Addition Of Alpha-fetoprotein Research Articles

New Insights on Potentiometric Immunosensor at Carbon Fiber Microelectrode for Alpha‐Fetoprotein in Hepatocellular Carcinoma

AbstractHerein, we investigated the analytical features of potentiometric immunosensors for detection of alpha‐fetoprotein (AFP) in hepatocellular carcinoma at different electrodes, such as carbon fiber microelectrode (CFME) and carbon‐disk electrode (CDE), respectively. To construct such an immunosensor, anti‐AFP capture antibodies were first conjugated covalently onto the activated electrodes through typical carbodiimide coupling. Thereafter, one‐step immunoreaction protocol was successfully introduced to develop a new potentiometric immunoassay upon addition of AFP. Accompanying the antigen‐antibody reaction, the surface charges of the modified electrodes were changed for the readout of electric potential. Results indicated that the linear range of CDE‐based immunosensor was 0.1–100 ng mL−1 AFP, whereas the assay sensitivity by using CFME could be further increased to 3.2 pg mL−1 with the linear range from 0.01 to 500 ng mL−1 AFP. Meanwhile, CFME‐based immunosensor showed high sensitivity, good reproducibility and specificity, and could be utilized for the analysis of human serum specimens with consistent results relative to commercialized ELISA kit.

Homogeneous immunoassay for alpha-fetoprotein based on the quenching of the fluorescence of quantum dots by antibody labelled with complexed copper ion tags.

A homogeneous fluorescent immunoassay is described for the determination of alpha fetoprotein (AFP) relying on the interaction between copper ion complex and quantum dots (QDs). Thecopper ion complex-labelled antibody can be employed as a quencher of fluorescence of QDs and capture probe of AFP in homogeneous solution. The labelled antibody is mixed with QDs to form the immune ensemble probe. Upon the addition of AFP, the labelled antibody is stripped away from QDs by antigen-antibody combination leading to the increase in the fluorescence signal. Thus, the determination of AFP can be realized by fluorometry (best measured at excitation/emission wavelengths of 360/520nm). The fluorescence intensity shows a good linear relationship with the AFP concentration ranging from 40 to 640ngmL-1, and the LOD is 26ngmL-1. The proposed method provides a new approach to incorporate metal complexes into QD-based biomolecule sensing. Graphical abstract Schematic presentation of a fluorescent probe comprised of quantum dots and antibody labelled with copper ion complex for homogeneous immunoassay of α-fetoprotein. The target antigen can break up the ground state QD/labelled antibody complex to set free the fluorescent QDs.

Electrochemical aptasensor for analyzing alpha-fetoprotein using RGO-CS-Fc nanocomposites integrated with gold-platinum nanoparticles.

Herein, a label-free electrochemical aptasensor for alpha-fetoprotein (AFP) analysis was established. The AFP aptamer (AFP-Apt), as the recognition molecule, was immobilized on the surface of a screen-printed carbon electrode, which was modified by gold-platinum metallic nanoparticles and reduced graphene oxide-chitosan-ferrocene nanohybrids (Au-Pt NPs/RGO-CS-Fc), to build the AFP electrochemical aptasensor. The construction process of the aptasensor was characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, Raman spectroscopy, and electrochemical impedance spectroscopy. With the addition of AFP, the formation of the AFP-aptamer conjugation blocked the electron transfer reaction, reducing the differential pulse voltammetric responses of the current of Fc in the RGO-CS-Fc nanohybrids. By optimizing the experimental parameters, AFP could be detected with the dynamic concentration range of 0.001 to 10.0 μg mL-1 and with a detection limit of 0.3013 ng mL-1. In addition, the approach was manifested to have good selectivity, reproducibility, and stability. The fabricated aptasensor had a good recovery rate of 102.36% to 118.09% in real human serum samples. This work demonstrates that the electrochemical aptasensor is a useful tool for analyzing AFP inexpensively, rapidly, and accurately.

Abstract 1567: A mass spectrometry based serum test for the detection of hepatocellular carcinoma (HCC) in high risk patients

Abstract Improved screening protocols (SP) for patients at high risk of developing HCC could lead to improved patient outcome and possibly cure if detected early. The current SPs, ultrasound with the possible addition of alphafetoprotein (AFP) measurement, suffer from insufficient sensitivity and specificity, and less than 30% of patients are diagnosed early enough to be suitable candidates for resection or transplantation. An easy to use biomarker for the early detection of HCC is clearly needed. We used mass spectrometry analysis of serum samples combined with specialized deep learning techniques to train and validate such a test. The development cohort consisted of patients undergoing transplant or resection for HCC (N = 52; median MELD = 14), and patients with advanced cirrhosis undergoing liver transplant (N = 53; median MELD = 25). Underlying cause of liver disease was 51% hepatitis C infection. The validation cohort consisted of patients with advanced HCC (N = 103; 70/26/7 Child-Pugh A/B/C) and liver disease but no HCC (N = 77; 68/7/2 Child-Pugh A/B/C), with 68% of patients having hepatitis B infection (HBV). The classifier was trained especially to avoid confounding by liver function. The resulting test showed a sensitivity/specificity of 73%/95% in cross validation in the development set overall. In the subset of T1 patients (or lesion size <2 cm) accuracy was 72% (75%). Performance was good across all represented causes of liver disease. The test was shown not to be confounded by liver function by classifying healthy patients (N = 17) as ‘no cancer’. In the validation set the performance was 89%/73%, with 91%/78% in the HBV subgroup and 90%/75% in the Child-Pugh A subgroup. The developed test shows promise in the early detection of HCC as a screening tool in high risk patients independent of the cause of underlying liver disease. While further validation will be necessary to conclusively prove its clinical validity, we believe such a test could substantially improve early detection of HCC. Citation Format: Devalingam Mahalingam, William K. Washburn, Glenn Halff, Leonidas Chelis, Stylianos Kakolyris, Stylianos Vradelis, Julia Grigorieva, Carlos Oliveira, Heinrich Roder, Joanna Roder. A mass spectrometry based serum test for the detection of hepatocellular carcinoma (HCC) in high risk patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1567. doi:10.1158/1538-7445.AM2015-1567

Abstract 3143: A functional interaction between alpha-fetoprotein and miRNA-29 modulates the HCC epigenome.

Abstract Globally, hepatocellular carcinoma (HCC) accounts for 70-85% of primary liver cancers and ranks second in the leading cause of male cancer death. Serum alpha-fetoprotein (AFP), normally expressed during fetal development, is reactivated in 60% of HCC tumors and associated with poor patient outcome. We hypothesize that AFP+ and AFP- tumors may differ biologically and have different prognosis in HCC patients. Using microarray-based global microRNA and mRNA profiling, we found that members of the miR-29 family are the most significantly (p<1E-5) down-regulated miRs in AFP+ tumors (n=242). Physiologically, during mouse embryonic development, miR-29 family expression is low but gradually increases after birth, contrary to AFP expression which dramatically decreases after birth. In addition, a member of the DNA methyltransferase (DNMT) family, DNMT3A, is one of the most significantly (p=8E-6) up-regulated genes in AFP+ HCC. Interestingly, there is a significant inverse correlation (p<1E-4) between miR-29 and DNMT gene expression, suggesting that they may be functionally antagonistic, which is consistent with the finding showing that DNMT3A and DNMT3B are direct targets of miR-29. Experimentally, we found that increased AFP expression or the addition of AFP+ media to AFP- cells inhibits miR-29a expression in HCC cell lines. We also found that AFP transcriptionally regulates the promoter of miR-29a. Moreover, global DNA methylation array profiling reveals increased methylation in HCC patients with high levels of serum AFP. These results support our hypothesis that AFP inhibits miR-29, which modulates epigenetic changes that contribute to poor outcome. Taken together, this study elucidates the mechanisms that link AFP and miR-29 expression to epigenetic alterations and may aid in the development of novel therapeutic agents to improve survival of HCC patients. Citation Format: Sonya Parpart, Stephanie Roessler, Fei Dong, Vinay Rao, Christopher Loffredo, Xin Wei Wang. A functional interaction between alpha-fetoprotein and miRNA-29 modulates the HCC epigenome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3143. doi:10.1158/1538-7445.AM2013-3143

The value of the Barcelona Clinic Liver Cancer and alpha-fetoprotein in the prognosis of hepatocellular carcinoma

presently, the reference staging system to evaluate the prognosis of hepatocellular carcinoma (HCC) patients is "The Barcelona Clinic Liver Cancer" (BCLC) system. The value of alpha-fetoprotein (AFP) has not been properly defined. The aim of this study was to evaluate the BCLC classification in our clinical practice and to know what the prognostic value of AFP is. 136 consecutive HCC patients were prospectively included in this study. The diagnosis of HCC was based on the recommendation of international guidelines. The patients were studied and managed according to usual clinical practice. Survival curves were estimated using the Kaplan-Meier method and predictors of survival were identified using the Cox model. 110 patients (80.9%) were male. The mean age of the patients was 66.62 + or - 11.68 years. Liver cirrhosis was present in 91.2%. The most frequent cause of liver disease was hepatitis C infection (38.97%). Serum AFP was - or = 20 ng/mL in the 57%, > 20-200 ng/mL in the 20%, and > 200 ng/mL in 23%. According to the BCLC staging system, 79 patients were classified as stage A (58.09%), 29 (21.32%) stage B, 17 (12.50%) stage C and 11 patients (8.09%) as stage D. The overall median survival time was 26.52 months (95% CI 16.7-36.3). The median survival according to BCLC system was: BCLC A 62.27, BCLC B 12.72, BCLC C 4.83, and BCLC D 0.62 months (p < 0.0001); and according to serum AFP was: AFP - or = 20: 62.27 months, > 20-200: 22.08 months, and > 200 ng/mL: 5.39 months (p < 0.0001). Multivariate analysis showed that AFP, BCLC classification and treatment were independent prognostic factors. our results confirm that the BCLC is a good prognostic system. The AFP has prognosis value in HCC patients. The addition of AFP could improve the BCLC system. Future studies are needed to confirm our results and also the best way to combine BCLC and AFP properly.

Purified human alpha fetoprotein inhibits growth factor-stimulated estradiol production by porcine granulosa cells in monolayer culture.

Purified alpha fetoprotein (AFP) synergizes with transforming growth factor alpha (TGF alpha) and insulin-like growth factor I (IGF-I) to enhance proliferation of porcine granulosa cells (pGC) in primary culture, suggesting a role for AFP in the modulation of growth factor-mediated cell growth. TGF alpha stimulates basal estrogen production by pGC and is in fact more potent than FSH in these cells. In this study, we investigated the effects of AFP on growth factor-stimulated estradiol (E2) production by pGC. Basal production of E2 was not altered by the addition of AFP. AFP dose-dependently inhibited TGF alpha-stimulated E2 production with statistically significant inhibition observed with 2.5 micrograms/ml. We have previously shown that the mitogenic effects of AFP are maximized with TGF alpha+IGF-I. E2 production was even more sensitive to AFP inhibition when the two growth factors were combined. Human serum albumin (HSA; 10 micrograms/ml) was without effect. AFP did not interfere with the E2 RIA, affect the uptake of or display specific in vitro binding of the androgen substrate. Furthermore, human AFP and HSA did not exhibit specific in vitro binding of E2, in contrast to purified rat AFP (positive control). These data indicate that physiological concentrations of purified AFP significantly and dose-dependently inhibit growth factor-stimulated E2 production by pGC in culture. Since AFP is known to increase TGF alpha+IGF-I mediated cell growth, these data suggest that AFP may be inhibiting the differentiated function (steroidogenesis) of pGC while enhancing the proliferation of these cells.

Synergistic Action of Purified α-Fetoprotein and Growth Factors on the Proliferation of Porcine Granulosa Cells in Monolayer Culture*

alpha-Fetoprotein (AFP) is present in high levels in fetal fluids, certain neoplasias, and regenerating liver. Although AFP's physiological role remains an enigma, we have recently demonstrated mitogenic activity for AFP. Using a primary monolayer culture system, we have further investigated the proliferative activity of purified AFP. Porcine granulosa cells from small ovarian follicles were attached for 2 days in Ham's F-12-Dulbecco's Modified Eagle's Medium (1:1) and 5% fetal calf serum, followed by 6 days of culture in medium containing 0.25% plasma-derived serum plus 25 micrograms/ml low density lipoprotein with or without growth factors and/or purified human AFP. In this system AFP alone does not stimulate proliferation. However, when combined with epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I; 10 ng/ml each), AFP (5 micrograms/ml) significantly (P less than 0.01) enhanced growth factor-mediated proliferation 4.5-fold over that of medium controls. Equivalent doses of purified human serum albumin or transferrin demonstrated no effect. The effects of AFP were dose dependent, with significant (P less than 0.05) enhancement of proliferation (2.7-fold over controls) observed with as little as 0.313 micrograms/ml AFP. Increased proliferation was noticed as early as 24 h after the addition of AFP and by 48 h AFP, EGF, and IGF-I had significantly (P less than 0.05) increased proliferation over that seen in medium controls, cells treated with EGF plus IGF-I, or cells treated with 10% fetal calf serum plus EGF, and this trend continued linearly over 5 days of culture. AFP (5 micrograms/ml) significantly increased the proliferative response observed with increasing doses of EGF, IGF-I, or EGF plus IGF-I, but did not appear to alter the dose-response curves. AFP dose-dependently (1.25-5 micrograms/ml) and significantly (P less than 0.05) increased proliferation of porcine granulosa cells in response to platelet-derived growth factor (PDGF) and EGF (25 and 10 ng/ml, respectively), but not to PDGF alone. In contrast, AFP produced no further proliferation of porcine thecal cells in response to PDGF plus EGF. Binding of EGF, IGF-I, or PDGF to purified AFP could not be demonstrated. These results demonstrate that physiological levels of AFP, although not mitogenic alone, can significantly enhance the mitogenic activity of EGF plus IGF-I/PDGF and may function to modulate growth factor-mediated cell proliferation during development and neoplasia.