CN105353127B - One kind of tumor can be detected more compositions and Its Application - Google Patents

One kind of tumor can be detected more compositions and Its Application Download PDF

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CN105353127B
CN105353127B CN201510789097.3A CN201510789097A CN105353127B CN 105353127 B CN105353127 B CN 105353127B CN 201510789097 A CN201510789097 A CN 201510789097A CN 105353127 B CN105353127 B CN 105353127B
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antibody
colloidal gold
solution
tumor
concentration
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CN105353127A (en
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徐家科
吴琨
徐丽婉
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暨赛再生医学科技有限公司
广州暨赛万德基因科技有限公司
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Abstract

本发明公开的一种可以检测多种肿瘤的组合物,作为抗体载体参加免疫浊度反应,可用于检测多种肿瘤,大大提高了检测的灵敏度。 One inventive compositions disclosed herein can detect a variety of tumors, to participate in an immune antibody carrier as turbidity of the reaction, may be used to detect a variety of tumors, greatly improving the detection sensitivity. 所述可以检测多种肿瘤的组合物,是由多种识别肿瘤特异性抗原的抗体、聚合物、胶体金溶液组成;其制备方法为:将所述多种识别肿瘤特异性抗原的抗体分别用胶体金标记后,与所述聚合物形成包含多种胶体金标记的用于识别不同肿瘤特异性抗原的抗体的共聚物,再作为抗体载体参加免疫浊度反应,用于检测多种肿瘤的免疫增强体外诊断试剂,可检测多种肿瘤,具有普遍适用性,且检测的灵敏度较高。 The composition may detect a variety of tumors, the antibody is a plurality of identification of tumor specific antigens, polymers, composed of colloidal gold solution; the preparation method: the plurality of antibodies which recognize tumor-specific antigens, respectively after colloidal gold label, with the polymer to form a copolymer comprising a plurality of colloidal gold labeled antibodies used recognize different tumor-specific antigens, and then participate in an immune antibody carrier as turbidity of the reaction, for the detection of various tumors immunity enhanced high in vitro diagnostic reagents, a variety of tumors can be detected, having a general applicability, and the detection sensitivity.

Description

一种可以检测多种肿瘤的组合物及其应用 One kind of tumor can be detected more compositions and Its Application

技术领域 FIELD

[0001] 本发明涉及医学检验技术领域,具体是涉及一种可以检测多种肿瘤的组合物及其应用。 [0001] The present invention relates to a technical field of medical testing, in particular, to a variety of tumor can be detected in the composition and its application.

背景技术 Background technique

[0002] 肿瘤已经成为严重危害人类生命健康的重要疾病之一,全世界恶性肿瘤的发病率和死亡率呈逐年上升的趋势。 [0002] the tumor has become one of the major diseases of serious harm to human life and health, worldwide cancer incidence and mortality showed an increasing trend. 而且与生态环境、生活方式相关的肿瘤呈现持续性增长势头。 Also related to the environment, lifestyle tumor continued to show growth momentum. 而对于肿瘤没有有效的治疗药物,尤其是晚期。 And there is no effective treatment for cancer drugs, especially late. 因此,肿瘤的早期检测和诊断,对于肿瘤的治疗具有关键作用。 Therefore, early detection and diagnosis of tumors, for the treatment of tumors having a key role.

[0003] 目前,肿瘤的临床诊断主要依据于患者的临床表现、各种影像学检查和实验室检测技术。 [0003] Currently, the clinical diagnosis based on clinical manifestations of tumors in patients with a variety of imaging studies and laboratory technology. 但是,临床上很多癌症患者在早期并无明显的症状,发现时往往已到了晚期。 However, many cancer patients in the clinical no obvious early symptoms are often found to have been late. 各种影像学检测技术,如X线、B超、CT、磁共振,作为重要的肿瘤辅助诊断手段,通常也不能发现较小的早期肿瘤,对早期胃癌诊断价值不高。 Imaging of various detection techniques, such as X-ray, B, CT, MRI, as an important means for tumor diagnosis, can not usually found in early tumors smaller, the value is not high in early diagnosis of gastric cancer.

[0004] 随着分子生物学技术的进步,越来越多的肿瘤标记物被发现,可用于肿瘤的检测, 而且特异性和灵敏度也很好。 [0004] With the progress of molecular biological techniques, more and more of the tumor marker is found and can be used to detect tumors, but also good sensitivity and specificity. 这些生物学检测方法包括免疫比浊法,酶联免疫发,荧光标记法等,其中,胶乳免疫透射比浊法是将抗体吸附在一种胶乳颗粒上,当遇到相应的抗原时, 抗原抗体结合而出现乳胶凝集。 These biological detection methods include immunoturbidimetric assay, enzyme-linked immunosorbent hair, fluorescent labeling method, wherein the latex turbidimetric immunoassay method is an antibody adsorbed on a latex particle, when faced with the respective antigens, antigen-antibody combined with the emergence of latex agglutination. 单个乳胶颗粒的大小在入射波长之内,光线可透过,当两个以上的乳胶颗粒凝集时,可阻碍光线透过,使得透射光减少,其减少程度与抗原的量成正比。 Size of the individual latex particles in the incident wavelength, the light can pass through, when two or more latex particle agglutination, can impede light transmittance, so that the transmitted light is reduced, which reduces the extent proportional to the amount of the antigen. 此法提高了检测的灵敏度和准确度,得到了广泛的应用。 This method increases the sensitivity and accuracy of detection, has been widely used. 然而,乳胶增强免疫比浊法反应后会产生沉淀,不利于生化仪的清洗,干扰试验结果,且乳胶制造成本较高,导致免疫比浊试剂盒价格和检测成本偏高。 However, latex-enhanced immune precipitate generated than nephelometry after the reaction, is not conducive to cleaning the biochemical analyzer, interference results, and high manufacturing cost the latex, resulting in higher turbidimetric immunoassay kit for the detection and cost price. 因此又出现了纳米颗粒增强的免疫比浊法,但是其应用也有一定的局限性,因此,在这方面还有待科学研宄工作者们进一步拓展。 So appeared nanoparticles enhanced immune turbidimetry, but its applications have some limitations, therefore, in this regard remains to be workers in the scientific study based on further expansion.

发明内容 SUMMARY

[0005] 本发明解决的技术问题是提供一种可以检测多种肿瘤的组合物,作为抗体载体参加免疫浊度反应,可用于检测多种肿瘤,大大提高了检测的灵敏度。 [0005] The technical problem underlying the present invention is to provide a composition of a variety of tumor can be detected as an immune antibody carrier participate in turbidity of the reaction, it may be used to detect a variety of tumors, greatly improving the detection sensitivity.

[0006] 本发明的技术方案是:如权利要求1所述,一种可以检测多种肿瘤的组合物,是由多种识别肿瘤特异性抗原的抗体、聚合物、胶体金溶液组成;其制备方法为:将所述多种识别肿瘤特异性抗原的抗体分别用胶体金标记后,与所述聚合物形成包含多种胶体金标记的用于识别不同肿瘤特异性抗原的抗体的共聚物。 [0006] aspect of the present invention is: Claim 1. A composition may detect a variety of tumors, the antibody is a plurality of identification of tumor specific antigens, polymers, composed of colloidal gold solution; preparation method: after identifying the plurality of tumor-specific antigens are labeled with colloidal gold, to form copolymers with the polymer comprise a plurality of colloidal gold labeled antibodies used recognize different tumor-specific antigens.

[0007] 进一步地,将所述包含多种胶体金标记的用于识别不同肿瘤特异性抗原的抗体的共聚物作为抗体载体参加免疫浊度反应,可作为检测多种肿瘤的免疫增强体外诊断试剂, 大大提高了检测的灵敏度,且可用于多种肿瘤的检测。 Copolymer [0007] Further, the plurality comprising a colloidal gold labeled antibodies used recognize different antigens as tumor-specific antibody carrier participate in immune turbidity of the reaction, can be enhanced as in vitro diagnostic reagents for immunological detection of various tumors , greatly improving the detection sensitivity, and can be used to detect a variety of tumors.

[0008]进一步地,所述聚合物为可生物降解的聚合物,其生物相容性较好,也便于后期处理。 [0008] Further, the polymer is a biodegradable polymer is preferably biocompatible, but also facilitate post-processing.

[0009]进一步地,所述可生物降解的聚合物选自聚羟基醋酸、聚羟基丁酸酯、乳酸-聚乙二醇共聚物、聚乳fe-聚乙二醇嵌段共聚物或聚羟基烷基醇酯中的任意一种。 [0009] Further, the biodegradable polymer is selected from polyhydroxy acid, polyhydroxybutyrate, lactic acid - polyethylene glycol copolymer, polylactic fe- polyethylene glycol block copolymer or polyhydroxy any alkyl alcohol ester.

[0010]进一步地,所述肿瘤包括胃癌、肝癌、结肠癌,当然也可以是其他癌症疾病。 [0010] Further, the tumor including gastric, liver, colon, of course, be other cancer diseases.

[0011]进一步地,所述用于识别胃癌特异性抗原的抗体为抗S100P蛋白多克隆抗体;所述用于识别肝癌特异性抗原的抗体为人肝癌组织AFU多克隆抗体;所述用于识别结肠癌特异性抗原的抗体为结肠癌抗原3抗体(SDCCAG3)。 [0011] Further, the antibodies recognize tumor specific antigens for anti S100P polyclonal antibody; hepatoma antibody recognizes a specific antigen for the human HCC AFU polyclonal antibody; means for identifying colon antibodies for colon cancer-specific antigen antibody antigen 3 (SDCCAG3).

[0012]进一步地,所述纳米金标记的识别肿瘤特异性抗原的抗体共聚物制备方法为: [0013] (1)制备胶体金溶液:用质量百分比浓度为1%的柠檬酸三钠水溶液与质量百分比浓度为0.01 %氯金酸水溶液,按1.5mL:100mL的体积比,将氯金酸溶液加热至沸腾,把柠檬酸三钠水溶液快速加入其中,搅拌反应至所得的反应液为酒红色,再加$10min,直至溶液透亮,冷至室温,然后用浓度为0 • 〇5〜0 • lm〇l/L的碳酸钾水溶液调节pH值为6.5〜8.0,即得胶体金溶液; _4] ⑵在步骤⑴所得的胶体金溶液中,在磁力搅拌下缓慢加入浓度为10〜50tlg/mL 的用于识别胃癌特异性抗原的抗体水溶液,混匀,37。 [0012] Further, an antibody preparation of the copolymer of gold nanoparticles labeled to identify tumor-specific antigens: [0013] (1) Preparation of colloidal gold solution: with a mass concentration of 1% trisodium citrate aqueous solution mass concentration of 0.01% chloroauric acid aqueous solution, by 1.5mL: 100mL volume ratio of the chloroauric acid solution was heated to boiling, the aqueous solution of trisodium citrate was added quickly, the reaction was stirred until the resultant reaction liquid is wine red, plus $ 10min, until a translucent solution was cooled to room temperature and then 0 • • 〇5~0 lm〇l / L aqueous potassium carbonate adjusted to pH 6.5~8.0, i.e., to obtain a concentration of colloidal gold solution; _4] ⑵ in the colloidal gold solution obtained in step ⑴, under magnetic stirring was slowly added an aqueous solution of a concentration of an antibody for recognizing tumor specific antigens 10~50tlg / mL, the mixing, 37. (:孵育30min,加封闭液继续37。(:孵育22〜25min,14000rpIn,4°C条件下离心20〜22min,去除上清液,稀释液重悬沉淀并洗涤一次,得到胶体金-抗S100P蛋白多克隆抗体标记物I; (: Incubation 30min, add 37 continues blocking solution (: incubation 22~25min, 14000rpIn, 4 ° C for centrifugation conditions 20~22min, remove the supernatant, and resuspend pellet dilution was washed once, to give colloidal gold - Anti S100P polyclonal antibody marker I;

[0015]另取步骤(1)所得的胶体金溶液,在磁力搅拌下缓慢加入浓度为10〜50ug/tnL的用于识别肝癌特异性抗原的抗体水溶液,按照上述制备胶体金-抗S100P蛋白多克隆抗体标记物I同样的操作方法,得到胶体金-人肝癌组织AFU多克隆抗体标记物n; [0015] Another step (1) colloidal gold solution obtained, under magnetic stirring was slowly added at a concentration 10~50ug / tnL antibody solution for identifying liver specific antigen, prepared as described above colloidal gold anti - proteoglycan S100P I monoclonal antibody markers similar method, to obtain colloidal gold - AFU human hepatocellular carcinoma tissue was polyclonal antibody labeled n;

[0016]另取步骤(1)所得的胶体金溶液,在磁力搅拌下缓慢加入浓度为10〜5〇ug/mL的用于识别结肠癌特异性抗原的抗体水溶液,按照上述制备胶体金-抗S100P蛋白多克隆抗体标记物I同样的操作方法,得到胶体金-结肠癌抗原3抗体(SDCCAG3)标记物DI; [0016] Another step (1) colloidal gold solution obtained, under magnetic stirring was slowly added an aqueous solution of a concentration of an antibody for identifying a colon cancer-specific antigen 10~5〇ug / mL of colloidal gold prepared in accordance with the above-described anti - S100P I polyclonal antibody markers similar method, to obtain colloidal gold - 3 colon cancer antigen antibody (SDCCAG3) the DI markers;

[0017] ⑶将所述聚合物按1:18〜30的重量比溶于二氯甲烷,加入上述步骤⑵制得的标记物I、标记物n、标记物m,用超声波处理30〜60s,得到胶体金标记的识别胃癌、肝癌、结肠癌三种肿瘤的特异性抗原的抗体共聚物。 [0017] ⑶ the polymer by 1: 18~30 weight ratio dissolved in dichloromethane and added to the above prepared steps ⑵ marker I, marker n, label m, 30~60s treated with ultrasound, colloidal gold labeled obtained recognition gastric cancer, liver cancer, colon specific antigen antibody copolymer of three tumor.

[0018]进一步地,所述的胶体金-抗S100P蛋白多克隆抗体标记物I,按质量比计算,即胶体金:抗S100P蛋白多克隆抗体为5:0 • 21〜0 • 23;所述的胶体金-人肝癌组织AFU多克隆抗体标记物n,按质量比计算,即胶体金:人肝癌组织AFU多克隆抗体为5:0.25〜0.28;所述的胶体金-结肠癌抗原3抗体(SDCCAG3)标记物HI,按质量比计算,即胶体金:结肠癌抗原3抗体(SDCCAG3)为5:0.23〜0.26。 [0018] Further, the colloidal gold - S100P polyclonal antibody anti-tag I, calculated mass ratio, i.e. colloidal gold: polyclonal antibody anti-S100P is 5: 0 • 21~0 • 23; the colloidal gold - the Tumor markers AFU polyclonal antibody n, calculated mass ratio, i.e., the colloidal gold: polyclonal human hepatoma tissue AFU 5: 0.25~0.28; the colloidal gold - 3 colon cancer antigen antibody ( SDCCAG3) the HI markers, calculate a mass ratio, i.e. colloidal gold: 3 colon cancer antigen antibody (SDCCAG3) 5: 0.23~0.26.

[0019]进一步地,所述超声波处理的频率为35〜55KHz,经超声波处理可以使形成的聚合物均匀。 [0019] Further, the frequency of the ultrasonic treatment is 35~55KHz, sonicated polymer formed can be made uniform.

[0020]进一步地,所述封闭液为含牛血清蛋白的洗涤液,其质量浓度为〇.3〜img/mL,加入封闭液可避免非特异性结合。 [0020] Further, the blocking buffer was bovine serum albumin-containing wash liquid, which concentration of 〇.3~img / mL, solution can be added to avoid blocking non-specific binding.

[0021]本发明的有益效果是:通过将多种识别肿瘤特异性抗原的抗体分别用胶体金标记后,与可生物降解的聚合物形成包含多种胶体金标记的用于识别不同肿瘤特异性抗原的抗体的共聚物,作为抗体载体参加免疫浊度反应,用于检测多种肿瘤的免疫增强体外诊断试剂,可检测多种肿瘤,具有普遍适用性;相对于现有技术中的各种影像学检测技术,如X线、B 超、CT、磁共振等重要的肿瘤辅助诊断手段来说,可以达到早期诊断的目的,且相比较于现有的免疫浊度法,也大大提高了检测的灵敏度。 [0021] Advantageous effects of the invention are: by more antibodies which recognize tumor-specific antigens are labeled with the colloidal gold, and the biodegradable polymer is formed may comprise a plurality of colloidal gold labeled for identifying different tumor specific copolymers of the antigen, as an immune antibody carrier participate turbidity of the reaction, for the detection of various tumors enhance immune diagnostic reagents in vitro, a variety of tumors can be detected, having general applicability; with respect to the prior art a variety of imaging Science detection techniques, such as the important tumor diagnosis means X ray, B, CT, MRI, etc., it can achieve the purpose of early diagnosis, and compared to conventional immunological nephelometry, also greatly enhance the detection of sensitivity.

具体实施方式[0022]实施例1: DETAILED DESCRIPTION [0022] Example 1:

[0023] —种可以检测胃癌、肝癌、结肠癌的组合物,是由用于识别胃癌特异性抗原的抗体、用于识别肝癌特异性抗原的抗体、用于识别结肠癌特异性抗原的抗体、聚合物、胶体金溶液组成;其中,所述用于识别胃癌特异性抗原的抗体为抗S100P蛋白多克隆抗体;所述用于识别肝癌特异性抗原的抗体为人肝癌组织AFU多克隆抗体;所述用于识别结肠癌特异性抗原的抗体为结肠癌抗原3抗体(SDCCAG3);所述聚合物为可生物降解的聚合物,选自聚羟基醋酸、聚羟基丁酸酯、乳酸-聚乙二醇共聚物、聚乳酸-聚乙二醇嵌段共聚物或聚羟基烷基醇酯中的任意一种,这些课生物降解的聚合物生物相容性较好,也便于后期处理。 [0023] - species can detect stomach cancer, liver cancer, colon cancer composition is an antibody for identifying tumor specific antigen, an antibody for a specific antigen recognition liver cancer, colon cancer antibody recognizes a specific antigen, polymer, colloidal gold solution composition; wherein the antibody recognizes a tumor specific antigen, polyclonal antibody anti S100P; for the antibody recognizes a human hepatoma liver cancer-specific antigen AFU polyclonal antibody; the antibodies for identifying colon cancer colon-specific antigen antibody antigen-3 (SDCCAG3); the polymer is a biodegradable polymer selected from polyglycolic acid, polyhydroxybutyrate, lactic acid - polyethylene glycol copolymers, polylactic acid - polyethylene glycol block copolymer or any of a polyhydroxy alkyl alcohol esters of these courses is preferably biocompatible biodegradable polymers, also facilitate post-processing.

[0024]该纳米金标记的识别肿瘤特异性抗原的抗体共聚物制备方法为: [0024] Antibodies prepared copolymers of the nano gold-labeled tumor-specific antigens identified as:

[0025] (1)制备胶体金溶液:用质量百分比浓度为1 %的柠檬酸三钠水溶液与质量百分比浓度为〇. 01 %氯金酸水溶液,按1 • 5mL: lOOmL的体积比,将氯金酸溶液加热至沸腾,把柠檬酸三钠水溶液快速加入其中,撹拌反应至所得的反应液为酒红色,再加热l〇min,直至溶液透亮,冷至室温,然后用浓度为0. 〇5mol/L的碳酸钾水溶液调节pH值为6.5,即得胶体金溶液; [0025] (1) Preparation of colloidal gold solution: with a mass concentration of 1% aqueous solution of trisodium citrate square mass concentration of 0.01% gold chloride acid solution, press 1 • 5mL:. LOOmL volume ratio of chlorine acid solution was heated to boiling, the aqueous solution of trisodium citrate was added quickly, the reaction stirred Jiao to the resulting reaction liquid was wine red, and then heated l〇min until the solution is translucent, cooled to room temperature, then treated with a concentration of 0.5 〇5mol / L aqueous potassium carbonate solution adjusted to pH 6.5, to obtain a colloidal gold solution;

[0026] (2)在步骤(1)所得的胶体金溶液中,在磁力搅拌下缓慢加入浓度为l〇ug/mL的用于识别胃癌特异性抗原的抗体水溶液,混匀,3 7 °C孵育3 0 min,加封闭液继续3 7 -C孵育22min,所述封闭液为含牛血清蛋白的洗涤液,其质量浓度为〇. 3mg/mL,加入封闭液可避免非特异性结合,再14000rpm,4t:条件下离心20min,去除上清液,稀释液重悬沉淀并洗涤一次,得到胶体金-抗S100P蛋白多克隆抗体标记物I,按质量比计算,胶体金:抗S100P蛋白多克隆抗体为5:0.21; [0026] (2) in step (1) in the resulting colloidal gold solution, under magnetic stirring was slowly added an aqueous solution of a concentration of the antibody l〇ug / mL for identifying tumor specific antigen, mixing, 3 7 ° C incubated for 3 0 min, blocking solution was added 3 7 -C continued 22min incubation, the blocking solution is a washing solution containing bovine serum albumin, the concentration of which is square. 3mg / mL, solution can be added to avoid blocking non-specific binding, then 14000rpm , a 4T: under centrifuged 20min, supernatant was removed and diluted pellet was resuspended and washed once to give a colloidal gold - S100P polyclonal antibody anti-tag I, calculated mass ratio, the colloidal gold: polyclonal antibody anti-S100P 5: 0.21;

[0027]另取步骤(1)所得的胶体金溶液,在磁力搅拌下缓慢加入浓度为10〜50ug/mL的用于识别肝癌特异性抗原的抗体水溶液,按照上述制备胶体金-抗S1 OOP蛋白多克隆抗体标记物I同样的操作方法,得到胶体金-人肝癌组织AFU多克隆抗体标记物n,按质量比计算,胶体金:人肝癌组织AFU多克隆抗体为5:0.25; [0027] Another step (1) colloidal gold solution obtained, under magnetic stirring was slowly added at a concentration 10~50ug / mL antibody solution for identifying liver cancer-specific antigen, prepared as described above colloidal gold anti - S1 OOP protein I polyclonal antibody markers similar method, to obtain colloidal gold - the Tumor markers AFU polyclonal antibody n, calculating the mass ratio, the colloidal gold: the Tumor AFU polyclonal antibody to 5: 0.25;

[0028]另取步骤(1)所得的胶体金溶液,在磁力搅拌下缓慢加入浓度为10iig/mL的用于识别结肠癌特异性抗原的抗体水溶液,按照上述制备胶体金-抗S100P蛋白多克隆抗体标记物I同样的操作方法,得到胶体金-结肠癌抗原3抗体(SDCCAG3)标记物m,按质量比计算,胶体金:结肠癌抗原3抗体(SDCCAG3)为5:0.23; [0028] Another step (1) colloidal gold solution obtained, under magnetic stirring was slowly added at a concentration 10iig / mL antibody solution for identifying colon cancer specific antigen, prepared as described above colloidal gold - polyclonal anti S100P antibody labels the same operation I, to give a colloidal gold - 3 colon cancer antigen antibody (SDCCAG3) markers m, the mass ratio calculated colloidal gold: 3 colon cancer antigen antibody (SDCCAG3) 5: 0.23;

[0029] (3)将所述聚合物按1:18的重量比溶于二氯甲烷,加入上述步骤(2)制得的标记物I、标记物II、标记物m,用超声波处理30s,所述超声波处理的频率为35KHz,经超声波处理可以使形成的聚合物均匀,得到胶体金标记的识别胃癌、肝癌、结肠癌三种肿瘤的特异性抗原的抗体共聚物。 [0029] (3) the polymer weight ratio of 1:18 was dissolved in dichloromethane, was added the above step (2) was obtained marker I, labeled class II, marker m, 30s treated with ultrasound, the frequency of 35KHz ultrasonic treatment, ultrasonic treatment can be made by forming a uniform polymer, are identified colloidal gold labeled gastric, liver, colon specific antigen antibody copolymer of three tumor.

[0030] 将所述胶体金标记的识别胃癌、肝癌、结肠癌三种肿瘤的特异性抗原的抗体共聚物作为抗体载体参加免疫浊度反应,作为检测胃癌、肝癌、结肠癌的免疫增强体外诊断试剂,大大提高了检测的灵敏度。 [0030] The identification of the colloidal gold labeled gastric cancer, liver cancer, colon specific antigen antibody copolymer as three tumor immune antibody carrier participate turbidity of the reaction, as the detection of gastric cancer, liver cancer, colon cancer immune enhancement vitro diagnostic reagents, greatly improving the detection sensitivity.

[0031] 实施例2: [0031] Example 2:

[0032] 一种可以检测胃癌、肝癌、结肠癌的组合物,是由用于识别胃癌特异性抗原的抗体、用于识别肝癌特异性抗原的抗体、用于识别结肠癌特异性抗原的抗体、聚合物、胶体金溶液组成;其中,所述用于识别胃癌特异性抗原的抗体为抗S100P蛋白多克隆抗体;所述用于识别肝癌特异性抗原的抗体为人肝癌组织AFU多克隆抗体;所述用于识别结肠癌特异性抗原的抗体为结肠癌抗原3抗体(SDCCAG3);所述聚合物为可生物降解的聚合物,选自聚羟基醋酸、聚羟基丁酸酯、乳酸-聚乙二醇共聚物、聚乳酸-聚乙二醇嵌段共聚物或聚羟基烷基醇酯中的任意一种,这些课生物降解的聚合物生物相容性较好,也便于后期处理。 [0032] A can detect stomach cancer, liver cancer, colon cancer composition is an antibody for recognizing tumor specific antigens for antibody recognizes a specific antigen hepatoma, colon cancer antibody recognizes a specific antigen, polymer, colloidal gold solution composition; wherein the antibody recognizes a tumor specific antigen, polyclonal antibody anti S100P; for the antibody recognizes a human hepatoma liver cancer-specific antigen AFU polyclonal antibody; the antibodies for identifying colon cancer colon-specific antigen antibody antigen-3 (SDCCAG3); the polymer is a biodegradable polymer selected from polyglycolic acid, polyhydroxybutyrate, lactic acid - polyethylene glycol copolymers, polylactic acid - polyethylene glycol block copolymer or any of a polyhydroxy alkyl alcohol esters of these courses is preferably biocompatible biodegradable polymers, also facilitate post-processing.

[0033]该纳米金标记的识别肿瘤特异性抗原的抗体共聚物制备方法为: [0033] Antibodies prepared copolymers of the nano gold-labeled tumor-specific antigens identified as:

[0034] (1)制备胶体金溶液:用质量百分比浓度为1%的柠檬酸三钠水溶液与质量百分比浓度为0 • 01 %氯金酸水溶液,按1.5mL: lOOmL的体积比,将氯金酸溶液加热至沸腾,把柠檬酸三钠水溶液快速加入其中,搅拌反应至所得的反应液为酒红色,再加热l〇min,直至溶液透亮,冷至室温,然后用浓度为0.075mol/L的碳酸钾水溶液调节pH值为7 • 0,即得胶体金溶液; [0034] (1) Preparation of colloidal gold solution: with a mass concentration of 1% trisodium citrate aqueous solution of the mass concentration of 0 • 01% chloroauric acid aqueous solution, by 1.5mL: lOOmL volume ratio, chloroauric acid solution was heated to boiling, the aqueous solution of trisodium citrate was added quickly, the reaction was stirred until the resultant reaction liquid is wine red, reheating l〇min until the solution is translucent, cooled to room temperature, then treated with a concentration of 0.075mol / L of aqueous potassium carbonate solution adjusting pH to 7 • 0, i.e., to obtain a colloidal gold solution;

[0035] ⑵在步骤⑴所得的胶体金溶液中,在磁力搅拌下缓慢加入浓度为30ug/mL的用于识别胃癌特异性抗原的抗体水溶液,混匀,37°C孵育30min,加封闭液继续37。 [0035] ⑵ ⑴ the colloidal gold solution obtained in step, under magnetic stirring was slowly added at a concentration of 30ug / antibody solution for identifying a tumor specific antigen mL, mix, 37 ° C incubated for 30min, added to continue blocking solution 37. (3孵育23 • 5mi n,所述封闭液为含牛血清蛋白的洗涤液,其质量浓度为〇• 65mg/mL,加入封闭液可避免非特异性结合,再14000rpm,4°C条件下离心21min,去除上清液,稀释液重悬沉淀并洗涤一次,得到胶体金-抗S100P蛋白多克隆抗体标记物I,按质量比计算,胶体金:抗S100P蛋白多克隆抗体为5:0.22; (3 were incubated for 23 • 5mi n, the blocking buffer was bovine serum albumin-containing wash liquid, which concentration of square • 65mg / mL, solution can be added to avoid blocking non-specific binding, then 14000 rpm, 4 ° C for centrifugation conditions 21min , remove the supernatant, and resuspend pellet dilution was washed once, to give colloidal gold - S100P polyclonal antibody anti-tag I, calculated mass ratio, the colloidal gold: polyclonal antibody anti-S100P 5: 0.22;

[0036]另取步骤⑴所得的胶体金溶液,在磁力搅拌下缓慢加入浓度为1〇〜5〇ug/mL的用于识别肝癌特异性抗原的抗体水溶液,按照上述制备胶体金-抗S100P蛋白多克隆抗体标记物I同样的操作方法,得到胶体金-人肝癌组织AFU多克隆抗体标记物n,按质量比计算,胶体金:人肝癌组织AFU多克隆抗体为5:0.265; [0036] Another step ⑴ resulting colloidal gold solution, under magnetic stirring was slowly added at a concentration 1〇~5〇ug / antibody solution for identifying liver cancer-specific antigen mL of colloidal gold prepared as described above - Anti S100P protein I polyclonal antibody markers similar method, to obtain colloidal gold - the Tumor markers AFU polyclonal antibody n, calculating the mass ratio, the colloidal gold: polyclonal human hepatoma tissue AFU 5: 0.265;

[0037]另取步骤(1)所得的胶体金溶液,在磁力搅拌下缓慢加入浓度为30ug/mL的用于识别结肠癌特异性抗原的抗体水溶液,按照上述制备胶体金-抗S100P蛋白多克隆抗体标记物I同样的操作方法,得到胶体金-结肠癌抗原3抗体(SDCCAG3)标记物HI,按质量比计算,胶体金:结肠癌抗原3抗体(SDCCAG3)为5:0.245; [0037] Another step (1) colloidal gold solution obtained, was slowly added under magnetic stirring at a concentration of 30ug / mL antibody solution for identifying colon cancer specific antigen, prepared as described above colloidal gold - polyclonal anti S100P antibody labels the same operation I, to give a colloidal gold - 3 colon cancer antigen antibody (SDCCAG3) the HI markers, calculated mass ratio, the colloidal gold: 3 colon cancer antigen antibody (SDCCAG3) 5: 0.245;

[0038] (¾将所述聚合物按1 :对的重量比溶于二氯甲烷,加入上述步骤(2)制得的标记物I、标记物n、标记物m,用超声波处理45s,所述超声波处理的频率为35〜55KHZ,经超声波处理可以使形成的聚合物均匀,得到胶体金标记的识别胃癌、肝癌、结肠癌三种肿瘤的特异性抗原的抗体共聚物。 [0038] (¾ the polymer by 1: weight ratio dissolved in dichloromethane and added to the above step (2) was obtained marker I, marker n, label m, 45s treated with ultrasonic waves, the said frequency ultrasonic treatment is 35~55KHZ, sonicated of the polymer can be uniformly formed, are identified colloidal gold labeled gastric, liver, colon specific antigen antibody copolymer of three tumor.

[0039] 将所述胶体金标记的识别胃癌、肝癌、结肠癌三种肿瘤的特异性抗原的抗体共聚物作为抗体载体参加免疫浊度反应,作为检测胃癌、肝癌、结肠癌的免疫增强体外诊断试剂,大大提局了检测的灵敏度。 [0039] The identification of the colloidal gold labeled gastric cancer, liver cancer, colon specific antigen antibody copolymer as three tumor immune antibody carrier participate turbidity of the reaction, as the detection of gastric cancer, liver cancer, colon cancer immune enhancement vitro diagnostic reagents, greatly enhanced detection sensitivity Board.

[0040] 实施例3: [0040] Example 3:

[0041] 一种可以检测胃癌、肝癌、结肠癌的组合物,是由用于识别胃癌特异性抗原的抗体、用于识别肝癌特异性抗原的抗体、用于识别结肠癌特异性抗原的抗体、聚合物、胶体金溶液组成;其中,所述用于识别胃癌特异性抗原的抗体为抗S100P蛋白多克隆抗体;所述用于识别肝癌特异性抗原的抗体为人肝癌组织AFU多克隆抗体;所述用于识别结肠癌特异性抗原的抗体为结肠癌抗原3抗体(SDCCAG3);所述聚合物为可生物降解的聚合物,选自聚羟基醋酸、聚羟基丁酸酯、乳酸-聚乙二醇共聚物、聚乳酸-聚乙二醇嵌段共聚物或聚羟基烷基醇酯中的任意一种,这些课生物降解的聚合物生物相容性较好,也便于后期处理。 [0041] A can detect stomach cancer, liver cancer, colon cancer composition is an antibody for recognizing tumor specific antigens for antibody recognizes a specific antigen hepatoma, colon cancer antibody recognizes a specific antigen, polymer, colloidal gold solution composition; wherein the antibody recognizes a tumor specific antigen, polyclonal antibody anti S100P; for the antibody recognizes a human hepatoma liver cancer-specific antigen AFU polyclonal antibody; the antibodies for identifying colon cancer colon-specific antigen antibody antigen-3 (SDCCAG3); the polymer is a biodegradable polymer selected from polyglycolic acid, polyhydroxybutyrate, lactic acid - polyethylene glycol copolymers, polylactic acid - polyethylene glycol block copolymer or any of a polyhydroxy alkyl alcohol esters of these courses is preferably biocompatible biodegradable polymers, also facilitate post-processing.

[0042]该纳米金标记的识别肿瘤特异性抗原的抗体共聚物制备方法为: [0042] Antibodies prepared copolymers of the nano gold-labeled tumor-specific antigens identified as:

[0043] (1)制备胶体金溶液:用质量百分比浓度为1 %的柠檬酸三钠水溶液与质量百分比浓度为0 • 01 %氯金酸水溶液,按1 • 5mL: lOOmL的体积比,将氯金酸溶液加热至沸腾,把柠檬酸二钠水溶液快速加入其中,搅拌反应至所得的反应液为酒红色,再加热lOmin,直至溶液透亮,冷至室温,然后用浓度为0 • lmol/L的碳酸钾水溶液调节pH值为8.0,即得胶体金溶液; [0044] (2)在步骤(1)所得的胶体金溶液中,在磁力搅拌下缓慢加入浓度为1〇〜5〇ug/mL 的用于识别胃癌特异性抗原的抗体水溶液,混匀,37 °C孵育30min,加封闭液继续37。 [0043] (1) Preparation of colloidal gold solution: with a mass concentration of 1% trisodium citrate aqueous solution of the mass concentration of 0 • 01% chloroauric acid aqueous solution, by 1 • 5mL: lOOmL volume ratio of chlorine acid solution was heated to boiling, the aqueous solution of disodium citrate quickly added thereto, the reaction was stirred until the resultant reaction liquid is wine red, reheating lOmin, until a translucent solution was cooled to room temperature, then treated with a concentration of 0 • lmol / L of aqueous potassium carbonate solution adjusted to pH 8.0, to obtain colloidal gold solution; [0044] (2) in step (1) in the resulting colloidal gold solution, under magnetic stirring was slowly added at a concentration 1〇~5〇ug / mL, antibody solution for identifying tumor specific antigen, mixing, 37 ° C incubation 30min, add 37 to continue blocking solution. (:孵育25min,所述封闭液为含牛血清蛋白的洗涤液,其质量浓度Slmg/mL,加入封闭液可避免非特异性结合,再1400〇rpm,4°C条件下离心22min,去除上清液,稀释液重悬沉淀并洗涤一次, 得到胶体金-抗S100P蛋白多克隆抗体标记物I,按质量比计算,胶体金:抗S100P蛋白多克隆抗体为5:0.23; (: Incubation 25min, the solution is washed with blocking solution containing bovine serum albumin, the concentration Slmg / mL, solution can be added to avoid blocking non-specific binding, then 1400〇rpm, centrifugation conditions of 4 ° C for 22min, the supernatant was removed , diluents and resuspend pellet was washed once, to give colloidal gold - S100P polyclonal antibody anti-tag I, calculated mass ratio, the colloidal gold: polyclonal antibody anti-S100P 5: 0.23;

[0045]另取步骤(1)所得的胶体金溶液,在磁力搅拌下缓慢加入浓度为50iig/mL的用于识别肝癌特异性抗原的抗体水溶液,按照上述制备胶体金-抗S100P蛋白多克隆抗体标记物I 同样的操作方法,得到胶体金-人肝癌组织AFU多克隆抗体标记物n,按质量比计算,胶体金:人肝癌组织AFU多克隆抗体为5:0.28; [0045] Another step (1) colloidal gold solution obtained, under magnetic stirring was slowly added at a concentration 50iig / mL antibody solution for identifying liver cancer-specific antigen, prepared as described above colloidal gold - polyclonal antibody anti-S100P I markers similar method, to obtain colloidal gold - the Tumor markers AFU polyclonal antibody n, calculating the mass ratio, the colloidal gold: the Tumor AFU polyclonal antibody to 5: 0.28;

[0046]另取步骤(1)所得的胶体金溶液,在磁力搅拌下缓慢加入浓度为50ug/tnL的用于识别结肠癌特异性抗原的抗体水溶液,按照上述制备胶体金-抗S100P蛋白多克隆抗体标记物I同样的操作方法,得到胶体金-结肠癌抗原3抗体(SDCCAG3)标记物HI,按质量比计算,胶体金:结肠癌抗原3抗体(SDCCAG3)为5:0.26; [0046] Another step (1) colloidal gold solution obtained, was slowly added under magnetic stirring at a concentration of 50ug / tnL antibody solution for identifying colon cancer specific antigen, prepared as described above colloidal gold - polyclonal anti S100P antibody labels the same operation I, to give a colloidal gold - 3 colon cancer antigen antibody (SDCCAG3) the HI markers, calculated mass ratio, the colloidal gold: 3 colon cancer antigen antibody (SDCCAG3) 5: 0.26;

[0047] ⑶将所述聚合物按1:30的重量比溶于二氯甲烷,加入上述步骤⑵制得的标记物I、标记物II、标记物m,用超声波处理60s,所述超声波处理的频率为55KHz,经超声波处理可以使形成的聚合物均匀,得到胶体金标记的识别胃癌、肝癌、结肠癌三种肿瘤的特异性抗原的抗体共聚物。 [0047] ⑶ the polymer weight ratio of 1:30 was dissolved in dichloromethane, added to the above prepared steps ⑵ marker I, labeled class II, marker m, 60s treated with ultrasonic waves, the ultrasonic treatment frequency of 55KHz, sonicated of the polymer can be uniformly formed, are identified colloidal gold labeled gastric, liver, colon specific antigen antibody copolymer of three tumor.

[0048]将所述胶体金标记的识别胃癌、肝癌、结肠癌三种肿瘤的特异性抗原的抗体共聚物作为抗体载体参加免疫浊度反应,作为检测胃癌、肝癌、结肠癌的免疫增强体外诊断试剂,大大提高了检测的灵敏度。 [0048] The identification of the colloidal gold labeled gastric cancer, liver cancer, colon specific antigen antibody copolymer as three tumor immune antibody carrier participate turbidity of the reaction, as the detection of gastric cancer, liver cancer, colon cancer immune enhancement vitro diagnostic reagents, greatly improving the detection sensitivity.

[0049] 实验数据 [0049] Experimental Data

[0050] 1、实验对象: [0050] 1, subjects:

[0051] 选择某医院自2009年1月至2010年12月收治的胃癌、肝癌、结肠癌患者共900名,其中胃癌患者300名,肝癌患者300名,结肠癌患者300名,所有病例均初次诊断为胃癌、肝癌或结肠癌,并且之前没有接受过放疗和化疗。 [0051] choose a hospital from stomach cancer from January 2009 to December 2010 admitted, liver cancer, colon cancer patients with a total of 900, of which 300 patients with gastric cancer, liver cancer 300 patients, 300 patients with colon cancer, in all cases for the first time diagnosis of gastric cancer, liver cancer or colon cancer, and had not received previous chemotherapy and radiotherapy.

[0052] 2、检测试剂: [0052] 2, detection reagents:

[0053]将本发明实施例1制备的胶体金标记的识别冑癌、肝癌、结肠癌三种肿瘤的特异性抗原的抗体共聚物作为检测试剂I,将本发明实施例2制备的胶体金标记的识别胃癌、肝癌、 结肠癌三种肿瘤的特异性抗原的抗体共聚物作为检测试剂n,将本发明实施例3制备的胶体金标记的识别胃癌、肝癌、结肠癌三种肿瘤的特异性抗原的抗体共聚物作为检测试剂m, 分别采集上述900名患者的血清,作为待测样品。 Colloidal gold labeled colloidal gold labeled [0053] Preparation Example 1 of the embodiment of the present invention to identify helmet cancer, liver cancer, colon cancer antibody copolymer three tumor-specific antigen as a detection reagent I, prepared in Example 2 of the embodiment of the present invention gastric colloidal gold labeled recognition identification gastric cancer, liver cancer, colon cancer antibody copolymer three tumor-specific antigen as a detection reagent n, prepared in Example 3 of the embodiment of the present invention, liver, colon three tumor specific antigens a copolymer of an antibody as a detection reagent m, sera were collected above 900 patients, as a test sample.

[0054] 3、实验方法: [0054] 3. Experimental Method:

[0055] 分组:将上述300名胃癌患者、300名肝癌患者、300名结肠癌患者分别平均分成3 组,每组各100人,分别记为:胃癌1组、胃癌2组、胃癌3组、肝癌1组、肝癌2组、肝癌3组、结肠癌1组、结肠癌2组、结肠癌3组。 [0055] Packet: above 300 gastric cancer patients, 300 patients with liver cancer, colon cancer patients were 300 were divided into 3 groups, each group 100, are denoted as follows: Group 1 gastric cancer, gastric cancer group 2, group 3 gastric cancer, group 1 hepatoma, liver cancer group 2, group 3 liver, colon group 1, group 2 colon cancer, colon cancer 3 groups. 胃癌1组、肝癌1组、结肠癌1组使用检测试剂I进行检测,胃癌2组、肝癌2组、结肠癌2组使用检测试剂II进行检测,冑癌3、肝癌3组、结肠癌3组使用检测试剂m进行检测。 Gastric group 1, a group of liver cancer, colon cancer groups using a detection reagent I is detected, gastric group 2, group 2 liver cancer, colon cancer groups using a detection reagent 2 to detect II, 3 helmet cancer, liver cancer group 3, group 3 colon cancer m is detected using a detection reagent.

[0056] 检测方法:分别在胃癌患者血清中提取胃癌抗原,在肝癌患者血清中提取肝癌抗原,在结肠癌患者血清中提取结肠癌抗原,分别稀释得到浓度分别1 • 〇Ug/L、7.5ug/L、15ug/ 1、30邱/1、60邱凡、120收凡的待测样本。 [0056] Detection: gastric cancer patients were extracted in gastric cancer antigen, a liver extract antigen in the serum of liver cancer patients, colon cancer extract antigen in the serum of patients with colon cancer, respectively, were diluted to give a concentration of 1 • 〇Ug / L, 7.5ug / L, 15ug / 1,30 Qiu / 1,60 where Qiu, 120 to close all of the test sample. 先检测试剂的空白吸光度000,采用6点定标方法, 在每500ul检测试剂中分别加入相应各组患者血清样本10此,混合混匀,使二者发生抗原抗体反应,置37°C,孵育3小时后,通过分光光度计读取波长为340nm处的对应6组吸光度值0D1、0D2、0D3、0D4、0D5、0D6。 First detection reagent blank absorbance 000, a 6-point calibration method, each detection reagent was added 500ul serum samples respectively corresponding to each of the 10 patients in this group, mixed mix the antigen-antibody reaction occurs between the two, opposing 37 ° C, incubation after 3 hours, read by a spectrophotometer at a wavelength of 340nm 6 groups corresponding to the absorbance value 0D1,0D2,0D3,0D4,0D5,0D6.

[0057] 检测仪器:OLYMPUS AU640全自动生化分析仪。 [0057] instrumentation: OLYMPUS AU640 automatic biochemical analyzer.

[0058] 4、检测数据统计结果: [0058] 4. Detection Statistics Results:

[0059] 300名胃癌患者中检测出阳性298例、300名肝癌患者检测出阳性296例、300名结肠癌患检测出阳性299例,总检出率为99.2%。 [0059] 300 gastric cancer patients detected positive for 298 cases, 300 detect positive hepatocellular carcinoma patients of 296 patients suffering from colon 300 299 cases detected positive, positive rate was 99.2%.

[0060] 5、结论:本发明用于检测多种肿瘤的组合物能够检测多种肿瘤,灵敏度高,检出率闻。 [0060] 5. Conclusions: the present invention for the detection of various tumors compositions capable of detecting a variety of tumors, high sensitivity, the detection rate of smell.

[0061] 最后应说明的是:以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明实施例技术方案的精神和范围。 [0061] Finally, it should be noted that: the above embodiments are intended to illustrate the present invention, rather than limiting;. Although the present invention has been described in detail embodiments, those of ordinary skill in the art should be understood: Examples which may still aspect of the described embodiment may be modified, or some technical features equivalents; as such modifications or replacements do not cause the essence of corresponding technical solutions to depart from the spirit and scope of the technical solutions of the embodiments of the present invention .

Claims (4)

1.一种可以检测多种肿瘤的组合物,其特征在于,是由多种识别肿瘤特异性抗原的抗体、聚合物、胶体金溶液组成;其制备方法为:将所述多种识别肿瘤特异性抗原的抗体分别用胶体金标记后,与所述聚合物形成包含多种胶体金标记的用于识别不同肿瘤特异性抗原的抗体的共聚物;将所述包含多种胶体金标记的用于识别不同肿瘤特异性抗原的抗体的共聚物作为抗体载体参加免疫浊度反应,可作为检测多种肿瘤的免疫增强体外诊断试剂;所述聚合物为可生物降解的聚合物;所述可生物降解的聚合物选自聚羟基醋酸、聚羟基丁酸酯、乳酸-聚乙二醇共聚物、聚乳酸-聚乙二醇嵌段共聚物或聚羟基烷基醇酯中的任意一种; 所述肿瘤包括胃癌、肝癌、结肠癌;所述用于识别胃癌特异性抗原的抗体为抗S100P蛋白多克隆抗体;所述用于识别肝癌特异性抗原的抗体为人肝癌组 A variety of tumors may be detected in the composition, wherein the plurality of recognition by an antibody is tumor-specific antigens, polymers, composed of colloidal gold solution; the preparation method: the plurality of tumor-specific identification antigen antibodies are labeled with the colloidal gold, and the polymer form a copolymer comprising a plurality of colloidal gold labeled antibodies used recognize different tumor-specific antigens; comprising a plurality of the gold colloid-labeled for recognize different tumor-specific antigens of antibody copolymer as a carrier to participate in an immune antibody turbidity of the reaction, can be enhanced in vitro diagnostic reagents as detection of various tumor immunity; the polymer is a biodegradable polymer; biodegradable the polymer is selected from polyhydroxy acid, polyhydroxybutyrate, lactic acid - any one of polyethylene glycol block copolymer or poly-hydroxy alkyl esters of - polyethylene glycol copolymer, a polylactic acid; the including gastric cancer, liver cancer, colon cancer; means for identifying tumor specific antibody is an anti-antigen polyclonal antibody S100P; the antibody for recognizing human hepatoma liver cancer-specific antigen group 织AFU多克隆抗体;所述用于识别结肠癌特异性抗原的抗体为结肠癌抗原3抗体(SDCCAG3);所述胶体金标记的识别肿瘤特异性抗原的抗体共聚物制备方法为: (1) 制备胶体金溶液:用质量百分比浓度为1%的柠檬酸三钠水溶液与质量百分比浓度为0 • 01 %氯金酸水溶液,按1 • 5mL: lOOmL的体积比,将氯金酸溶液加热至沸腾,把柠檬酸三钠水溶液快速加入其中,搅拌反应至所得的反应液为酒红色,冷至室温,然后调节pH值为6.5〜8.0,即得胶体金溶液; (2) 在步骤(1)所得的胶体金溶液中在磁力搅拌下缓慢加入浓度为10〜50iig/mL的用于识别胃癌特异性抗原的抗体水溶液,混匀,37 °C孵育30min,加封闭液继续37。 AFU woven polyclonal antibody; the antibody for identifying colon cancer colon-specific antigen antibody antigen 3 (SDCCAG3); SYNTHESIS antibody colloidal gold labeled the identification of tumor specific antigens: (1) preparation of colloidal gold solution: with a mass concentration of 1% trisodium citrate aqueous solution of the mass concentration of 0 • 01% chloroauric acid aqueous solution, by 1 • 5mL: lOOmL volume ratio of the chloroauric acid solution was heated to boiling , an aqueous solution of the trisodium citrate was added quickly, the reaction was stirred until the resultant reaction liquid is wine red, cooled to room temperature, then adjusted to pH 6.5~8.0, to obtain colloidal gold solution; (2) in step (1) obtained colloidal gold solution was slowly added under magnetic stirring an aqueous solution of a concentration of an antibody for recognizing tumor specific antigens 10~50iig / mL and mixed, 37 ° C incubation 30min, add 37 to continue blocking solution. (:孵育22〜 25min,14000rpm,4°C条件下离心20〜Mmin,去除上清液,稀释液重悬沉淀并洗涤一次,得到胶体金-抗S100P蛋白多克隆抗体标记物I; 另取步骤⑴所得的胶体金溶液,加入浓度为10〜5〇Ug/mL的用于识别肝癌特异性抗原的抗体水溶液,按照上述制备胶体金-抗S100P蛋白多克隆抗体标记物I同样的操作方法,得到胶体金-人肝癌组织AFU多克隆抗体标记物n; 另取步骤(1)所得的胶体金溶液,加入浓度为10〜50ug/mL的用于识别结肠癌特异性抗原的抗体水溶液,按照上述制备胶体金-抗S1 OOP蛋白多克隆抗体标记物I同样的操作方法, 得到胶体金-结肠癌抗原3抗体(SDCCAG3)标记物HI; (3) 将所述聚合物溶于一氯甲烧,加入上述步骤(2)制得的标记物I、标记物n、标记物m,用超声波处理3〇〜6〇s,得到胶体金标记的识别胃癌、肝癌、结肠癌三种肿瘤的特异性抗原的抗体共聚物;所 (: Incubation 22~ 25min, centrifuged 20~Mmin at 14000rpm, 4 ° C conditions, remove the supernatant, and resuspend pellet dilution was washed once, to give colloidal gold - S100P polyclonal antibody anti-marker I; Another step the resulting ⑴ colloidal gold solution was added at a concentration 10~5〇Ug / antibody solution for identifying liver cancer-specific antigen mL of colloidal gold prepared as described above - to obtain an anti-tag polyclonal antibody S100P similar method I, colloidal gold - AFU human hepatocellular carcinoma tissue was polyclonal antibody labeled n; another step colloidal gold solution (1) obtained, was added at a concentration of 10~50ug / antibody solution for identifying mL colon specific antigen prepared as described above colloidal gold anti - S1 OOP I polyclonal antibody markers the same operation, to give a colloidal gold - 3 colon cancer antigen antibody (SDCCAG3) the HI markers; (3) the polymer is dissolved in a burn chloroformate was added the above step (2) was obtained marker I, marker n, label m, 3〇~6〇s ultrasonic treatment, to obtain colloidal gold labeled recognition gastric, liver, colon three tumor specific antigens antibodies copolymers; the 的胶体金-抗S100P蛋白多克隆抗体标记物I,按质量比计算,即胶体金:抗S100P蛋白多克隆抗体为5:0 • 21〜0 • 23;所述的胶体金-人肝癌组织AFU多克隆抗体标记物n,按质量比计算,即胶体金:人肝癌组织AFU多克隆抗体为5:0 • 25〜0 • 28;所述的胶体金-结肠癌抗原3抗体(SDCCAG3)标记物m,按质量比计算,即胶体金:结肠癌抗原3抗体(SDCCAG3)为5:0•23〜0.26;所述超声波处理的频率为35〜55KHz;所述封闭液为含牛血清蛋白的洗潘液,其质量浓度为〇. 3〜lmg/mL。 Colloidal gold - S100P polyclonal antibody anti-tag I, calculated mass ratio, i.e. colloidal gold: polyclonal antibody anti-S100P is 5: 0 • 21~0 • 23; the colloidal gold - the Tumor AFU polyclonal antibody marker n, calculated mass ratio, i.e., the colloidal gold: the Tumor AFU polyclonal antibodies 5: 0 • 25~0 • 28; the colloidal gold - 3 colon cancer antigen antibody (SDCCAG3) markers m, the mass ratio is calculated, i.e. the colloidal gold: 3 colon cancer antigen antibody (SDCCAG3) is 5: 0 • 23~0.26; the frequency of the ultrasonic treatment is 35~55KHz; the blocking solution containing bovine serum albumin in wash Pan solution, the concentration of which is square. 3~lmg / mL.
2.—种可以检测胃癌、肝癌、结肠癌的组合物,是由用于识别胃癌特异性抗原的抗体、 用于识别肝癌特异性抗原的抗体、用于识别结肠癌特异性抗原的抗体、聚合物、胶体金溶液组成;其中,所述用于识别胃癌特异性抗原的抗体为抗S100P蛋白多克隆抗体;所述用于识别肝癌特异性抗原的抗体为人肝癌组织AFU多克隆抗体;所述用于识别结肠癌特异性抗原的抗体为结肠癌抗原3抗体(SDCCAG3);所述聚合物为可生物降解的聚合物,选自聚羟基醋酸、聚羟基丁酸酯、乳酸-聚乙二醇共聚物、聚乳酸-聚乙二醇嵌段共聚物或聚羟基烷基醇酯中的任意一种; 该胶体金标记的识别肿瘤特异性抗原的抗体共聚物制备方法为: (1) 制备胶体金溶液:用质量百分比浓度为1 %的柠檬酸三钠水溶液与质量百分比浓度为0.01 %氯金酸水溶液,按1.5mL: lOOmL的体积比,将氯金酸溶液加热至沸腾,把 2.- species can detect stomach cancer, liver cancer, colon cancer composition is an antibody for recognizing tumor specific antigens for antibody recognizes a specific antigen hepatoma, colon cancer antibody recognizes a specific antigen for the polymerization was composed of a colloidal gold solution; wherein said antibody recognizes a tumor specific antigen, polyclonal antibody anti S100P; for the antibody recognizes a human hepatoma liver cancer-specific antigen polyclonal antibody AFU; with the antibody recognizes a specific antigen on the colon is colon cancer antigen 3 antibody (SDCCAG3); the polymer is a biodegradable polymer selected from polyglycolic acid, polyhydroxybutyrate, lactic acid - polyethylene glycol copolymer was polylactic acid - polyethylene glycol block copolymer or any of a polyhydroxy alkyl alcohol esters; antibody preparation of the copolymer of colloidal gold labeled tumor-specific antigens are identified: (1) preparation of colloidal gold solution: mass percent concentration with a 1% aqueous solution of trisodium citrate and the mass concentration of 0.01% chloroauric acid aqueous solution, by 1.5mL: lOOmL volume ratio of the chloroauric acid solution was heated to boiling, the 柠檬酸三钠水溶液快速加入其中,搅拌反应至所得的反应液为酒红色,再加热lOrain,直至溶液透亮, 冷至室温,然后用浓度为0 • 05m〇l/L的碳酸钾水溶液调节pH值为6.5,即得胶体金溶液; (2) 在步骤(1)所得的胶体金溶液中,在磁力搅拌下缓慢加入浓度为l〇Ug/mL的用于识别胃癌特异性抗原的抗体水溶液,混匀,37°C孵育30min,加封闭液继续37°C孵育22min,所述封闭液为含牛血清蛋白的洗涤液,其质量浓度为0.3mg/mL,加入封闭液可避免非特异性结合,再14〇〇〇rpm,4°C条件下离心20min,去除上清液,稀释液重悬沉淀并洗涤一次,得到胶体金-抗S100P蛋白多克隆抗体标记物I,按质量比计算,胶体金:抗S100P蛋白多克隆抗体为5:0.21; 另取步骤(1)所得的胶体金溶液,在磁力搅拌下缓慢加入浓度为10〜50ug/mL的用于识别肝癌特异性抗原的抗体水溶液,按照上述制备胶 Trisodium citrate solution was added quickly, the reaction was stirred until the resultant reaction liquid is wine red, Lorain reheated, until a translucent solution was cooled to room temperature and then the concentration is 0 • The 05m〇l / L aqueous potassium carbonate solution to adjust the pH 6.5, i.e., to obtain a colloidal gold solution; (2) in step (1) in the resulting colloidal gold solution, under magnetic stirring was slowly added an aqueous solution of a concentration of an antibody for recognizing tumor specific antigens l〇Ug / mL in an admixture uniform, 37 ° C for 30min, blocking solution was added to continue 37 ° C for 22min, the solution is washed with blocking solution containing bovine serum albumin, the concentration of 0.3mg / mL, solution can be added to avoid blocking non-specific binding, and then 14〇〇〇rpm, centrifuged 4 ° C for 20min, supernatant was removed and diluted pellet was resuspended and washed once to give a colloidal gold - S100P polyclonal antibody anti-tag I, calculated mass ratio, the colloidal gold: S100P polyclonal antibody anti-5: 0.21; another step (1) colloidal gold solution obtained, under magnetic stirring was slowly added at a concentration 10~50ug / antibody solution for identifying mL of liver-specific antigen, as described above preparation of glue 金-抗S100P蛋白多克隆抗体标记物I 同样的操作方法,得到胶体金-人肝癌组织AFU多克隆抗体标记物n,按质量比计算,胶体金:人肝癌组织AFU多克隆抗体为5:0 • 25;另取步骤(1)所得的胶体金溶液,在磁力搅拌下缓慢加入浓度为lOwg/mL的用于识别结肠癌特异性抗原的抗体水溶液,按照上述制备胶体金-抗S100P蛋白多克隆抗体标记物I同样的操作方法,得到胶体金-结肠癌抗原3抗体(SDCCAG3)标记物m,按质量比计算,胶体金:结肠癌抗原3抗体(SDCCAG3)为5:0 • 23; (3)将所述聚合物按1: 1S的重量比溶于二氯甲烷,加入上述步骤(2)制得的标记物I、标记物n、标记物m,用超声波处理30s,所述超声波处理的频率为35KHZ,经超声波处理可以使形成的聚合物均匀,得到胶体金标记的识别胃癌、肝癌、结肠癌三种肿瘤的特异性抗原的抗体共聚物。 Gold - Anti S100P I polyclonal antibody markers the same operation, to give colloidal gold - the Tumor markers AFU polyclonal antibody n, calculating the mass ratio, the colloidal gold: Polyclonal human hepatoma tissue is AFU 5: 0 • 25; another step (1) colloidal gold solution obtained, under magnetic stirring was slowly added an aqueous solution of a concentration of an antibody for identifying a colon cancer-specific antigen lOwg / mL of colloidal gold prepared as described above - polyclonal anti S100P antibody labels the same operation I, to give a colloidal gold - 3 colon cancer antigen antibody (SDCCAG3) markers m, the mass ratio calculated colloidal gold: 3 colon cancer antigen antibody (SDCCAG3) is 5: 0 • 23; (3 ) the polymer according to 1: 1 weight ratio of 1S dissolved in dichloromethane and added to the above step (2) was obtained marker I, marker n, label m, 30s treated with ultrasonic waves, the ultrasonic treatment frequency of 35KHZ, sonicated of the polymer can be uniformly formed, are identified colloidal gold labeled gastric, liver, colon specific antigen antibody copolymer of three tumor.
3.—种可以检测胃癌、肝癌、结肠癌的组合物,其特征在于由用于识别胃癌特异性抗原的抗体、用于识别肝癌特异性抗原的抗体、用于识别结肠癌特异性抗原的抗体、聚合物、胶体金溶液组成;其中,所述用于识别冑癌特异性抗原的抗体为抗S100P蛋白多克隆抗体;所述用于识别肝癌特异性抗原的抗体为人肝癌组织AFU多克隆抗体;所述用于识别结肠癌特异性抗原的抗体为结肠癌抗原3抗体(SDCCAG3);所述聚合物为可生物降解的聚合物,选自聚羟基醋酸、聚羟基丁酸酯、乳酸-聚乙二醇共聚物、聚乳酸-聚乙二醇嵌段共聚物或聚羟基烷基醇酯中的任意一种; 该胶体金标记的识别肿瘤特异性抗原的抗体共聚物制备方法为: (1) 制备胶体金溶液:用质量百分比浓度为1%的柠檬酸三钠水溶液与质量百分比浓度为0 • 01 %氯金酸水溶液,按1 • 5mL: 100mL的体积比,将氯金酸溶液加 3.- species can detect stomach cancer, liver cancer, colon cancer composition comprising an antibody for recognizing tumor specific antigens for antibody recognizes a specific antigen hepatoma, colon cancer antibody recognizes a specific antigen , a polymer, colloidal gold solution; wherein said antibody recognizes a cancer-specific antigen helmet anti S100P polyclonal antibody; hepatoma antibody recognizes a specific antigen for the human HCC AFU polyclonal antibody; the antibodies used to identify colon cancer colon-specific antigen antibody antigen 3 (SDCCAG3); the polymer is a biodegradable polymer selected from polyglycolic acid, polyhydroxybutyrate, lactic acid - polyethylene glycol copolymers, polylactic acid - polyethylene glycol block copolymer or any of a polyhydroxy alkyl alcohol esters; antibody preparation of the copolymer of colloidal gold labeled tumor-specific antigens are identified: (1) preparation of colloidal gold solution: with a mass concentration of 1% trisodium citrate aqueous solution of the mass concentration of 0 • 01% chloroauric acid aqueous solution, by 1 • 5mL: 100mL volume ratio of the chloroauric acid solution was added 热至沸腾,把柠檬酸三钠水溶液快速加入其中,搅拌反应至所得的反应液为酒红色,再加热lOmin,直至溶液透亮, 冷至室温,然后用浓度为0 • 〇75mol/L的碳酸钾水溶液调节pH值为7 • 0,即得胶体金溶液; (2) 在步骤(1)所得的胶体金溶液中,在磁力搅拌下缓慢加入浓度为30ug/mL的用于识别胃癌特异性抗原的抗体水溶液,混匀,37°C孵育30min,加封闭液继续37°C孵育23.5min, 所述封闭液为含牛血清蛋白的洗涤液,其质量浓度为0.65mg/mL,加入封闭液可避免非特异性结合,再14000rpm,4t:条件下离心21min,去除上清液,稀释液重悬沉淀并洗涤一次,得到胶体金-抗S100P蛋白多克隆抗体标记物I,按质量比计算,胶体金:抗S100P蛋白多克隆抗体为5:0.22; 另取步骤(1)所得的胶体金溶液,在磁力搅拌下缓慢加入浓度为10〜50ug/mL的用于识别肝癌特异性抗原的抗体水溶液,按照 Heat to boiling, the aqueous solution of trisodium citrate was added quickly, the reaction was stirred until the resultant reaction liquid is wine red, reheating lOmin, until a translucent solution was cooled to room temperature and then the concentration is 0 • The 〇75mol / L potassium carbonate solution adjusting pH to 7 • 0, i.e., to obtain a colloidal gold solution; (2) in step (1) in the resulting colloidal gold solution, under magnetic stirring was slowly added at a concentration of 30ug / mL for identifying tumor specific antigen antibody solution, mixed, 37 ° C for 30min, blocking solution was added to continue 37 ° C for 23.5 min, the solution is washed with blocking solution containing bovine serum albumin, the concentration of 0.65mg / mL, solution can be added to avoid blocking non-specific binding, then 14000rpm, 4t: 21min under centrifugation, the supernatant was removed and diluted pellet was resuspended and washed once to give a colloidal gold - S100P polyclonal antibody anti-tag I, calculated mass ratio, the colloidal gold: S100P polyclonal antibody anti-5: 0.22; another step (1) colloidal gold solution obtained, under magnetic stirring was slowly added at a concentration 10~50ug / antibody solution for identifying liver cancer-specific antigen mL, in accordance with 上述制备胶体金-抗S100P蛋白多克隆抗体标记物I 同样的操作方法,得到胶体金-人肝癌组织AFU多克隆抗体标记物n,按质量比计算,胶体金:人肝癌组织AFU多克隆抗体为5:0 •况5;另取步骤⑴所得的胶体金溶液,在磁力搅拌下缓慢加入浓度为3〇yg/mL的用于识别结肠癌特异性抗原的抗体水溶液,按照上述制备胶体金-抗S100P蛋白多克隆抗体标记物I同样的操作方法,得到胶体金-结肠癌抗原3抗体(SDCCAG3)标记物m,按质量比计算,胶体金:结肠癌抗原3抗体(SDCCAG3)为5:0.245; ⑶将所述聚合物按1:24的重量比溶于二氯甲烷,加入上述步骤⑵制得的标记物I、标记物n、标记物m,用超声波处理45s,所述超声波处理的频率为35〜55KHz,经超声波处理可以使形成的聚合物均匀,得到胶体金标记的识别胃癌、肝癌、结肠癌三种肿瘤的特异性抗原的抗体共聚物。 Colloidal gold prepared as described above - an anti-tag polyclonal antibody S100P I the same operation, to give colloidal gold - the Tumor markers AFU polyclonal antibody n, calculating the mass ratio, the colloidal gold: the Tumor AFU polyclonal antibody 5: 0 • condition 5; another step ⑴ resulting colloidal gold solution, under magnetic stirring was slowly added at a concentration 3〇yg / antibody solution for identifying mL of colon-specific antigen, prepared as described above colloidal gold anti - S100P I polyclonal antibody markers similar method, to obtain colloidal gold - 3 colon cancer antigen antibody (SDCCAG3) markers m, the mass ratio calculated colloidal gold: 3 colon cancer antigen antibody (SDCCAG3) 5: 0.245; ⑶ the polymer weight ratio of 1:24 was dissolved in dichloromethane, added to the above prepared steps ⑵ marker I, marker n, label m, 45s treated with ultrasonic waves, the frequency of the ultrasonic treatment is 35~55KHz, sonicated polymer formed can be made uniform, to obtain colloidal gold labeled recognition gastric, liver, colon specific antigen antibody copolymer of three tumor.
4.一种可以检测胃癌、肝癌、结肠癌的组合物,其特征在于:由用于识别胃癌特异性抗原的抗体、用于识别肝癌特异性抗原的抗体、用于识别结肠癌特异性抗原的抗体、聚合物、 胶体金溶液组成;其中,所述用于识别胃癌特异性抗原的抗体为抗S100P蛋白多克隆抗体; 所述用于识别肝癌特异性抗原的抗体为人肝癌组织AFU多克隆抗体;所述用于识别结肠癌特异性抗原的抗体为结肠癌抗原3抗体(SDCCAG3);所述聚合物为可生物降解的聚合物,选自聚羟基醋酸、聚羟基丁酸酯、乳酸-聚乙二醇共聚物、聚乳酸-聚乙二醇嵌段共聚物或聚羟基烷基醇酯中的任意一种; 该胶体金标记的识别肿瘤特异性抗原的抗体共聚物制备方法为: (1) 制备胶体金溶液:用质量百分比浓度为1%的柠檬酸三钠水溶液与质量百分比浓度为0.01 %氯金酸水溶液,按1 • 5mL: lOOmL的体积比,将氯金酸溶液 A can detect stomach cancer, liver cancer, colon cancer composition, comprising: an antibody to identify tumor specific antigens for antibody recognizes a specific antigen hepatoma, colon cancer-specific antigens used to identify an antibody, a polymer, colloidal gold solution composition; wherein the antibody recognizes a tumor specific antigen, polyclonal antibody anti S100P; for the antibody recognizes a human hepatoma liver cancer-specific antigen AFU polyclonal antibody; the antibodies used to identify colon cancer colon-specific antigen antibody antigen 3 (SDCCAG3); the polymer is a biodegradable polymer selected from polyglycolic acid, polyhydroxybutyrate, lactic acid - polyethylene glycol copolymers, polylactic acid - polyethylene glycol block copolymer or any of a polyhydroxy alkyl alcohol esters; antibody preparation of the copolymer of colloidal gold labeled tumor-specific antigens are identified: (1) preparation of colloidal gold solution: with a mass concentration of 1% aqueous solution of trisodium citrate concentration of 0.01% by mass percentage aqueous chloroauric acid solution, press 1 • 5mL: lOOmL volume ratio, chloroauric acid solution 加热至沸腾,把柠檬酸三钠水溶液快速加入其中,搅拌反应至所得的反应液为酒红色,再加热lOmin,直至溶液透亮, 冷至室温,然后用浓度为0. ltnol/L的碳酸钾水溶液调节pH值为8.0,即得胶体金溶液; (2) 在步骤(1)所得的胶体金溶液中,在磁力搅拌下缓慢加入浓度为10〜50ug/mL的用于识别胃癌特异性抗原的抗体水溶液,混匀,37°C孵育30min,加封闭液继续37°C孵育25min,所述封闭液为含牛血清蛋白的洗涤液,其质量浓度为lmg/mL,加入封闭液可避免非特异性结合,再14000rPm,4°C条件下离心22min,去除上清液,稀释液重悬沉淀并洗涤一次, 得到胶体金-抗S100P蛋白多克隆抗体标记物I,按质量比计算,胶体金:抗S100P蛋白多克隆抗体为5:0.23; 另取步骤(1)所得的胶体金溶液,在磁力搅拌下缓慢加入浓度为50ug/mL的用于识别肝癌特异性抗原的抗体水溶液,按照上述制备 Heated to boiling, the aqueous solution of trisodium citrate was added quickly, with stirring potassium carbonate aqueous solution was wine red, and then heated lOmin, until a translucent solution was cooled to room temperature, then treated with a concentration of 0. ltnol / L of the reaction to the resulting reaction solution adjusting pH to 8.0, to obtain a colloidal gold solution; the resulting colloidal gold solution, under magnetic stirring (2) in step (1) was slowly added at a concentration 10~50ug / mL antibody for recognizing tumor specific antigens aqueous mix, 37 ° C for 30min, blocking solution was added to continue 37 ° C for 25min, the solution is washed with blocking solution containing bovine serum albumin, the concentration of lmg / mL, solution can be added to prevent blocking of nonspecific binding , then 14000 rpm, 4 ° C for centrifugation conditions 22min, supernatant was removed and diluted pellet was resuspended and washed once to give a colloidal gold - S100P polyclonal antibody anti-tag I, calculated mass ratio, the colloidal gold: anti-S100P polyclonal antibody was 5: 0.23; another step (1) colloidal gold solution obtained, was slowly added under magnetic stirring at a concentration of 50ug / mL for aqueous hepatoma antibody recognizes a specific antigen, prepared as described above 体金-抗S100P蛋白多克隆抗体标记物I同样的操作方法,得到胶体金-人肝癌组织AFU多克隆抗体标记物n,按质量比计算,胶体金:人肝癌组织AFU多克隆抗体为5:0.28;另取步骤(1)所得的胶体金溶液,在磁力搅拌下缓慢加入浓度为5〇Ug/mL的用于识别结肠癌特异性抗原的抗体水溶液,按照上述制备胶体金-抗S100P蛋白多克隆抗体标记物I同样的操作方法,得到胶体金-结肠癌抗原3抗体(SDCCAG3) 标记物IH,按质量比计算,胶体金:结肠癌抗原3抗体(SDCCAG3)为5:0 • 26; (3) 将所述聚合物按1:30的重量比溶于二氯甲烷,加入上述步骤⑵制得的标记物I、标记物n、标记物m,用超声波处理60s,所述超声波处理的频率为55KHZ,经超声波处理可以使形成的聚合物均匀,得到胶体金标记的识别胃癌、肝癌、结肠癌三种肿瘤的特异性抗原的抗体共聚物。 Gold body - Anti S100P I polyclonal antibody markers similar method, to obtain colloidal gold - the Tumor markers AFU polyclonal antibody n, calculating the mass ratio, the colloidal gold: Polyclonal human hepatoma tissue AFU 5: 0.28; another step (1) colloidal gold solution obtained, under magnetic stirring was slowly added at a concentration 5〇Ug / antibody solution for identifying colon specific antigen mL of colloidal gold prepared as described above - antibody protein S100P monoclonal antibody markers the same operation I, to give a colloidal gold - 3 colon cancer antigen antibody (SDCCAG3) markers of the IH, calculated mass ratio, the colloidal gold: 3 colon cancer antigen antibody (SDCCAG3) is 5: 0 • 26; ( frequency 3) to the polymer weight ratio of 1:30 was dissolved in dichloromethane, added to the above prepared steps ⑵ marker I, marker n, label m, sonicated for 60s, the ultrasonic treatment is 55KHz, the polymer can be sonicated uniformly formed, are identified colloidal gold labeled gastric, liver, colon specific antigen antibody copolymer of three tumor.
CN201510789097.3A 2015-11-16 2015-11-16 One kind of tumor can be detected more compositions and Its Application CN105353127B (en)

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