Chemiluminescent immunosensor for CA19-9 based on antigen immobilization on a cross-linked chitosan membrane

A novel chemiluminescent immunosensor for carbohydrate antigen 19-9 (CA19-9) based on the immobilization of CA19-9 on the cross-linked chitosan membrane was developed. The different membranes were characterized by atomic force microscopy (AFM) and infrared spectrum, respectively. Based on a noncompetitive immunoassay format, this proposed chemiluminescent immunosensor enabled a low-cost, flexible and rapid determination for CA19-9 in combination with flow injection analysis (FIA). After an off-line incubation of the analyte CA19-9 with horseradish peroxidase (HRP)-labeled anti-CA19-9, the mixture was injected into the immunosensor, which led to the trapping of free HRP-labeled anti-CA19-9 by the immobilized antigen in the immunosensor. The trapped HRP-labeled antibody was detected by chemiluminescence due to its catalytic activity following the reaction of luminol and H2O2. Under optimal conditions, the decreased chemiluminescent signal of the immunosensor was proportional to the CA19-9 concentration in the range of 2.0-25 U/ml with a detection limit of 1.0 U/ml. The immunosensor showed an acceptable accuracy and good reproducibility. The results of 20 human serum samples detected by this method were in acceptable agreement with those obtained by immunoradiometric assay. The proposed immunosensor provided a new promising tool for practical clinical detection of the serum CA19-9 level.     

2例老年非肿瘤患者血清糖原抗原19-9水平明显升高

1病例介绍1.1病例1男,86岁,患者于1989年体检时发现肝及肾多发囊肿,无呕吐、呕血及便血等情况,此后定期复查,未治疗.1999年发现肝脏肿物明显增大,并伴右上腹胀痛,腹部可触及肿物,无黄疸及发热,遂于我院行肝囊肿穿刺抽液治疗,治疗后腹部胀痛缓解.     

A one-step immunoassay for carbohydrate antigen 19-9 by biosensor based on imaging ellipsometry

Cancer markers can offer much more information for developing or developed cancers. Simple and short-time assay of cancer biomarker is essential for clinic diagnosis and management. In this study, a one-step immunoassay for carbohydrate antigen 19-9 (CA19-9) by biosensor based on imaging ellipsometry (BIE) is described and its analysis performances are evaluated. Anti-CA19-9 monoclonal antibody as ligand covalently immobilized on carboxyl-modified silicon substrate and its immobilization concentration concurrently screened. Then, bovine serum albumin (BSA) is used to block for acquisition of sensing layer. The detection linear range is from 15.0 to 120.0 U/mL with a minimum limit of 10.0 U/mL (S/N = 3). The intra-slide and inter-slide reproducibility, expressed as coefficient variation (CV%), are 9.9, 5.9, 5.4% and 9.6, 7.6, 5.3% for 31.9, 57.4, and 99.2 U/mL of CA19-9, respectively. Accuracy determined by spiked-recovery is among 95.1–106.7%. Specificity expressed as cross-reaction rate are all below 5.8% evaluated by three analogous biomarkers alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and carbohydrate antigen 242 (CA242). Application of BIE to 25 clinic samples demonstrated that the results are in high correlate with electrochemiluminescent immunoassay (ECLIA, R ² = 0.997). The proposed immunoassay has the potential for clinic application.     

糖类抗原19-9生物半衰期监测原发性肝癌转移的评价

近十年来糖类抗原ＣＡ１９－９主要用于消化道恶性肿瘤和胰腺癌的诊断与治疗监测＾～１～３〕。因大多数肿瘤标志物缺乏特异性,故其临床应用的另一重要方面是对疗效或预后和预后的监测与评估及其半衰期在复发、转移中的意义＾〔４,５〕。但经５年的检索未见有关ＣＡ１９－９半衰期在恶性肿瘤监测中的应用报道,为此,作者检测了原发性肝细胞癌（ｐｒｉｍａｒｙ ｈｅｐａｔｏｃｅｌｌｕｌａｒ ｃａｒｃｉｎｏｍａ,ＰＨＣ）患者ＣＡ１９－９生物半衰期,现报告如下。     

Diagnostic value of carcinoembryonic antigen and carcinoma antigen 19-9 for colorectal carcinoma

Objective: The purpose of this study was to evaluate the diagnostic efficiency of colorectal carcinoma (CRC) with the tumor markers Carcinoembryonic Antigen (CEA) and Carbohydrate Antigen 19-9 (CA 19-9), in addition to investigating whether CA 19-9 can be used to screen the disease process in patients with CRC who had no elevation of CEA levels. Methods: Serum levels of CEA and CA 19-9 were measured in: 138 patients with CRC; 111 patients with benign colorectal diseases. The diagnostic value was performed using the logistic regression equation and receiver operating characteristic curves (ROC). Results: The serum levels of CEA and CA 19-9 in the patients with CRC were significantly higher than those in the patients with benign colorectal diseases (P < 0.001). Receiver operating characteristic curves (ROC) in the patients with CRC versus those with benign colorectal disease yielded the optimal cut-off value of 3.36 ng/ml for CEA and 23.9 U/ml for CA 19-9, respectively. The area under ROC curve (AUC) was 0.789 for CEA, 0.690 for CA 19-9 and 0.900 for the combination of the two tumor markers. The combination resulted in a higher Youden index and a sensitivity of 85.3%. Conclusion: The combined detection of serum CEA and CA 19-9 could play a pivotal role in the diagnosis of CRC, and could drastically improve the sensitivity for the diagnosis of CRC. CA 19-9 might be a tumor biomarker in addition to CEA for CRC.     

Diagnostic utility of clinical and biochemical parameters in pancreatic head malignancy patients with normal carbohydrate antigen 19-9 levels

BackgroundCarbohydrate antigen (CA)19-9 that is the most widely used biomarker for pancreatic cancer has certain limitations in diagnosis, which results in a tough job to distinguish pancreatic cancer from benign tumors with normal CA19-9. The aim of this study was to investigate the diagnostic utility of clinical parameters and serum markers in patients with pancreatic head masses but without elevated CA19-9.MethodsRetrospectively, 106 (69 malignant, 37 benign) of 487 patients admitted for pancreatic head masses were enrolled with CA19-9 level of <37u/ml. Clinical parameters and serum biomarkers were assessed. Among the patients with pancreatic head mass, male individuals (p=0.025) and elder individuals (p<0.001) were more likely to have cancer; and cancer patients were more likely to present with abdominal-pain (p=0.023), weight-loss (p=0.013) and jaundice (p<0.001). Serum bilirubin levels among malignancies, including total bilirubin (p<0.001), direct bilirubin (p<0.001) and indirect bilirubin (p<0.001), were considerably higher than those of benign ones. Logistic regression further concluded that age-distribution, abdominal-pain and direct-bilirubin were three independent factors correlating with final diagnosis. However, CEA (p=0.156) was not sufficient enough to exclude pancreatic cancer.ConclusionsIn patients with pancreatic head masses and CA19-9 of <37u/ml, age-distribution, abdominal-pain and direct bilirubin might be helpful in differential diagnosis. CEA was insufficient for exclusion of malignancy.     

Serum carbohydrate 19-9 Tm and bronchial cancer. Comparative study with another tumor marker, carcinoembryonic antigen

Carbohydrate antigen 19-9 Tm (CA 19-9 Tm) level was measured in 3 groups of sera: 138 control subjects affected by bronchogenic no malignant diseases, 117 subjects with primary bronchogenic carcinoma, 43 with pulmonary metastasis of different cancers. Serum level CA 19-9 Tm was compared with serum level CEA. Control subjects bring us to state the discrimination standard between bronchogenic carcinoma and no carcinoma to 40 U/ml of CA 19-9 Tm. In primary bronchogenic carcinoma, a simultaneous increase of the two markers was noted in some subjects whereas in others only one tumor marker was increased pulmonary metastasis are considered. In these two cases, CEA increases more often in serum of subjects affected by adenocarcinomas CA 19-9 Tm and CEA are thus two different tumor associated markers.     

Chronic bronchitis with fungal infection presenting with marked elevation of serum carbohydrate antigen 19-9: a case report

Carbohydrate antigen 19-9 (CA19-9) is the most frequently applied serum tumor marker for diagnosis of cancers in the digestive organs. However, some patients with benign diseases can have elevated serum levels of CA19-9 as well. The current study presents a 55-year-old female who was admitted to our hospital for further evaluation of a nodular cavity shadow in the right lower lobe and clarification of the cause of the marked elevation of serum CA19-9 levels. Abdominal MRI and gastrointestinal endoscopy did not find any malignancy. As lung cancer cannot be excluded in this patient, a video-assisted thoracoscopic surgery was carried, intraoperative and postoperative biopsy analysis both suggested chronic bronchitis with fungal infection (due to Histoplasma capsulatum or Penicillium marneffei) and organization. Immunohistochemistry showed marked positive staining for CA19-9 in the damaged lung tissue. The CA19-9 levels quickly returned to the normal range following lobe resection. Therefore, the marked elevation of serum CA19-9 levels, in this case, may have resulted from the chronic bronchitis with fungal infection.     

Immunohistochemical staining of colorectal tissues with monoclonal antibodies to ras oncogene p21 product and carbohydrate determinant antigen 19-9.

Two monoclonal antibodies were applied to benign, dysplastic, and malignant human colorectal tissues using immunohistochemical techniques on formalin fixed paraffin embedded material. RAP-5 antibody is directed against a synthetic peptide, reflecting an amino acid sequence of the ras oncogene p21 protein product. Despite using several different techniques and antibody dilutions differential staining between the various epithelial populations was not obtained. RAP-5 also showed other tissue components such as plasma cells, histiocytes, fibroblasts, smooth muscle and vascular endothelium. CA19-9 antibody recognizes an epithelial surface carbohydrate antigen originally derived from a human colorectal carcinoma cell line: it did not stain normal colorectal mucosa or adenomatous polyps, but showed focal expression of variable strength in regenerative, dysplastic, and cancerous mucosa in ulcerative colitis, and in non-colitic colorectal carcinoma. Neither antibody was found to be a reliable marker of the evolution of malignant mucosal changes, although CA19-9 may be of limited use in confirming adenocarcinoma of gastrointestinal origin.     

Carbohydrate antigen 19-9 and carcinoembryonic antigen immunostaining in benign multicystic mesothelioma of the peritoneum.

A 58-year-old Italian man was incidentally discovered to have an elevated carbohydrate antigen 19-9 (CA-19-9) level of 132 U/mL on routine blood testing. Multisystem imaging studies revealed multiple benign-appearing cysts of the liver and single cysts in the pancreas and kidney parenchyma. Throughout 14 months, fluctuations were observed in the elevated serum CA-19-9 levels from 99 to 450 U/mL. Serum carcinoembryonic antigen (CEA) and other tumor markers were normal. Laparoscopy disclosed multiple cystic lesions on the surface of the liver, on the serosal surface of the ileum, and in the mesentery. Electron microscopy characterized the cells as mesothelial. The pathologic diagnosis was benign multicystic mesothelioma of the peritoneum (BMMP). Aspirated fluid from the liver cyst revealed CA-19-9 levels at 28 500 U/mL, strongly linking the elevated serum CA-19-9 levels with mesothelial cyst secretion. Immunostaining was positive for CA-19-9, CEA, and cancer antigen 125 (CA-125). We believe this is the first documented instance of CA-19-9 and CEA secretion in BMMP.     

Cell kinetic analyses and expression of carcinoembryonic antigen,carbohydrate antigen 19-9 and DU-PAN-2 in hyperplastic,pre-neoplastic and neoplastic lesions of intrahepatic bile ducts in livers with hepatoliths

Summary We evaluated cell proliferative activity and expression of carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA 19-9) and DU-PAN-2 in various bile duct lesions in livers with hepatoliths, using histochemical and immunohistochemical methods. Histologically, the bile duct lesions were divisible into hyperplasia, dysplasia, adenocarcinoma in situ and invasive adenocarcinoma. All cases showed mucosal hyperplasia in stone-bearing bile ducts. Livers with invasive adenocarcinoma frequently contained adenocarcinoma in situ and dysplasia, and livers with adenocarcinoma in situ occasionally harboured dysplasia. Proliferating cell nuclear antigen (PCNA) labelling index was low in hyperplasia (mean±SD=20.5±8.7%), intermediate in dysplasia (35.4±15.9%), and high in adenocarcinoma in situ (46.4±9.3%). The mean number of argyrophilic nucleolar organizer regions (AgNORs) was low in hyperplasia (1.52), intermediate in dysplasia (2.26) and high in adenocarcinoma in situ (2.69). There was a significant positive correlation between PCNA labelling index and AgNORs count. CEA was expressed on invasive adenocarcinoma cells and adenocarcinoma in situ cells in most cases and on dysplastic cells in about a half, while CEA was never present in hyperplastic epithelia. Expression of CA 19-9 was low in adenocarcinoma, intermediate in dysplasia and rather high in hyperplasia. There was no significant difference in DU-PAN-2 expression among these bile duct lesions. These data suggest that cell replicative activity is low in hyperplasia, intermediate in dysplasia and high in adenocarcinoma in situ, and that CEA appears in the following order: dysplasia, adenocarcinoma in situ, invasive adenocarcinoma. We suggest that carcinogenesis in biliary epithelia in livers with stones is a multi-step process through hyperplasia, dysplasia and adenocarcinoma in situ to invasive adenocarcinoma.     

CA19-9时间分辨荧光免疫分析方法建立及EDTA处理血浆对其测定结果的影响

建立双抗体夹心法CA19-9时间分辨荧光免疫分析(TRFIA),探讨抗凝剂EDTA对其检测结果的影响,并对本法的稳定性和与Abbort Axsym测定结果的相关性等指标进行评价.分别按一步法和二步法对CA19-9进行测定,并比较血清标本和用EDTA处理的血浆标本对CA19-9结果的影响.结果表明本试剂盒的线性范围为0.19～600U/mL,灵敏度为0.19U/mL,批内和批间CV(%)分别为1.1%～5.4%,1.2%～5.6%.对3230份标本用本试剂盒检测与Abbort Axsym试剂盒检测结果相关系数为0.931.一步法中EDTA处理血浆标本的检测结果明显低于血清标本,二步法中两者无显著性差异.本试剂盒各项指标达到规定的要求,可用于临床血清CA19-9检测.使用中采用血清或血浆标本时要用二步法进行测定,以减少EDTA对结果的影响.     

CA 50 and CA 19-9 antigen expression in normal, hyperplastic, and neoplastic thyroid tissue

The occurrence of the tumor-associated carbohydrate antigens defined by the monoclonal antibodies (moabs) C 50 and 19-9 has been studied by immunoperoxidase staining of formalin-fixed and paraffin-embedded tissue specimens from normal, hyperplastic, adenomatous, and carcinomatous thyroid tissues. Epithelial expression of these antigens was observed neither in normal nor in hyperplastic thyroid tissue. The antigens were expressed in only 1 of 26 follicular adenomas and the staining in this case was weak and restricted to a few cells. In contrast, the expression of this antigens is marked and progressive in carcinomatous tissues. A high proportion, 48 of 52 papillary carcinomas demonstrated C 50 reactivity, whereas 25 of these tumors expressed the CA 19-9 antigen. Of 25 follicular carcinomas, 15 gave a positive staining for the CA 50 and 6 for the CA 19-9 antigen. CA 50 antigen expression was still detected in tumor cells lacking the CA 19-9 antigen and C 50 reactive material was found in all tissue specimens from medullary carcinomas tested, whereas CA 19-9 antigen staining was consistently negative. This indicates that the moab C 50 which reacts, like the moab 19-9, with the sialylated Lewisa (Lea) blood group determinant also binds to other antigens apart from the sialylated Lea in CA 19-9 antigen negative tumor cells. Although, the functional significance of CA 50 and CA 19-9 antigen expression remains to be investigated, these results suggest that the demonstration of these antigens could provide additional differential diagnostic parameters for the characterization of hyperplastic and neoplastic lesions of the thyroid gland. Further clinical studies will show whether these carbohydrate antigens are useful serum markers for the monitoring of thyroid carcinomas.     

利用组织芯片技术研究CA19-9在直肠癌组织中表达

目的探讨CA19-9在直肠癌中的表达,为临床直肠癌的病理诊断和鉴别诊断提供参考。方法应用组织芯片技术和免疫组化染色SP法,检测50例直肠癌直肠粘膜中CA19-9的表达情况,对照组为39例正常直肠粘膜中CA19-9的表达。结果CA19-9在50例的直肠癌组织芯片中阳性率62%,在39例正常直肠粘膜的阳性率为15.4%。两者结果比较相差显著(P<0.05)。结论CA19-9对直肠癌的病理诊断有重要的参考价值。组织芯片技术的信息容量大,实验操作简便,可供临床参考使用。     

Urinary bladder urothelial carcinoma with concurrent plasmacytoid and micropapillary differentiations: A report of two cases with an emphasis on serum carbohydrate antigen 19‐9

We report two cases of urinary bladder urothelial carcinoma (UC). In both, histological examination of a transurethral resection specimen of the bladder tumor revealed UC with plasmacytoid and micropapillary differentiations. In Case 1, residual plasmacytoid UC deeply invaded the extravesical fat tissue of the radical cystectomy specimen, and metastatic carcinoma was found in almost all the dissected lymph nodes. Despite adjuvant chemotherapy and radiotherapy, the patient died 25 months postdiagnosis. Elevated serum carbohydrate antigen 19‐9 (CA19‐9) returned to near normal levels after radical cystectomy, but they increased shortly before death. In Case 2, no residual carcinoma was found in the radical cystectomy specimen or lymph nodes. Postoperative serum CA19‐9 was maintained at normal levels, and the patient remains alive without recurrence or metastasis. Although plasmacytoid and micropapillary UC are known aggressive variants of UC, plasmacytoid UC may be more aggressive. Serum CA19‐9 could serve as a useful biomarker to monitor progression of plasmacytoid UC.     

肿瘤相关抗原CA 19－9免疫放射分析（IRMA）与免疫酶标分析（IEMA）

以抗ＣＡ１９－９单克隆抗体Ｃ２４１为捕捉抗体，以Ｃ１９２为标记抗体建立了ＣＡ１９－９免疫放射分析（ＩＲＭＡ）和ＣＡ１９－９免疫酶标分析（ＩＥＭＡ），ＣＡ１９－９ＩＲＭＡ的标准曲线斜率Ｂ３００ｕ／Ｂ０为３７．１倍，可测定下限在４Ｕ／ｍｌ以下，批内ＣＶ５．１％，批间ＣＶ９．４％，平均回收率９８．６％。ＣＡ１９－９ＩＥＭＡ的标准曲线斜率Ｂ１５０ｕ／Ｂ０为８８．２倍，批内ＣＶ为２．４７％，批间ＣＶ为５．４％，回收率９９．５％。以ＩＲＭＡ方法检测正常血清９９例，ＣＡ１９．９为１２．９±８．５Ｕ／ｍｌ；胰腺癌患者３０例，血清ＣＡ１９－９为１６５．３±１１７．２Ｕ／ｍｌ。以３０Ｕ／ｍｌ为正常上限，则对胰腺癌检测的灵敏度为８６．６％，特异性９６．９７％，说明该方法在胰腺癌诊断中有重要应用价值。本文中的ＣＡ１９－９ＩＲＭＡ已成为第一个国产ＣＡ１９－９试剂盒，开始在全国推广应用。     

Diagnostic usefulness of Ca 19-9 antigen in cancer of the pancreas and neoplasms of the pancreato-biliary intersection

Antigen CA 19-9 was measured in the serum of a group of 16 patients with pancreatic cancer, in 19 patients with non-pancreatic gastrointestinal malignancies, and in 19 others with benign pancreatobiliary disease. The test showed a high sensitivity with a low specificity and a positive predictive value for diagnosis of pancreatic cancer when confronted with a group of controls that included both benign and malignant gastrointestinal disease. The low specificity was due to high CA 19-9 values in other gastrointestinal cancers, especially in carcinoma of the ampulla and the biliary tract. When the latter were added to the group of patients with pancreatic cancer, and the diagnostic accuracy of the test was re-evaluated, sensitivity, specificity and both positive and negative predictive values increased. Values of CA 19-9 over 100 U/ml are highly predictive of malignancy in patients with pancreato-biliary disease.     

Analysis of a serological determinant of H-Y antigen: evidence for carbohydrate specificity using an H-Y specific monoclonal antibody

Using a monoclonal antibody to H-Y antigen we characterized the molecular determinant responsible for H-Y reactivity. H-Y-positive cells were treated with enzymes which alter carbohydrate structure and then were tested for their ability to absorb monoclonal anti-H-Y antibody. Indications are that the serological determinant recognized by this antibody is a glycoconjugate containing terminal non-reducing and internal galactosyl residues.     

Sodium desoxycholate-extracted treponemal antigen in an enzyme-linked immunosorbent assay for syphilis.

The extraction of Treponema pallidum antigen with sodium desoxycholate, based on a previously described procedure (J. Portnoy and H.J. Magnuson, J. Immuno. 75:348-355, 1955), was used in an enzyme-linked immunosorbent assay (ELISA) test for syphilis. The antigen was prepared from T. pallidum street strain no. 14, and its overall sensitivity and specificity was compared with those of sonicated antigen preparations made with phosphate-buffered saline. The optimum serum dilution for testing and the significant absorbance reading at 490 nm were selected by examination of quantitative dilutions of 91 sera from presumably normal individuals and 92 sera from syphilitics. The time and temperature of serum and conjugate incubations were also examined. With an absorbance reading of greater than or equal to 0.2 at the 1:80 serum dilution, 88 (95.8%) of 92 sera from syphilitics were reactive in the ELISA test with desoxycholate-extracted antigen, and 82 (89.1%) were reactive with the sonicated antigen. Only one nonsyphilitic serum was reactive with each antigen. Greater sensitivity without loss in specificity was obtained with longer serum and conjugate incubations. We concluded that an ELISA test with sodium desoxycholate-extracted antigen is more sensitive than and equally specific to an ELISA with sonicated treponemal antigen.