Simultaneous multianalysis for tumor markers by antibody fragments microarray system

It is often necessary to measure a spectrum of tumor markers in oncology. We have developed a simultaneous multiplexing immunoassay method to determine six tumor markers: -fetoprotein (AFP), carcinoma embryonic antigen (CEA), beta-human chorionic gonadotropin (β-HCG), carbohydrate antigen 125 (CA 125), carbohydrate antigen 19-9 (CA 19-9) and carbohydrate antigen 15-3 (CA 15-3). F(ab′)₂ fragments of six capture antibodies were prepared and printed as microarrays on silylated slides to perform sandwich immunoassays with the use of an avidin–biotin system for amplified fluorescence signals. Each antigen with different concentrations was detected to assemble its calibration curve, and combinations of different markers were determined to examine the specificity of simultaneous detection based on the F(ab′)₂ microarrays. Some clinical samples were analyzed to compare with results obtained with the use of immunoradiometric assay (IRMA) method. Wide range calibration curve and its R-value were obtained for each analyte. Calibration curves concentrations were 0–640 μg/l for CEA, AFP and β-HCG, and 0–1280 kU/l for CA 125, CA 19-9 and CA 15-3. The antibody fragments microarray system bears comparison with conventional immunoassays and may find feasible application in measurement of series markers in oncology and other areas of medicine.     

增强CT联合血清指标诊断结直肠癌的价值

本文探讨癌胚抗原(CEA)、糖类抗原19-9(CA19-9)、胰岛素样生长因子结合蛋白-3(IGFBP-3)及增强CT在结直肠癌中的联合诊断效果。选取100例结直肠癌患作为观察组,同时选取结直肠良性肿瘤患者60例作为对照组,检测血清CEA、CA19-9、IGFBP-3,同时给予增强CT检查。观察组血清CEA和CA19-9高于对照组(P<0.05),而IGFBP-3低于对照组(P<0.05);TNM分期Ⅲ~Ⅳ和Ⅰ~Ⅱ患者血清CEA、CA19-9和IGFBP-3差异比较有统计学意义(P<0.05);增强CT联合血清指标联合诊断灵敏性为89.00%、特异性为90.00%;增强CT诊断结直肠癌T分期、N分期与病理结果Kappa值分别为0.696和0.790(P<0.05)。CEA、CA19-9、IGFBP-3联合增强CT是一种具有较高效能的结直肠癌诊断方法。     

Multianalyte immunoassay chip for detection of tumor markers by chemiluminescent and colorimetric methods

Most cancers developed an elevation of the level of at least two markers associated with their incidence. Simultaneous detection of multi-tumor markers associated with a particular type of cancer plays an important role in cancer diagnostic. Here, a multianalyte immunoassay chip for simple and sensitive detection of tumor markers with chemiluminescent and colorimetric methods was proposed, in which carcinoembryonic antigen (CEA) and carbohydrate antigen (CA19-9) that associated with colorectal cancer were detected as model. The immunoassay chip was fabricated by co-immobilization of CEA/CA19-9 antibody on a glass slide with γ-glycidoxypropyltrimethoxysilane as linkage. Through sandwiched immunoreactions, CEA, CA19-9, and their corresponding enzyme tracers, alkaline phosphatase-labeled anti-CEA and horseradish peroxidase-labeled anti-CA19-9, were introduced on the chip. Then, they were sequentially detected by chemiluminescent method in the range of 0.5–80 μg/L and 0.5–80 kU/L with the detection limits of 0.41 μg/L and 0.36 kU/L at 3σ for CEA and CA19-9, respectively. They could also be detected by colorimetric method in the range of 1–200 μg/L and 5–200 kU/L with the detection limits of 0.25 μg/L and 1.25 kU/L at 3σ for CEA and CA19-9, respectively. All these results demonstrated that the present work provided a promising analytical method for tumor markers’ analysis with the advantages of simple analytical procedure, small sample volume and lower cost, which made the proposed method potential for high-throughput detection.     

纳米金修饰电流型CA19-9免疫传感器的制备及应用

通过固定辣根过氧化酶（HRP）标记CA19-9抗体在纳米金修饰的碳糊电极制备了CA19-9安培免疫传感器。该免疫传感器在舍有CA19-9抗原的磷酸盐缓冲溶液中培育后,溶液中CA19-9抗原分子和HRP标记CA19-9抗体分子免疫结合导致了传感器电流的降低。在优化的实验条件下,样品中CA19-9浓度在2～30U／mL范围内与电流降低成线性关系,方法的检出限为1．4U／mL。该免疫传感器表现出较好的稳定性、准确性和重现性,为临床免疫分析提供了一种快速便捷方法。     

血清CEA、CA19-9检测诊断老年胃癌的价值探讨

目的对应用血清CEA、CA19-9联合检测方式对患有胃癌疾病的老年患者的病情实施诊断的临床价值进行研究。方法选择广丰县中医院收治的患有胃癌疾病和胃部良性疾病的老年患者各60例,再抽取同期接受健康体检的健康老年人资料60例,分别将其定义为研究1组、研究2组、对照组。采用化学发光法对三组研究对象的血清CEA、CA19-9水平进行测定,对比分析检测结果和两项指标的阳性率。结果研究1组的血清CEA、CA19-9水平明显高于对照组和研究2组,组间数据比较差异显著(P0.05);研究2组的血清CEA、CA19-9水平明显高于对照组,组间数据比较差异显著(P0.05)。研究1组的CEA、CA19-9水平检测阳性人数明显多于对照组和研究2组,组间数据比较差异显著(P0.05);研究2组的CEA、CA19-9水平检测阳性人数明显多于对照组,组间数据比较差异显著(P0.05)。结论患有胃癌疾病的老年患者的血清CEA、CA19-9水平会异常升高,临床上可以将其作为老年胃癌疾病诊断的重要依据。     

Reagentless amperometric carbohydrate antigen 19-9 immunosensor based on direct electrochemistry of immobilized horseradish peroxidase

A reagentless immunosensor for rapid determination of carbohydrate antigen 19-9 (CA19-9) in human serum was proposed. This strategy was based on the immobilization of antibody in colloidal gold nanoparticle modified carbon paste electrode and the direct electrochemistry of horseradish peroxidase (HRP) that was labeled to a CA19-9 antibody. The nanoparticles were efficient for preserving the activity of immobilized biomolecules. Thus, the immobilized HRP displayed its direct electrochemistry with a rate constant of 1.02 s⁻¹. The incubation of the immunosensor in phosphate buffer solution (PBS) including CA19-9 antigen leading to the formation of antigen-antibody complex, which made the block of electron transfer of HRP toward electrode and resulted in significant peak current decrease of HRP. Under the optimal conditions, the current decrease was proportional to CA19-9 concentrations ranging from 2 to 30 U/ml with a detection limit of 1.37 U/ml at a current decrease by 10%. The immunosensor showed an acceptable accuracy compared with those obtained from immunoradiometric assays, with intra-assay coefficient of 7.3 and 6.9% at CA19-9 concentrations of 5 and 15 U/ml, respectively, and inter-assay coefficient of 9.6% at a CA19-9 concentration of 20 U/ml. The storage stability was acceptable in a pH 7.0 PBS at 4 °C for 10 days. This method avoids the addition of electron transfer mediator, thus simplifies the immunoassay procedure and decreases the analytical time. It provides a new promising platform for clinical immunoassay.     

Electrochemical Immunoassay for Tumor Marker CA19-9 Detection Based on Self-Assembled Monolayer

A CA19-9 electrochemical immunosensor was constructed using a hybrid self-assembled membrane modified with a gold electrode and applied to detect real samples. Hybrid self-assembled membranes were selected for electrode modification and used to detect antigens. First, the pretreated working electrodes were placed in a 3-mercaptopropionic acid (MPA)/β-mercaptoethanol (ME) mixture for 24 h for self-assembly. The electrodes were then placed in an EDC/NHS mixture for 1 h. Layer modification was performed by stepwise dropwise addition of CA19-9 antibody, BSA, and antigen. Differential pulse voltammetry was used to characterize this immunosensor preparation process. The assembled electrochemical immunosensor enables linear detection in the concentration range of 0.05–500 U/mL of CA19-9, and the detection limit was calculated as 0.01 U/mL. The results of the specificity measurement test showed that the signal change of the interfering substance was much lower than the response value of the detected antigen, indicating that the sensor has good specificity and strong anti-interference ability. The repeatability test results showed that the relative standard deviations were less than 5%, showing good accuracy and precision. The CA19-9 electrochemical immunosensor was used for the actual sample detection, and the experimental results of the standard serum addition method showed that the RSD values of the test concentrations were all less than 10%. The recoveries were 102.4–115.0%, indicating that the assay has high precision, good accuracy, and high potential application value.     

Poly-L-lysine/hydroxyapatite/carbon nanotube hybrid nanocomposite applied for piezoelectric immunoassay of carbohydrate antigen 19-9

Hybrid composites are of special scientific interest for biochemical applications wherein the abilities to modulate the morphology and property of the hybrid material are important. In this paper, the formation of poly-L-lysine/hydroxyapatite/carbon nanotube (PLL/HA/CNT) hybrid nanoparticles is described and a general design strategy for an immunosensing platform has been proposed on the basis of PLL/HA/CNT nanocomposite adsorption of antibodies. Quartz crystal microbalance (QCM) used as a model transducer and the detection performances of the resulting immunosensor were investigated by use of the immuno-system of carbohydrate antigen 19-9 (CA19-9), an important indicator in the diagnosis of clinical cancers. The hybrid nanocomposite was characterized by the transmission electron microscope (TEM), scanning electron microscope (SEM) and Fourier transform-infrared (FT-IR) spectrum measurements. The frequency response characteristics for the processes of immobilization and immunoreaction of anchored anti-CA19-9 antibodies were studied in detail. It was found that the developed sensing interface has some advantages such as the activation-free immobilization and the high antigen-binding activities of antibodies. The as-prepared immunosensor can allow for the determination of CA19-9 in the concentration range of around 12.5-270.0 U ml(-1). Such an interface design with the hybrid nanocomposite should be tailored as a new alternative used for biosensor design.     

Electron paramagnetic resonance, photo-acoustic spectroscopy and thermally stimulated luminescence for identification of thermally stimulated processes in gamma-irradiated LaBa2F7

Prostate specific antigen (PSA) is a reliable biochemical marker used in screening for prostate carcinoma. Immunoradiometric assays (IRMA) are generally used for the estimation of total/free PSA in serum samples. Radiolabeled antibody, an important reagent of IRMA was prepared and characterized using an in-house anti-PSA monoclonal antibody (Mab), Mab-2S. Mab-2S was radiolabeled with ¹²⁵I and characterized for immunoreactivity and radiochemical purity. The usability of the radioiodinated Mab as tracer in IRMA was ascertained using authentic reagents for IRMA of PSA.     

Serum N-glycan fingerprint helps to discriminate intrahepatic cholangiocarcinoma from hepatocellular carcinoma

Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are two main types of primary liver cancer, and reliable discrimination is important for optimal treatment. Aberrant glycosylation was detected in HCC and ICC. Both cross-sectional and follow-up studies were performed to establish a differential diagnosis model using N-glycans. A total of 420 participants were enrolled, with 310 patients in training cohort and 110 patients in validation cohort. The follow-up cohort was used to assess the prognosis of ICC. As the results, the diagnostic efficacy of the model was superior to alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9) when identifying ICC from HCC (AUC of the nomogram: 0.845, 95%CI: 0.788–0.902; AFP: 0.793, 95%CI: 0.732–0.854; CEA: 0.592, 95%CI: 0.496–0.687; CA 19-9: 0.674, 95%CI: 0.582–0.767) in training cohort. In validation cohort, this model (AUC: 0.810, 95% CI: 0.728–0.891) also demonstrated high efficacy in distinguishing ICC from HCC. Furthermore, the nomogram helps to stratify ICC into two subgroups with high or low risk of survival and recurrence. Therefore, a nomogram integrating six N-glycans [NGA2FB(Peak2), NG1A2F (Peak3), NA2 (Peak5), NA2F (Peak6), NA3 (Peak8) and NA4 (Peak11)] was established for ICC and HCC differentiation, and for prognosis assessment in ICC patients.     

The Comparison of CA 125 Immunoassay Methods

CA 125 is an antigen defined by monoclonal antibody OC 125 and has been widely applied for clinical diagnosis of ovarian cancer Methods based on IRMA, ELISA and CLEIA for detecting CA 125 have been reported. Many instruments for CA 125 assay in clinical practice have been estabilished. However, there have been many discrepancies among different assay methods. In this work, we study the discrepancies in precision, positivity rate, assay linearity and reproducibility between commercially available CA 125 CLEIA and ELISA techniques by comparing with IRMA. The results would provide a scientific basis for choosing a method of CA 125 immunoassay in the diagnosis of ovarian carcinoma and monitoring of response to therapy,with no need of radioactive reagents.     

Simultaneous detection of four biomarkers with one sensing surface based on redox probe tagging strategy

In this paper, a simple and sensitive amperometric immunosensor for simultaneous detection of four biomarkers by using distinguishable redox-probes as signal tags was proposed for the first time. In sandwich immunoassay format, four kinds of capture antibodies (C-Ab) were immobilized by gold nanoparticles (AuNPs) electro-deposited on the surface of glass carbon electrode (GCE); four kinds of detection antibodies (D-Ab) labeled with different redox probes (including anthraquinone 2-carboxylic acid (Aq), thionine (Thi), ferrocenecarboxylic acid (Fc) and tris(2,2’-bipyridine-4,4’-dicarboxylic acid) cobalt(III) (Co(bpy)₃³⁺)), were combined with 3,4,9,10-perylenetetracarboxylic acid (PTCA), poly(diallyldimethylammonium chloride) (PDDA) and AuNPs functionalized carbon nanotubes, and served as signal tracer. When four target antigens were present, differential pulse voltammetry (DPV) scan exhibited four well-resolved peaks, each peak indicated one antigen, and its intensity was quantitative correlational to the concentration of corresponding analyte. To verify the strategy, four biomarkers for diagnosis of colorectal carcinoma, including carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9 CA125, and CA242, were used as model analytes, the immunosensor exhibited high selectivity and sensitivity, and peak current displayed good linear relationship to logarithm concentration in the ranges from 0.016 to 15 ng mL⁻¹ for CEA; 0.008 to 10 ng mL⁻¹ for CA19-9; 0.012 to 12 ng mL⁻¹ for CA125; 0.010 to 10 ng mL⁻¹ for CA242, and low detection limits of 4.2, 2.8, 3.3 and 3.8 pg mL⁻¹, respectively.     

Lateral flow assay for carbohydrate antigen 19–9 in whole blood by using magnetized carbon nanotubes

The authors describe a rapid, low-cost and sensitive approach for the determination of carbohydrate antigen 19–9 (CA 19–9) in whole blood by using magnetized carbon nanotubes (MCNTs) and a lateral flow strip biosensor (LFSB). MCNTs were synthesized by depositing magnetite (Fe₃O₄) nanoparticles on multiwalled carbon nanotubes (CNTs) via co-precipitation of ferric and ferrous ions within a dispersion of shortened multiwalled CNTs. Antibody against CA 19–9 (Ab₁) was covalently immobilized on the MCNTs and were used to capture CA 19–9 in blood. After magnetic separation, the MCNT-Ab₁-CA 19–9 complexes are applied to the LFSB, in which a capture antibody (Ab₂) and a secondary antibody (Ab₃) are immobilized on the test zone and control zone of the LFSB, respectively. The captured MCNTs on the test zone and control zone are producing characteristic brown bands, and this enables CA 19–9 to be visually detected. Quantitation is accomplished by reading the intensities of the bands with a portable strip reader. Under optimized conditions, the assay has a detection limit as low as 30 U·mL^?1 of CA19–9 in blood. This is below the cutoff value (37 U mL^?1) of CA 19–9. The assay duration for blood samples is 35 min. In our perception, the assay represents a rapid and low-cost tool for rapid determination of CA19–9 in blood that holds promise for clinical applications, particularly in limited resource settings.

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Graphical abstract Schematic of a rapid, low-cost and sensitive approach to detect carbohydrate antigen 19–9 (CA 19–9) in whole blood by using magnetized carbon nanotubes (MCNT) and a lateral flow strip biosensor. The approach offers new opportunities for detecting protein markers in whole blood avoiding sample purification and pre-treatment. This may lead to a new tool for disease diagnosis and monitoring disease recurrence.

     

Protein complexes/aggregates as potential cancer biomarkers revealed by a nanoparticle aggregation immunoassay

Protein–protein interactions and protein complex/aggregate formation play an essential role in almost all biological functions and activities. Through a nanoparticle aggregation immunoassay, we discovered that some proteins are substantially more complexed/aggregated in cancer tissues than normal tissues. This study examined four biomarkers proteins, CA125, CEA (carcinoembryonic antigen), CA19-9 and PAP (prostatic acid phosphatase) in ovarian, colon and prostate tissue lysates. The most exciting results were observed from the PAP assay of prostate tissues: prostate cancer can be clearly distinguished from normal prostate and prostate with benign conditions such as BPH (benign prostate hyperplasia) based on the complex/aggregation level of PAP in prostate tissue lysates. The complex/aggregate level of a protein can be potential biomarkers for cancer detection and diagnosis.     

Amperometric carbohydrate antigen 19-9 immunosensor based on three dimensional ordered macroporous magnetic Au film coupling direct electrochemistry of horseradish peroxidase

A sandwich-type electrochemical immunosensor for the detection of carbohydrate antigen 19-9 (CA 19-9) antigen based on the immobilization of primary antibody (Ab₁) on three dimensional ordered macroporous magnetic (3DOMM) electrode, and the direct electrochemistry of horseradish peroxidase (HRP) that was used as both the label of secondary antibody (Ab₂) and the blocking reagent. The 3DOMM electrode was fabricated by introducing core–shell Au–SiO₂@Fe₃O₄ nanospheres onto the surface of three dimensional ordered macroporous (3DOM) Au electrode via the application of an external magnet. Au nanoparticles functionalized SBA-15 (Au@SBA-15) was conjugated to the HRP labeled secondary antibody (HRP-Ab₂) through the Au–SH or Au–NH₃⁺ interaction, and HRP was also used as the block reagent. The formation of antigen–antibody complex made the combination of Au@SBA-15 and 3DOMM exhibit remarkable synergistic effects for accelerating direct electron transfer (DET) between HRP and the electrode. Under the optimal conditions, the DET current signal increased proportionally to CA 19-9 concentration in the range of 0.05 to 15.65 U mL⁻¹ with a detection limit of 0.01 U mL⁻¹. Moreover, the immunosensor showed high selectivity, good stability, satisfactory reproducibility and regeneration. Importantly, the developed method was used to assay clinical serum specimens, achieving a good relation with those obtained from the commercialized electrochemiluminescent method.     

Unlabeled multi tumor marker detection system based on bioinitiated light addressable potentiometric sensor

Multi biomarkers' assays are of great significance in clinical diagnosis. A label-free multi tumor markers' parallel detection system was proposed based on a light addressable potentiometric sensor (LAPS). Arrayed LAPS chips with basic structure of Si(3)N(4)-SiO(2)-Si were prepared on silicon wafers, and the label-free parallel detection system for this component was developed with user friendly controlling interfaces. Then the l-3,4-dihydroxyphenyl-alanine (L-Dopa) hydrochloric solution was used to initiate the surface of LAPS. The L-Dopa immobilization state was investigated by the theoretical calculation. L-Dopa initiated LAPS' chip was biofunctionalized respectively by the antigens and antibodies of four tumor markers, α-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen 19-9 (CA19-9) and Ferritin. Then unlabeled antibodies and antigens of these four biomarkers were detected by the proposed detection systems. Furthermore physical and measuring principles in this system were described, and qualitative understanding for experimental data were given. The measured response ranges were compared with their clinical cutoff values, and sensitivities were calculated by OriginLab. The results indicate that this bioinitiated LAPS based label-free detection system may offer a new choice for the realization of unlabeled multi tumor markers' clinical assay.     

Ultrasensitive electrochemiluminescence immunoassay for tumor marker detection using functionalized Ru-silica@nanoporous gold composite as labels

In this work, we reported a simple and sensitive sandwich-type electrochemiluminescence (ECL) immunosensor for carcinoembryonic antigen (CEA) on a gold nanoparticles (AuNPs) modified glassy carbon electrode (GCE). The Ru-silica (Ru(bpy)(3)(2+)-doped silica) capped nanoporous gold (NPG) (Ru-silica@NPG) composite was used as an excellent label with amplification techniques. The NPG was prepared with a simple dealloying strategy, by which silver was dissolved from silver/gold alloys in nitric acid. The primary antibody was immobilized on the AuNPs modified electrode through l-cysteine and glutaraldehyde, and then the antigen and the functionalized Ru-silica@NPG composite labeled secondary antibody were conjugated successively to form a sandwich-type immunocomplex through the specific interaction. The concentrations of CEA were obtained in the range from 1 pg mL(-1) to 10 ng mL(-1) with a detection limit of 0.8 pg mL(-1). The as-proposed ECL immunosensor has the advantages of high sensitivity, specificity and stability and could become a promising technique for tumor marker detection.     

A Signal‐Amplified Electrochemical Immunosensor Based on Prussian Blue and Pt Hollow Nanospheres as Matrix

Through electrodepositing Prussian blue (PB) and chitosan (CS), then casting Pt hollow nanospheres (HN‐Pt) and assembling CA19‐9 antibody on the electrode surface, an immunosensor was achieved. A new signal amplification strategy based on PB and HN‐Pt toward the electrocatalytic reduction of H₂O₂ was employed when performing the determination. The resulting immunosensor showed a high sensitivity, broad linear response to carbohydrate antigen 19‐9 (CA19‐9) in two ranges from 0.5 to 30 and 30 to 240 U mL^?1 with a low detection limit of 0.13 U mL^?1 (S/N=3). Moreover, it displayed good reproducibility and stability, and would be potentially attractive for clinical immunoassay of CA19‐9.     

Multiplex detection of tumor markers with photonic suspension array

A novel photonic suspension array was developed for multiplex immunoassay. The carries of this array were silica colloidal crystal beads (SCCBs). The codes of these carriers are the characteristic reflection peak originated from their structural periodicity, and therefore they do not suffer from fading, bleaching, quenching, and chemical instability. In addition, because no dyes or materials related with fluorescence are included, the fluorescence background of SCCBs is very low. With a sandwich format, the proposed suspension array was used for simultaneous multiplex detection of tumor markers in one test tube. The results showed that the four tumor markers, α-fetoprotein (AFP), carcinoembryonic antigen (CEA), carcinoma antigen 125 (CA 125) and carcinoma antigen 19-9 (CA 19-9) could be assayed in the ranges of 1.0-500 ng mL⁻¹, 1.0-500 ng mL⁻¹, 1.0-500 U mL⁻¹ and 3.0-500 U mL⁻¹ with limits of detection of 0.68 ng mL⁻¹, 0.95 ng mL⁻¹, 0.99 U mL⁻¹ and 2.30 U mL⁻¹ at 3σ, respectively. The proposed array showed acceptable accuracy, detection reproducibility, storage stability and the results obtained were in acceptable agreement with those from parallel single-analyte test of practical clinical sera. This technique provides a new strategy for low cost, automated, and simultaneous multiplex immunoassay.     

Immunoradiometric assay for in-vitro determination of prolactin hormone in human serum or plasma using solid phase anti-PRL cellulose particles

The objective of the present study was oriented to produce and purify polyclonal anti-PRL antibody as the main key store in immunoradiometric assay (IRMA) using solid phase cellulose particles for determination of PRL in human sera. The preparation of ¹²⁵I-PRL was carried out by lactoperoxidase method for estimation of the titre of antibody production. The preparation of standards was undertaken. The activation of cellulose particles using 1,1-carbonyl diimidazole (CDI) and coupling of these solid phase particles with purified Rabbit anti-PRL were carried out. Optimization and validation of the assay were carried out. Results revealed that the produced PRL polyclonal antibodies have high titre. Cellulose particles IRMA system was highly sensitive and specific. The intra- and inter-assay variations were satisfactory. The recovery and dilution tests indicated accurate calibration and appropriate matrix. The present technique agreed well with IRMA commercial kit. These cellulose particles retained their characteristics during storage for 6 months at 4 °C. In conclusion, this low cost assay could be used as a useful diagnostic tool for diagnosis and follow up of galactorrhea, infertility and pituitary adenoma.