YKL-40与前列腺癌临床分期、病理分级的相关性研究

目的 了解YKL-40与前列腺癌的发生、发展及转移的关系,旨在探讨YKL-40作为肿瘤标记物用于前列腺癌早期诊断、病情监测的价值.方法收集新诊断且未经治疗的前列腺癌患者血液标本40例,前列腺增生患者血液标本21例,健康对照组患者血液标本19例,采用酶联免疫吸附（ELISA）双抗体夹心法分别检测其血清YKL-40浓度.结果前列腺癌患者组、前列腺增生患者组和健康对照组的血清YKL-40浓度分别为（167.74±59.61） ng/ml,（118.84±25.81） ng/ml,（72.29±15.00） ng/ml,血清YKL-40浓度与临床分期有相关性（P＜0.01）,且在前列腺癌患者转移阶段呈高度表达,与Gleason评分无相关性.YKL-40截断点为120.89 ng/ml时,灵敏度为86.7％,特异度为71.4％.结论YKL-40在前列腺癌患者血清中的浓度显著高于前列腺增生患者和正常患者,提示检测YKL-40对前列腺癌诊断有一定的临床意义;血清YKL-40浓度对预测前列腺癌患者转移可能有重要的价值,可能参与了前列腺癌患者转移的过程.     

氯霉素单克隆抗体ELISA检测方法的建立

目的 建立CAP间接和直接ELISA检测方法.方法 用重氮化法分别偶联氯霉素(Chloramphenicol,CAP)与BSA和OVA作为免疫原和检测原,腹腔注射8周健康雌BALB/c小鼠大量制备抗CAP的单克隆抗体并优化条件,建立了CAP的快速、灵敏、简便的直接和间接竞争ELISA方法.结果 两种检测方法的回归方程和相关系数分别为:y=-25.411x 97.041,R2=0.9889和y=-27.768x 103.91,R2=0.9889,线性范围分别为1.87-75ng/mL和0.93-25ng/mL,对CAP的最低检出浓度为0.97ng/mL和0.35ng/mL.并利用所建立的两种ELISA方法对鸡肉模拟样品进行了检测,其检测样品的回收率分别为98.607%,85.152%.结论 为下步CAP的试纸条或试剂盒建立提供基础.     

血清肿瘤标志物检测在肝癌诊断中的应用价值研究

目的探讨血清肿瘤标志物检测在肝癌诊断中的临床意义。方法选取2013年1月至2016年7月该院收治的40例肝癌患者作为观察组,另选取同期健康体检者40例作为对照组,比较两组血清甲胎蛋白(AFP)、碱性磷酸酶(ALP)、γ-谷氨酰转移酶(GGT)、癌胚抗原(CEA)和糖类抗原125(CA125)水平,并分析各单项标志物检测对肝癌诊断的灵敏性、特异性和准确性。结果观察组血清AFP、ALP、GGT、CEA、CA125水平分别为(2 212.61±456.49)ng/mL、(253.58±62.19)U/L、(252.58±70.93)U/L、(86.45±38.07)ng/mL、(159.08±61.79)U/mL,均明显高于对照组(P0.05);各项血清肿瘤标志物检测的灵敏度、特异度和准确度差异无统计学意义(P0.05)。结论血清肿瘤标志物联合检测可为肝癌诊断提供参考依据。     

BHMT用于检测急性中毒性肝损伤的价值

目的分析甜菜碱高半胱氨酸甲基转移酶(BHMT)用于检测急性中毒性肝损伤的临床价值。方法采用ELISA法检测血清样本中的BHMT,检测样本为健康体检者血清和急性中毒患者血清。结果 ELISA法检测健康体检者血清样本300例,300例健康体检血清中BHMT的平均水平为9.02ng/mL(6.19~12.84ng/mL)。健康人群血清中BHMT水平的95%医学参考值范围为0.00~28.01ng/mL;ELISA法检测43例急性中毒患者的血清样本,经Spearman秩相关分析,提示BHMT和丙氨酸氨基转移酶(ALT)之间有较好的正相关关系(rs=0.757 62,P0.001);ELISA法检测20例肾损伤病例和20例健康体检者血清中的BHMT水平,提示肾损伤患者与健康体检者血清中BHMT水平的差异无统计学意义(P0.05)。结论健康人群血清中BHMT水平的医学参考值范围为0.00~28.01ng/mL;急性中毒患者血清中的BHMT与其对应的ALT之间有很好的正相关关系;BHMT水平的变化可能与肾损伤无关,其反映肝损伤的特异性较好,因此推测BHMT可能是更好的急性肝损伤的血清学标志物。     

夹心酶联免疫吸附试验检测人表皮生长因子的方法建立及其应用研究

目的:建立临床适用的表皮生长因子(epidermalgrowthfactor,EGF)酶联免疫检测方法,探讨EGF在临床常见肿瘤患者血清中的含量变化规律。方法:采用EGF单抗包被酶标板,兔抗EGF多抗为二抗,以辣根过氧化物酶标记的羊抗兔IgG作为酶标抗体建立夹心法ELISA检测临床常见肿瘤患者及健康体检人群的血清EGF含量。结果:建立的EGF酶联免疫吸附法最小检测限达15.6ng/L,批内精密度为8.7%,批间精密度为11.6%,平均回收率为94.7%,符合临床检测要求。血清EGF含量在31名正常人为(1.305±0.382)ng/mL,24名乳腺癌病人术前值为(0.729±0.347)ng/mL,术后为(0.711±0.332)ng/mL,17名结直肠癌病人术前为(0.678±0.311)ng/mL,术后为(0.658±0.298)ng/mL,19名胃癌病人术前为(0.598±0.224)ng/mL,术后为(0.614±0.257)ng/mL。各肿瘤组患者血清EGF水平较健康对照组明显降低(P<0.01)。结论:建立了适用于临床检测的EGF酶联免疫检测方法,肿瘤患者血清中EGF含量较健康对照组明显下降,该检...     

人血清哇巴因UPLC-MS/MS检测方法的建立和方法比对

目的建立超高效液相色谱串联质谱(UPLC-MS/MS)检测人血清哇巴因的方法。方法采用高特异性的UPLC-MS/MS,以氘标记的哇巴因-d3作为内标。样本采用固相萃取(SPE)前处理方法,以反相色谱柱负离子模式及电喷雾电离源(ESI)检测血清哇巴因水平。对建立的方法进行方法学(基质效应、回收率、准确度、批内精密度、批间精密度及稳定性)验证。采用建立的UPLC-MS/MS方法检测20名体检健康者及40例高血压患者血清哇巴因水平,并与酶联免疫吸附试验(ELISA)进行比较。结果 UPLC-MS/MS检测血清哇巴因的标准曲线范围为0.02～5.0 ng/mL,最低定量检测限(LLOQ)为0.02ng/mL。采用ABN固相萃取小柱进行样本前处理的基质效应较小,且回收率较高,达85%。LLOQ和低值(0.06 ng/mL)、中值(0.6 ng/mL)、高值(4 ng/mL)质控品的准确度分别为108.0%、89.2%、101.0%、103.0%。3个水平质控品的批内变异系数(CV)分别为2.87%、1.95%、0.56%,批间CV分别为5.98%、1.90%、0.75%。样本室温过夜放置16 h及样本前处理后室温放置自动进样器48 h的偏差均15%。采用UPLC-MS/MS检测哇巴因,正常对照者及高血压患者血清中均未检测到哇巴因。采用ELISA测定血清哇巴因,高血压患者为0.096 ng/mL,正常对照者为0.062 ng/mL。UPLC-MS/MS检测5个水平(0.02、0.05、0.10、0.20、0.50 ng/mL)的哇巴因标准品,其测定结果与对应的哇巴因标准品浓度呈正相关,且线性较好(r20.99),准确度较高;而ELISA检测5个水平哇巴因标准品的结果均很接近(0.024 9～0.029 6 ng/mL)。结论建立了检测人血清哇巴因的UPLC-MS/MS方法,未检测到正常人及高血压患者的血清哇巴因。UPLC-MS/MS与ELISA检测血清哇巴因的结果存在较大差异。     

血清肿瘤标志物PSA检测助力前列腺癌有效管理

正日前,在北京举办的肿瘤标志物大师班交流会上,西班牙巴塞罗那临床医院生化实验室癌症研究中心主任Rafael Molina教授就PSA的研究发展、医学价值及其对前列腺癌管理的应用进行了深入探讨。WHO 1999年发布的PSA校准方法(WHO96/670)将截断(cut-off)值定为3.1 ng/mL,一般超过10 ng/mL考虑罹患前列腺癌的可能。但血清PSA浓度在前列腺增生和前列腺炎中均有升高,前列腺良性疾病与前列腺癌在2~10 ng/ml时有交叉,存在很高比例的假阴性和假阳性结果。若假阴性出现在2~4 ng/mL,可导致15%癌症漏检;假阳性在4~10 ng/     

血清ENAH在HBV相关肝癌患者中的表达及其临床意义

摘要:目的探讨血清肌动蛋白调节剂(enablehomolog,ENAH)在乙型肝炎病毒相关性肝癌(HBV-HCC)中的临床意义。方法采用ELISA法检测67例HBV-HCC患者、20例非HBV相关肝癌患者( nonHBV-HCC)、60例慢性乙型肝炎(CHB)患者以及20例健康人对照(HC)血清ENAH水平,采用Spearman分析血清ENAH与患者肿瘤特征及实验室指标的相关性,绘制ENAH、甲胎蛋白(AFP)单独及二者联合检测( Logistic回归模型)的ROC曲线,并评估其对HBV-HCC的临床诊断价值。结果HBV-H0C、nonHBV-HCC、CHB患者和HC组血清ENAH的表达水平分别为8.5(7.0,8.9)ng/ mL.5.5(4.0, 6.0) ng/ mL .7.3(5.0,7.9)ng/mL和2.9(1.8 ,4.9)ng/mL,各组间差异有统计学意义(x2=71.36,P<0.001);血清ENAH与BCLC分期及肿瘤最大直径呈正相关(r分别为0.261和0.322 ,P值分别为0.035和0.008);血清ENAH、AFP单独及二者联合检测诊断HBV-HCC 的ROC曲线下面积( AUCROC )分别为0.808( 95%CI:0.735 ~ 0.868)、0.782( 95%CI:0.707 ~0.846)和0.838( 95%CI :0.768 ~0.894) ;血清ENAH诊断HBV-HCC敏感性和特异性分别为62. 69% ( 95% CI: 50.0% ~74.2%)和90.0% (95% Cl:81.2%~95.6%)。结论血清ENAH可作为一种潜在的HBV-HCC诊断和评估的血清学标志物。     

胃癌患者外周血高迁移率族蛋白B-1水平的检测及临床意义

目的探讨高迁移率族蛋白B-1(HMGB1)在胃癌患者血清中的变化及临床意义。方法采用双抗体夹心酶联免疫吸附试验(ELISA)检测101例胃癌患者血清中HMGB1水平,同时用电化学发光法(ECL)测定血清癌胚抗原(CEA)含量,并与31例胃良性病变患者、40名正常对照者比较,数据用中位数(范围)表示。结果胃癌组患者血清HMGB1和CEA水平分别为15.58(3.05～125.63)ng/mL、11.75(0.85～850.23)ng/mL,胃良性病变组患者血清HMGB1和CEA水平分别为4.46(2.61～12.06)ng/mL、2.75(0.52～11.23)ng/mL,正常对照组血清HMGB1和CEA水平分别为3.24(2.50～5.38)ng/mL、1.23(0.45～4.56)ng/mL,胃癌组患者血清HMGB1和CEA水平显著高于胃良性病变组和正常对照组(P<0.01)。血清HMGB1水平与胃癌患者肿瘤大小、浸润深度、TNM分期、淋巴结转移密切相关(P均<0.05)。与传统的肿瘤标志物CEA相比,血清HMGB1在早期胃癌患者中显示良好的诊断敏感性(72.2%)和特异性(74.6%)。结论 HMGB1与胃癌的发生、发展有关,检测血清HMGB1含量对于提高胃癌的早期诊断水平、监测病情变化、判断预后具有重要的临床价值。     

人再生基因蛋白Ⅳ在晚期胃癌患者血清中的表达及意义

[目的]探讨人再生基因蛋白Ⅳ(REG Ⅳ)在胃癌的诊断和预后判断中的价值.[方法]收集61例晚期胃癌患者及35例同期健康成年人的血清学标本,采用酶联免疫吸附试验(ELISA)定量检测胃癌患者化疗前后及健康者REG Ⅳ的水平.同时查阅患者病历资料结合胃癌传统肿瘤标志物癌胚抗原(CEA)、CA199、CA724的水平进行比较.[结果]胃癌组血清REG Ⅳ水平显著高于健康对照组[P<0.01,5.15(2.14~22.55)ng/mL vs.2.22(0.02~12.29)ng/mL].在胃癌患者中,有淋巴转移患者的REG Ⅳ水平高于无淋巴结转移者(P<0.05).REG Ⅳ检测胃癌的灵敏度为78.7%,明显高于CEA、CA199、CA724(P<0.05).血清REG Ⅳ水平阳性与阴性患者的总生存期相比较差异无显著性(P>0.05).[结论]REG Ⅳ作为血清标志物对胃癌的诊断具有较高的敏感性和特异性,有一定的诊断价值.     

Prostate targeting: PSP94 gene promoter/enhancer region directed prostate tissue-specific expression in a transgenic mouse prostate cancer model

To date, only a few prostate-specific vector genes have been tested for prostate targeting in gene therapy of prostate cancer (CaP). Current clinical trials of gene therapy of CaP utilize the only two available vector genes with a combination of a rat probasin promoter and a human PSA promoter sequence in an adenovirus vector to target CaP. There is an urgent need to establish additional vector gene systems to sustain and propagate the current research. Since PSP94 (prostate secretory protein of 94 amino acids) is one of the three most abundant proteins secreted from the human prostate and is generally considered to be prostate tissue-specific in both human and rodents, we performed a transgenic experiment to assess the promoter/enhancer region of PSP94 gene-directed prostate targeting. Firstly, a series of progressive deletion mutants of a 3.84 kb PSP94 gene promoter/enhancer region (including parts of the intron 1 sequence) linked with a reporter LacZ gene was constructed and assessed in vitro in cell culture. Next, transgenic mice were generated with two transgene constructs using the SV40 early region (Tag oncogene) as a selection marker. PSP94 gene promoter/enhancer region-directed SV40 Tag expression specifically in the mouse was demonstrated in three breeding lines (A, B, C, n = 374) by immunohistochemistry staining of Tag expression. Specific targeting to the prostate in the PSP94 gene-directed transgenic CaP model was characterized histologically by correlation of SV40 Tag-induced tumorigenesis (tumor grading) with puberty and age (10-32 weeks). Prostatic hyperplasia was observed as early as 10 weeks of age, with subsequent emergence of prostatic intraepithelial neoplasia (PIN) and eventually high grade carcinoma in the prostate. The PSP94 transgenic mouse CaP model was further characterized by its tumor progression and metastatic tendency at 20 weeks of age and also by its responsiveness and refractoriness to androgen manipulation. This study indicates that the PSP94 gene promoter/enhancer has the potential for prostate specific targeting and may ultimately be of use in gene therapy of CaP.     

Development of a microplate assay for serum chromogranin A (CgA): establishment of normal reference values and detection of elevated CgA in malignant diseases

Chromogranin A (CgA), a marker for neuroendocrine cells, is associated with poor prognosis when detected by immunohistochemical technique in prostate tumors. We have developed an ELISA on microplates for serum CgA and established the normal reference range. We also attempted to find out whether elevated serum CgA levels could be found in patients with various malignant diseases. Because of non-Gaussian distribution, both medians and 97.5 percentiles of serum CgA levels for men and women of four different age groups were determined. For women, the median and 97.5 percentiles are 20.7 and 63.9 ng/mL for ages 20 to 50, and 32 and 93.8 for 50 to 80 years of age, respectively; for men, they are 27.9 and 78.4 ng/mL for ages 18 to 40 and 41.6 and 92 for 40 to 80 years old, respectively. Elevated serum concentrations of CgA were detectable in patients with prostate cancer not undergoing hormonal treatment, and in patients with various malignant diseases including nonendocrine carcinomas. Most elevated serum CgA levels were associated with sera containing highly elevated serum tumor markers. Drugs targeting neuroendocrine cells should be administered for cancer patients with elevated serum CgA levels.     

Sandwich ELISAs for soluble immunoglobulin superfamily receptor translocation-associated 2 (IRTA2)/FcRH5 (CD307) proteins in human sera.

BACKGROUND: The immunoglobulin superfamily receptor translocation-associated 2 (IRTA2) gene encodes both membrane and secreted forms of the B-cell differentiation antigen (also identified as FcRH5). The membrane form is highly expressed on the surface of hairy cell leukemia (HCL) cells from patients. This study aimed to develop immunoassays for soluble IRTA2/FcRH5 proteins in human serum. METHODS: Two sandwich ELISAs for soluble IRTA2/FcRH5 were designed using two pairs of monoclonal antibodies specific to four different epitopes on IRTA2/FcRH5. RESULTS: In both ELISAs, the lower limit of quantitation for soluble IRTA2/FcRH5 in human serum was 30 ng/mL. The analytical recovery of 0.3-2.1 ng/mL of IRTA2/FcRH5-Fc used as the standard, from a 100-fold dilution of IRTA2/FcRH5-free sera, was 94-106% for ELISA #1 and 89-97% for ELISA #2. Between-assay coefficients of variance were 7.7-17.6% for ELISA #1 and 7.7-32.7% for ELISA #2. Both ELISAs detected soluble IRTA2/FcRH5 protein in sera from normal donors (median 169 ng/mL in ELISA #1 and 146 ng/mL in ELISA #2, n=123) without correlations to gender or age. A marked increase in soluble IRTA2/FcRH5 levels was observed in samples from patients with HCL (medians 719 ng/mL in ELISA #1 and 754 ng/mL in ELISA #2, n=44). CONCLUSIONS: These ELISAs may be useful for monitoring soluble IRTA2/FcRH5 in HCL and other B-cell malignancies.     

Human kallikrein 10 ELISA development and validation in breast cancer sera

BACKGROUND: Human kallikrein 10 (hK10) is a putative secreted serine protease belonging to the same gene family as prostate specific antigen (hK3; PSA). There is significant interest in measuring hK10 which may act as a tumor suppressor in some cancers. We have developed an ELISA for hK10 and determined its analytical and clinical performance in normal and breast cancer sera. METHODS: The assay used a previously described pair of monoclonal anti-hK10 antibodies and recombinant hK10 protein. Serum hK10 was detected quantitatively in a 2-step sandwich ELISA with colorimetric detection. The assay was analytically validated and used to determine serum levels of hK10 in a set of breast cancer, benign breast disease and normal samples. RESULTS: The assay covered a linear range of 0.2 to 15 ng/mL and had a detection limit of 0.08 ng/mL. The within-run and between-run imprecision was <9%. The average spike and dilution linearity recoveries were 96 and 103% respectively. Mean hK10 concentration in normal female sera was 0.79+/-0.26 ng/mL. Concentrations were not age related and were not significantly different from benign fibrocystic disease or breast cancer. However, in a subset of breast cancer patients with both early and late stage disease, serum hK10 levels were elevated, at >1.55 ng/mL, above all normal female and benign disease samples. CONCLUSIONS: We report in detail the analytical performance of a colorimetric hK10 ELISA validated in human serum and report for the first time the hK10 serum concentration in benign and breast cancer samples.     

Enzyme linked immunosorbent assays (ELISA) for the quantitative determination of human leukocyte collagenase and gelatinase

A competitive and a sandwich enzyme linked immunosorbent assay (ELISA) were developed for human leukocyte collagenase and gelatinase. The competitive assay could detect 0.5 ng collagenase and 0.05 ng gelatinase. The detection limit of the sandwich ELISA was 0.05 ng for collagenase and 0.02 ng for gelatinase. No cross reactivity between human leukocyte collagenase and gelatinase was detected. The sandwich ELISA was used to determine plasma levels of these enzymes. The 90% range for collagenase was between 0 and 50 micrograms/l; the 90% range for gelatinase was between 27 and 94 micrograms/l.     

Development of a sensitive and specific enzyme-linked immunosorbent assay for thymosin beta15, a urinary biomarker of human prostate cancer

OBJECTIVES: In tissue-based assays, thymosin beta15 (Tbeta15) has been shown to correlate with prostate cancer (CaP) malignancy and with future recurrence. To be clinically effective, it must be shown that Tbeta15 is released by the tumor into body fluids in detectable concentrations. Toward this end, we have worked to develop a quantitative high-throughput assay that can accurately measure clinically relevant concentrations of Tbeta15 in human urine. DESIGN AND METHODS: Sixteen antibodies were raised against recombinant Tbeta15 and/or peptide conjugates. One antibody, having stable characteristics over the wide range of pH and salt concentrations found in urine and minimal cross-reactivity with other beta thymosins, was used to develop a competitive enzyme-linked immunosorbent assay (ELISA). Urinary Tbeta15 concentration was determined for control groups; normal (N = 52), prostate intraepithelial neoplasia (PIN, N = 36), and CaP patients; untreated (N = 7) with subsequent biochemical failure, radiation therapy (N = 17) at risk of biochemical recurrence. RESULTS: The operating range of the competition ELISA fell between 2.5 and 625 ng/mL. Recoveries exceeded 75%, and the intra- and inter-assay coefficients of variability were 3.3% and 12.9%, respectively. No cross-reactivity with other urine proteins was observed. A stable Tbeta15 signal was recovered from urine specimens stored at -20 degrees C for up to 1 year. At a threshold of 40 (ng/dL)/mug protein/mg creatinine), the assay had a sensitivity of 58% and a specificity of 94%. Relative to the control groups, Tbeta15 levels were greater than this threshold in a significant fraction of the CaP patients (P < 0.001), including 5 of the 7 patients who later experienced PSA recurrence. CONCLUSIONS: We have established an ELISA that is able to detect Tbeta15 at clinically relevant concentrations in urine from patients with CaP. The assay will provide a tool for future clinical trials to validate urinary Tbeta15 as a predictive marker for recurrent CaP.     

高迁移率族蛋白B1单克隆抗体制备与竞争ELISA测定法的建立

目的制备高迁移率族蛋白B1(HMGB1)单克隆抗体和建立HMGB1的直接竞争ELISA测定方法。方法采用PEG细胞融合技术、有限稀释法和间接ELISA法制备和筛选杂交瘤细胞。直接竞争ELISA法测定大鼠血清HMGB1水平。结果共获得7株HMGB1特异性杂交瘤细胞株。建立的直接竞争ELISA方法的测定范围为1~320 ng/ml,回收率为97%~106%,变异系数为2.6%~7.3%。用该方法测定大鼠血清HMGB1水平发现,糖尿病大鼠模型HMGB1水平高于正常大鼠(P<0.001)。结论直接竞争ELISA可用于HMGB1的测定,该方法较双抗体夹心ELISA法更简便、快速。     

Serum chromogranin A: Early detection of hormonal resistance in prostate cancer patients

We monitored both chromogranin A (CgA) and neuron specific enolase (NSE) in serial serum specimens from 14 patients with prostate cancer (CAP patients) showing resistance to hormonal treatment. Elevated serum CgA was detected in 10 out of these 14 patients (71%) during treatment, and an early appearance of elevated serum CgA was found in 6 of 14 (43%) of these patients when serum tPSA levels were still in the normal range. If patients with radical prostatectomy were not included, the percentage of patients showing an early appearance of elevated serum CgA would have been much higher. Elevated serum CgA levels also were found in patients not subject to hormonal therapy. Serial specimens from two out of three prostate cancer patients, randomly selected, contained elevated serum CgA. Serum NSE was not detectable in any of the serial specimens we studied, suggesting that CgA, not NSE, should be used as a marker for neuroendocrine differentiation. We also compared the serum CgA in random serum specimens between patients with BPH (benign prostate hyperplasia) and with prostate cancer in the concentration range of serum tPSA between 3–15 ng/mL. Although serum CgA concentrations in BPH patients overlapped considerably with those levels in patients with prostate cancer, levels > 100 ng/mL should suggest prostate cancer. The early appearance of elevated serum CgA allows an early change of therapy to be made and can lead to the effective prevention of any further development of metastases. J. Clin. Lab. Anal. 12:20–25, 1998. © 1998. Wiley-Liss, Inc.     

Development of an enzyme-linked immunosorbent assay for metallothionein-I and -II in plasma of humans and experimental animals

BackgroundAn easy and specific enzyme-linked immunoassay (ELISA) for the determination of metallothinein-1 (MT-1) and 2 (MT-2) simultaneously in serum and other biological specimens in humans and experimental animals has not been developed yet.MethodsWe developed a competitive ELISA, a specific polyclonal antibody against rat MT-2. The epitope mapping of the antibody was conducted using MTs in mouse, rat, rabbit, human and the fragment peptides of human MT-2. MT1/2 and MT-3 knock-out mice and cadmium treated mice were used for the evaluation of the ELISA. Pretreatment method of serum was examined to deplete blocking factors for this assay.ResultsThe antibody used for this ELISA had the same cross-reactivity with MT in humans and experimental animals. NH₂ terminal peptide of MT with acetylated methionine was proved to be the epitope of this antibody. The reactivity of this ELISA system with liver, kidney and brain in MT1/2 knock-out mice was significantly low, but was normal in MT-3 knock-out mouse. The lowest detection limit of this ELISA was 0.6 ng/ml and the added MT-1 was fully recovered from serum. The mean MT concentration in our preliminary study was 23 ± 4.6 ng/ml in human serum. Cadmium treatment to mice induced significantly higher amount of MT in serum, liver, kidney and spleen as reported previously by different established methods.ConclusionThe proposed competitive ELISA is an easy and specific method for practical use, determining total MT-1 and -2 simultaneously in serum and other biological specimens of human and experimental animals.     

Characterization of the oncogenic activity of the novel TRIM59 gene in mouse cancer models

A novel TRIM family member, TRIM59 gene was characterized to be upregulated in SV40 Tag oncogene-directed transgenic and knockout mouse prostate cancer models as a signaling pathway effector. We identified two phosphorylated forms of TRIM59 (p53 and p55) and characterized them using purified TRIM59 proteins from mouse prostate cancer models at different stages with wild-type mice and NIH3T3 cells as controls. p53/p55-TRIM59 proteins possibly represent Ser/Thr and Tyr phosphorylation modifications, respectively. Quantitative measurements by ELISA showed that the p-Ser/Thr TRIM59 correlated with tumorigenesis, whereas the p-Tyr-TRIM59 protein correlated with advanced cancer of the prostate (CaP). The function of TRIM59 was elucidated using short hairpin RNA (shRNA)-mediated knockdown of the gene in human CaP cells, which caused S-phase cell-cycle arrest and cell growth retardation. A hit-and-run effect of TRIM59 shRNA knockdown was observed 24 hours posttransfection. Differential cDNA microarrray analysis was conducted, which showed that the initial and rapid knockdown occurred early in the Ras signaling pathway. To confirm the proto-oncogenic function of TRIM59 in the Ras signaling pathway, we generated a transgenic mouse model using a prostate tissue-specific gene (PSP94) to direct the upregulation of the TRIM59 gene. Restricted TRIM59 gene upregulation in the prostate revealed the full potential for inducing tumorigenesis, similar to the expression of SV40 Tag, and coincided with the upregulation of genes specific to the Ras signaling pathway and bridging genes for SV40 Tag-mediated oncogenesis. The finding of a possible novel oncogene in animal models will implicate a novel strategy for diagnosis, prognosis, and therapy for cancer.