-20;Her2呈中度表达的LYX、KYC,Her2无表达的MCF-7、QYC、A431、RENCA细胞。 By comparing the fluorescence intensity obtained Her2 antigen is highly expressed D2F2 / E2, SK-BR-3, HTB-20; Her2 showed moderate expression LYX, KYC, Her2 no expression of MCF-7, QYC, A431, RENCA cells . 上述几种细胞呈现不同表达强度的流式细胞检测结果显示在图2中。 Flow cytometry showed different results of the expression of several cell is shown in FIG.

实施例2表达Her2抗原均一性肿瘤组织的制备制备方法:1)选取实施例1中获得的Her2分别呈现高度、中度、无表达的D2F2/E2、LYX及MCF-7三种肿瘤细胞,于含有10%胎牛血清的RPMI-1640完全培养基中传代扩增,培养条件为5% CO2,37℃及饱和湿度;2)对数生长期的三种细胞,PBS洗涤,200g×5分钟离心,弃上清,细胞沉淀重悬于PBS中,计数,调整细胞密度至1×107/mL;3)将肿瘤细胞悬液分别接种于Balb/c裸鼠颈部皮下,定期观察接种局部肿瘤生长情况及受试动物的一般情况;4)待肿瘤长至0.5cm3左右大小(肿瘤体积计算公式为x2y/2,其中x为肿瘤长径,y为肿瘤短径),颈椎脱臼法处死受试动物,无菌摘取肿瘤组织;5)将肿瘤组织箭碎,研磨通过200目钢网;6)过钢网的细胞悬液用PBS洗涤,200g×5分钟离心,弃上清,细胞沉淀重悬于含有10%胎牛血清的RPMI-1640完全培养基,进行原代肿 Example 2 Preparation method of preparing an antigen Her2 expressing tumor tissue homogeneity: Her2 obtained in Example 11) were selected embodiment exhibits high, moderate, no expression of D2F2 / E2, LYX and three tumor MCF-7 cells, in RPMI-1640 complete medium containing 10% fetal bovine serum were passaged, the culture conditions were 5% CO2,37 ℃ and saturated humidity; 2), washed with PBS three types of cells in logarithmic growth phase, 200g × 5 minutes centrifugation the supernatant was discarded, the cell pellet was resuspended in PBS, counted, adjusted to a cell density of 1 × 107 / mL; 3) tumor cell suspensions were inoculated in Balb / c nude mice subcutaneously, local tumor growth observed regularly vaccinated general conditions and subject animal; 4) the tumor grew to about 0.5cm3 size (tumor volume calculated as x2y / 2, where x is the tumor size, y is shorter tumor diameter), the test animals were sacrificed by cervical dislocation sterile removal of tumor tissue; 5) tumor tissue broken arrows, milled through a 200 mesh steel; 6) through the steel mesh cell suspensions were washed with PBS, 200g × 5 minutes centrifugation, the supernatant was discarded, the cell pellet was resuspended RPMI-1640 containing 10% fetal calf serum complete medium, for primary swelling 细胞体外培养,培养条件为5%CO2,37℃及饱和湿度;7)肿瘤细胞长至对数生长期,如上法,再次接种Balb/c裸鼠,定期观察接种局部肿瘤生长情况及受试动物的一般情况;8)待肿瘤长至0.5cm3左右大小(肿瘤体积计算公式为x2y/2,其中x为肿瘤长径,y为肿瘤短径),颈椎脱臼法处死受试动物,无菌摘取肿瘤组织,再次进行原代培养,结果获得的三种肿瘤细胞D2F2/E2、LYX及MCF-7在Balb/c裸鼠体内的成瘤率已达100%;9)将前述获得的肿瘤细胞体外培养扩增,至对数生长期后接种于Balb/c裸鼠,定期观察接种局部肿瘤生长情况及受试动物的一般情况;10)待肿瘤长至0.5cm3左右大小(肿瘤体积计算方法与前述相同),颈椎脱臼法处死受试动物,摘取肿瘤组织,固定于多聚甲醛。 Cultured in vitro, the culture conditions were 5% CO2,37 ℃ and saturated humidity; 7) tumor cells grown to logarithmic phase method as described above, revaccination Balb / c nude mice, vaccinated regularly observe local tumor growth and test animals general case; 8) the tumor grew to about 0.5cm3 size (tumor volume calculated as x2y / 2, where x is the tumor size, y is shorter tumor diameter), the test animals were sacrificed by cervical dislocation, sterile removal tumor tissue, primary culture again, the results obtained in three tumor cells D2F2 / E2, LYX and rate of MCF-7 tumor in Balb / c nude mice reached 100%; 9) tumor cells obtained in the foregoing culture expansion to logarithmic growth phase were inoculated in Balb / c nude mice were inoculated generally regular observation of local tumor growth and the subject animal; 10) the tumor grew to about 0.5cm3 size (tumor volume was calculated with the the same), the test animals were sacrificed by cervical dislocation, the removal of tumor tissue, fixed in paraformaldehyde.

实施例3肿瘤组织病理切片的制备及表面Her2抗原表达的检测1.制片1)多聚甲醛磷酸缓冲液的配制:多聚甲醛40g,0.1mol/L pH7.4的PBS500mL,两者混合加热至60℃,搅拌并滴加1N NaOH至溶液清晰为止,冷却后加PBS至总体积为1000mL。 Expression of surface preparation and formulation of Example 3 of tumor tissue biopsy antigen Her2 1. Producer 1) paraformaldehyde in phosphate buffer: paraformaldehyde 40g, 0.1mol / L pH7.4 The PBS500mL, heating a mixture of both to 60 ℃, 1N NaOH was added dropwise with stirring to the solution until the clear, cooled PBS was added to a total volume of 1000mL.

2)摘取肿瘤组织后,尽快处理取材的组织,力求保持组织新鲜,不干燥,组织块不可过大或过厚,尤其厚度不要超过1cm。 2) After the removal of the tumor tissue, tissue drawn as soon as possible, striving to maintain fresh tissue, without drying, tissue mass is not too large or too thick, in particular a thickness not more than 1cm.

3)取材的组织固定于多聚甲醛磷酸缓冲液,固定液量要充足,至少20倍于组织的量,否则会造成固定液浓度降低,固定时间以完全浸透组织为宜(48小时),固定时间过久可能会影响染色效果。 3) is drawn and fixed with paraformaldehyde phosphate buffer, the amount of fixing solution should be sufficient, at least 20 times the amount of tissue, which will cause reduced concentration of fixative, a fixed time is appropriate tissue completely soaked (48 hours), the fixed for too long it may affect staining results.

4)组织的脱水、透明、浸蜡:整个操作过程中要掌握的原则是脱水透明要够而不过,浸蜡的温度不宜超过64℃,否则可能使抗原活性下降,降低检出率,或是造成组织焦脆,切片困难,具体程序如下表1。 4) The dehydrated tissue, transparent, waxing: to master the whole operation in principle to be transparent and dehydration, however, the temperature should not exceed dipping wax 64 deg.] C, or it may decrease the activity of antigen, the detection rate decreased, or cause tissue Jiaocui difficult sections, specific procedures are as follows in table 1.

5)从蜡缸拿出后用石蜡进行包埋。 5) embedded in paraffin wax from the cylinder out.

6)载玻片的预处理:载玻片的处理是做好免疫组化染色的重要步骤,本发明采用多聚左旋赖氨酸处理载玻片。 6) pretreated slides: slide processing is an important step to do immunohistochemical staining, the present invention employs poly-L-lysine treated slides.

切片:用石蜡切片机将包埋有组织的蜡块切成薄片,切片厚度4μm,45℃水温展片,要尽量展平,否则皱褶处易出现非特异染色。 Sections: the paraffin microtome organized paraffin embedded sliced, slice thickness 4μm, 45 ℃ flatting water, to try to flatten the folds or non-specific staining prone. 55℃温箱烤片2小时,防水密封,低温保存备用。 55 ℃ baking sheet incubator for 2 hours waterproof seal, cryopreservation standby.

表1组织脱水、透明、浸蜡程序 Table 1 tissue dehydration, transparent, dipping wax process

7)石蜡切片的脱蜡至水:石蜡切片脱蜡至水是组织脱水、透明、浸蜡和包埋的逆向过程,具体操作流程如下:a)4μm石蜡切片,55℃温箱烤片18小时后开始脱蜡;b)二甲苯I、II和III级各10分钟;c)无水乙醇、90%乙醇、80%乙醇及70%乙醇各2分钟;d)自来水洗2-3分钟,进入IHC染色。 7) Paraffin sections were dewaxed to water: Paraffin sections were deparaffinized tissue is dehydrated to water, transparent, dipping wax embedding and reverse the process, the specific operation process is as follows: a) 4μm paraffin, baking sheet 55 deg.] C incubator 18 hours dewaxing after the start; b) xylene I, II and III for 10 minutes each; c) ethanol, 90% ethanol, 80% ethanol and 70% ethanol for 2 minutes each; D) water washing 2-3 minutes into the IHC staining.

2.免疫组化(IHC)染色(1)热诱导的抗原修复1)热修复方法:100℃水浴,10分钟×2;2)热修复的介质:0.01mol/L pH6.0柠檬酸缓冲液。 Immunohistochemistry (IHC) staining (1) heat-induced antigen retrieval 1) hot repair method: 100 ℃ water bath for 10 minutes × 2; 2) repair of heat medium: 0.01mol / L pH6.0 citrate buffer .

通过抗原修复处理,可以提高抗原抗体阳性检测率。 By antigen retrieval process, can improve the antigen-antibody-positive detection rate.

(2)ABC法染色ABC法的特点是利用亲和素分别连接生物素标记的第二抗体和生物素标记的酶。 (2) Characteristics of ABC ABC staining method is the use of avidin and antibodies, respectively ligase second biotin-labeled biological labeled. 染色步骤如下:1)切片常规脱蜡至水,PBS洗3×3分钟;2)3%过氧化氢(H2O2)孵育5分钟,用以清除内源性过氧化物酶反应;3)蒸馏水洗,PBS浸泡5分钟;4)稀释的正常小牛血清(阻断剂),室温孵育20分钟;5)吸去多余的小牛血清;6)抗Her2人源化单克隆抗体或鼠源抗Her2单克隆抗体(获自第二军医大学国际合作肿瘤研究所)稀释至10μg/mL,37℃孵育60分钟或4℃过夜;7)PBS洗3×3分钟;8)生物素标记的抗人IgG或抗鼠IgG(购自晶美公司),37℃孵育30-60分钟;9)PBS洗3×3分钟;10)ABC复合物(购自晶美公司),37℃孵育30-60分钟;11)PBS洗3×3分钟;12)DAB-H2O2显色5-10分钟,水洗;13)苏木素复染、脱水、封固。 Dyeing procedure as follows: 1) The sections were deparaffinized to water, PBS washed 3 × 3 min; 2) 3% hydrogen peroxide (H2O2) for 5 min to remove the endogenous peroxidase reaction; 3) of distilled water , PBS soak for 5 minutes; 4) diluted in normal calf serum (blocking agent), at room temperature for 20 min; 5) to absorb excess calf serum; 6) humanized anti-Her2 antibody or a murine monoclonal anti-Her2 monoclonal antibodies were diluted to 10μg / mL (obtained from cancer Institute international cooperation second Military Medical University), incubated for 37 ℃ 60 minutes or overnight at 4 ℃; 7) PBS washed 3 × 3 min; 8) of biotin-labeled anti-human IgG or anti-mouse IgG (commercially available from crystal Corp.), incubated for 37 [deg.] C for 30-60 minutes; 9) PBS washed 3 × 3 minutes; 10) ABC complex (commercially available from crystal Corp.), incubated for 30-60 min 37 ℃; 11) PBS washed 3 × 3 minutes; 12) DAB-H2O2 color for 5-10 minutes, washing with water; 13) hematoxylin, dehydrated, sealing.

将制备的病理组织切片抽样检测,染色结果证实:D2F2/E2、LYX及MCF-7肿瘤组织经如上所述的ICH染色法检测,其Her2抗原的表达水平分别呈高、中及无不同的水平,即显色呈强阳性、弱阳性及阴性。 The histopathology sampling preparation, staining confirmed: D2F2 / E2, LYX and MCF-7 tumors in ICH staining detected as described above, which are the expression levels of Her2 antigen was high, and no different levels that significant Secheng strong positive, weak positive and negative. 不同切片位置的组织染色结果均匀,表明肿瘤细胞在接种动物体内形成的肿瘤组织具有良好的均质性。 Stained tissue slice position different uniform results, showed that tumor tissue tumor cells in animals vaccinated formed with good homogeneity. 因此,该组切片可用于检测肿瘤患者肿瘤组织Her2抗原的表达水平。 Thus, the set of slices may be used to detect the expression level in tumor tissue of patients with cancer antigens Her2.

实施例4肿瘤患者肿瘤组织Her2抗原表达水平的检测获取10位肿瘤患者手术切除的肿瘤组织或活检获得的肿瘤组织,经与前述相同的方法固定、脱水、透明、浸蜡、包埋、切片后,将切片贴附于已制备好的检测用对照切片载片的另一端,而后利用相同的方法进行ICH的染色检测。 After detecting Example 4 Her2 levels of tumor antigen expression of tumor tissue of a patient acquiring embodiment 10 tumors resected tumor tissue or tumor tissue obtained by biopsy, by fixing the same with the foregoing method, dehydration, clearing, waxing, embedded, sliced , the sections were attached to the other end has been prepared with the control sections of the detection slide, and then using the same staining method of ICH.

结果判断: Results to determine:

D2F2/E2、LYX及MCF-7肿瘤组织染色情况须为强阳性、弱阳性及阴性,实验结果有效,按下述步骤进行判断,否则实验无效;患者肿瘤组织染色情况与D2F2/E2、LYX及MCF-7肿瘤组织染色情况进行比对,染色情况≤MCF-7,判为阴性(-);染色情况介于MCF-7、LYX之间,判为弱阳性(+);染色情况介于LYX、D2F2/E2之间,判为中强阳性(++);染色情况≥D2F2/E2,判为强阳性(+++),结果见表2。 D2F2 / E2, LYX and MCF-7 tumor tissues shall staining strongly positive, weakly positive and negative, effective results, the determination according to the following steps, or the test is invalid; patient tumor tissue staining with D2F2 / E2, LYX and MCF-7 tumor staining for comparison, staining ≤MCF-7, judged as negative (-); staining between MCF-7, between LYX, judged weak positive (+); staining between LYX between D2F2 / E2, judged as strongly positive (++); staining ≥D2F2 / E2, judged as strong positive (+++) (Table 2).

表2 Table 2

部分受试样品的切片的染色情况见图3,图3中可见,A为MCF-7,Her2无表达;B为LYX,Her2中度表达;C为D2F2/E2,Her2高度表达;D为样品3(+);E为样品5(++);F为样品7(+++)。 Staining test sample slice portion shown in Figure 3, seen in Figure 3, A is a MCF-7, Her2 no expression; B is LYX, Her2 moderate expression; C was D2F2 / E2, Her2 highly expressed; D is sample 3 (+); E is a sample 5 (++); F is sample 7 (+++).

应理解,在阅读了本发明的上述公开内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请的权利要求书所限定的范围。 It should be understood, after reading the above disclosure of the present invention, those skilled in the art that various changes or modifications may be made to the present invention, and these equivalents also fall within the present application claims the claimed scope defined.

Claims (10)

  1. 1.一种检测乳腺组织中Her2表达的测试片,其特征在于,所述的测试片的一个主表面上具有以下区域:(a)Her2高表达对照区,所述的高表达对照区设有Her2高表达的肿瘤组织切片;(b)Her2中表达对照区,所述的中表达对照区设有Her2中表达的肿瘤组织切片;(c)Her2无表达对照区,所述的无表达对照区设有Her2无表达的组织切片;(d)Her2检测区,所述的检测区用于放置待检测的乳腺组织。 1. A method of detecting breast tissue Her2 expression test piece, wherein a region having the main surface of the test piece: (a) Her2 expression control zone, the control zone provided with high expression tumor tissue sections high expression of Her2; (B) Her2 expression control region, the expression control region is provided in the tissue sections in Her2 expressing tumors; (C) no Her2 expression control zone, the control zone and no expression tissue sections with no expression of Her2; (d) Her2 detection zone, the detection zone for the detection of breast tissue to be placed.
  2. 2.根据权利要求1所述的检测乳腺组织中Her2表达的测试片,其特征在于,所述Her2高表达的肿瘤组织切片选自:D2F2/E2、HTB-20或SK-BR-3肿瘤组织切片。 2. Detection of breast tissue of claim 1, Her2 expression in the test piece according to claim, characterized in that, the high expression of Her2 tumor tissue sections were selected: D2F2 / E2, HTB-20, or SK-BR-3 tumor slice.
  3. 3.根据权利要求1所述的检测乳腺组织中Her2表达的测试片,其特征在于,所述Her2中表达的肿瘤组织切片选自:KYC或LYX肿瘤组织切片。 The test piece is detected in the breast tissue of claim 1 Her2 expression claim, wherein the tumor tissue is selected from Her2 expression slices: KYC LYX or tumor tissue sections.
  4. 4.根据权利要求1所述的检测乳腺组织中Her2表达的测试片,其特征在于,所述Her2无表达的组织切片选自:A431、QYC、MCF-7、RENCA肿瘤组织切片或Her2无表达的正常组织切片。 The detection of breast tissue of claim 1 in Her2 expression test piece as claimed in claim, wherein said non-expression of Her2 selected tissue sections: A431, QYC, MCF-7, RENCA tumor tissue sections or no expression of Her2 normal tissue sections.
  5. 5根据权利要求1-4中任一所述的检测乳腺组织中Her2表达的测试片,其特征在于,所述的组织切片为石蜡包埋的切片。 5 test piece according to any one of the breast tissue is detected in Her2 expression of claims 1-4, wherein said tissue section is a paraffin-embedded sections.
  6. 6.一种乳腺癌的Her2免疫组化诊断试剂盒,其特征在于,包含权利要求1所述的检测乳腺组织中Her2表达的测试片。 A breast Her2 immunohistochemical diagnostic kit comprising the test strip of claim 1, said detecting breast tissue in Her2 expression.
  7. 7.根据权利要求6所述的乳腺癌的Her2免疫组化诊断试剂盒,其特征在于,所述试剂盒中还包含免疫组化染色试剂,所述免疫组化染色试剂包含:非特异性结合位点的阻断剂,与组织抗原连接的初始抗体,与初始抗体连接的第二抗体,酶,底物,显色剂。 The breast Her2 immunohistochemical diagnostic kit according to claim 6, wherein said kit further comprises immunohistochemical staining agent, the immunohistochemical staining agent comprising: non-specific binding blockers point, the initial tissue antigens linked antibody, a second antibody linked to the initial antibody, enzymes, substrates, color-developing agent.
  8. 8.根据权利要求7所述的乳腺癌的Her2免疫组化诊断试剂盒,其特征在于,所述的初始抗体为抗Her2人源化单克隆抗体或鼠源抗Her2单克隆抗体,所述的第二抗体为生物素标记的抗人IgG或抗鼠IgG。 The breast Her2 immunohistochemical diagnostic kit according to claim 7, characterized in that the initial anti-Her2 antibody is a humanized monoclonal antibody or a murine anti-Her2 monoclonal antibody, said the second antibody is biotin-labeled anti-human IgG or anti-mouse IgG.
  9. 9.根据权利要求7所述的乳腺癌的Her2免疫组化诊断试剂盒,其特征在于,非特异性结合位点的阻断剂选自:动物血清,白蛋白;所述的酶为辣根过氧化物酶;所述的底物为H2O2;或者所述的显色剂选自:DAB-H2O2,AEC-H2O2,CN-H2O2。 According to claim breast Her2 immunohistochemical diagnostic kit of claim 7, wherein the non-specific binding blocker is selected sites: animal serum albumin; through the enzyme is horseradish peroxidase; said substrate is by H2O2; or said color agent is selected from: DAB-H2O2, AEC-H2O2, CN-H2O2.
  10. 10.一种体外检测样品中Her2抗原表达水平的检测方法,所述的样品是肿瘤组织,其特征在于,该方法包括步骤:将肿瘤组织制成切片;将所述切片置于权利要求1所述的检测乳腺组织中Her2表达的测试片中的Her2检测区,进行免疫组化染色检测,将样品的染色情况与测试片上Her2高表达、中表达、无表达的组织切片的染色情况进行比对,结果判定如下:染色情况≤Her2低表达区,判为阴性(-);染色情况介于Her2低表达区和Her2中表达区之间,判为弱阳性(+);染色情况介于Her2中表达区和Her2高表达区之间,判为中强阳性(++);染色情况≥Her2高表达区,判为强阳性(+++)。 10. An in vitro expression levels of the test sample Her2 antigen detection method, the sample is a tumor tissue, characterized in that the method comprises the steps of: tumor tissue sections were prepared; the sections were placed as claimed in claim 1 said detecting Her2 detection zone test pieces expressed in breast tissue Her2, immunohistochemical staining, the upper staining of the test piece samples of Her2 expression, expression, staining no expression of tissue sections for comparison the results determined as follows: staining ≤Her2 low expression region, judged as negative (-); staining region between Her2 expression and low Her2 expression region, judged weak positive (+); Her2 in staining between area between expression and high expression area Her2, judged as strongly positive (++); staining ≥Her2 expression area, judged as strongly positive (+++).
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