Cancer NanomedicineView this Special Issue
Single-Molecule Sandwich Immunoassay for Quantification of Alpha-Fetoprotein Based on Evanescent Field-Enhanced Fluorescence Imaging
A highly sensitive immunosensor based on a gold nanopatterned chip was developed for accurate determination of alpha-fetoprotein (AFP) via total internal refection fluorescence microscopy (TIRFM). The surface of the gold nanopatterned chips was modified with dithiobis(succinimidyl propionate) and protein A/G for immobilization of the AFP antibody. The immunoassay created a sandwich of antigen between the AFP antibody on the chip that was modified with protein A/G, and the secondary antibody, a monoclonal anti-human-AFP labeled with biotin (biotin-labeled anti-AFP). AFP concentration was determined based on evanescent field fluorescence signal, which was generated by interaction between biotin-labeled anti-AFP and a streptavidin-labeled fluorescence dye. AFP concentration could be measured in a wide dynamic linear range of 720 zM–10 nM with a detection limit of 720 zM. A significant enhanced sensitivity (~40,000-fold) was achieved with the AFP-nanoarray chip compared to conventional chemiluminescence immunosensors. The immunoassay exhibited a wide detection range and high sensitivity and accuracy, qualities valuable for clinical assay of AFP.
Since the introduction of sandwich assays using monoclonal antibodies, various immunoassays have been introduced with automated analysis and increased specificity . However, many immunoassay methods such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and single radial immunodiffusion have disadvantages such as being time-consuming procedures using expensive instrumentation with complicated separation and labeling steps, and potential dangers (e.g., radiation hazards) or expensive materials . New techniques such as electrochemistry , chemiluminescence , piezoelectricity , surface plasmon resonance (SPR) , and sandwich immunoassays based on nanoarray protein chips [7, 8] have attracted interests because of their characteristics. Specifically, nanoarray protein chips offer a sensitive, accurate, quantitative, and simple alternative methodology for determination of tumor markers.
Alpha-fetoprotein (AFP) is a 70 kDa oncofetal glycoprotein of 591 amino acids. It contains a single asparagine-linked (Asn233) carbohydrate chain that is a known biomarker for hepatocellular carcinoma (HCC) [9–12]. The association between serum AFP and HCC has been extensively described [13–16]. AFP has been a diagnostic test for HCC since the 1970s, when most patients with HCC were diagnosed at an advanced stage with clinical symptoms . AFP is suggested to function as a transport molecule for several different ligands and various drugs [18, 19] and to have immunosuppressive activity and a role in regulation of cell proliferation . The first conditionally quantitative serum assays for AFP were introduced in 1971 . Several approaches have attempted to enhance the techniques of SPR detection  such as bioluminescent sandwich immunoassays , electrochemical immunoassays , and chemiluminescence resonance energy transfer . However, the sensitivities for these assays are still poor .
In this study, a sandwich immunoassay using gold nanopatterned protein chips was developed for quantitative detection of tumor markers such as AFP in serum. A total internal reflection fluorescence microscopy (TIRFM) technique based on evanescent field fluorescence imaging was applied for trace analysis of AFP with a wide dynamic linear range to use in clinical diagnosis. The relationship between evanescent field-fluorescence signal and AFP concentration showed excellent and extensive linearity. The method was successfully applied to determine AFP in human serum.
2. Experimental Details
2.1. Reagent Preparation
Dimethyl sulfoxide (DMSO) and glycine were from Sigma-Aldrich Inc. (St. Louis, MO, USA). Dithiobis(succinimidyl propionate) (DSP) and protein A/G were from Pierce (Rockford, IL, USA). Tris (base) was from Mallinckrodt Baker, Inc. (Phillipsburg, NJ, USA). StabilGuard was from SurModics (Eden Prairie, MN, USA). Alexa Fluor 488 streptavidin was obtained from Molecular Probes (Eugene, OR, USA). Human AFP antigen, monoclonal antibody to human AFP (5H7), and monoclonal antibody to human AFP labeled with biotin (biotin-labeled anti-AFP, 4A3), were from Biodesign International (ME, USA). Normal human serum samples were isolated from blood by centrifugation at 2,000 rpm for 15 min at 2°C. To mimic clinical conditions, a standard AFP sample of 20 fM was spiked into 1 : 104 diluted normal human serum at a three-to-one ratio. Before use, 1× PBS buffer solution (pH 7.4; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4) was filtered through a 0.2-μm membrane filter and photobleached overnight using a UV-B lamp (G15TE, 280–315 nm, Philips, The Netherlands).
2.2. Gold Nanopatterned Chips
A gold nanopatterned substrate was designed as shown in Figure 1(a) and fabricated by the National Nanofab Center (Daejeon, Republic of Korea). Four-inch soda-lime glass wafers from Winwin Tech (Bucheon, Republic of Korea) were used to make 4 × 5 nanoarrays with 100 nm diameter spots (SEM in Figure 1(a)) with a 10 μm pitch. Gold spots were deposited on the glass substrate by an electron beam evaporator. Substrates were coated with a 5 nm adhesive layer of chromium (99.997% purity) at a rate of 0.1 nm/s, followed by deposition of a 20 nm layer of gold (99.997% purity) at a rate of 0.1 nm/s. Before linker deposition, chips were immersed in acetone (99.5% purity) for 30 s, followed by isopropyl alcohol (99.9% purity) for 30 s. Gold nanopatterned chips were exposed to piranha solution (1 : 1 = H2SO4 : 30% H2O2) for 30 min, rinsed with deionized water, and dried under a stream of nitrogen. Before use, chips were stored in a desiccator.
2.3. AFP Single-Molecule Sandwich Immunoassay on Gold Nanopatterned Chips
The analytical procedure for sandwich immunoassay of AFP on a gold substrate is schematically depicted in Figure 1(b). Gold patterned chips were immersed in 4 mg/mL DSP in DMSO for 30 min, then rinsed with DMSO and deionized water. Addition of 0.1 mg/mL of protein A/G, which binds to the heavy chains of the antibody Fc region, was used to uniformly orient the antibodies for 1 h. Unreacted succinimide groups were blocked with 10 mM Tris (pH 7.5) and 1 M glycine for 30 min. Chips were incubated with StabilGuard for 30 min to stabilize bound proteins, then rinsed briefly with a few drops of deionized water. Chips were incubated with 20 μL of 2 μg/mL monoclonal antibody to human AFP (5H7) in PBS (pH 7.4) for 1 h. After washing, AFP standard protein that was diluted to various concentrations, or normal or spiked clinical samples were incubated on chips for 1 h. The incubation time of the sample with 720 zM AFP was increased to 5 h to allow sufficient time for Brownian motion. Reaction with 20 μL of 2 μg/mL biotin-labeled anti-AFP (4A3) was for 1 h. To detect biotin-labeled anti-AFP, 20 μL of Alexa Fluor 488 streptavidin (2 μg/mL) was added to chips and incubated for 30 min. Chips were washed in 100 mL 1× PBS for 2 min and rinsed briefly with deionized water at each step. All reactions were carried out at room temperature with agitation.
2.4. Total Internal Reflection Microscopic System
A schematic diagram of the TIRFM system is in Figure 1(c). An upright Olympus BX51 microscope (Olympus Optical Co., Ltd., Tokyo, Japan) with an Olympus 100× UPLFL objective lens (oil type, 1.3 N.A., W.D. 0.1 mm) was used. A 520/10 nm band-pass filter from Semrock (Rochester, NY, USA) was coordinated with the use of 473 nm laser excitation during imaging. Fluorescence images were captured by an electron-multiplying, cooled charge-coupled device (EM-CCD) camera (QuantEM 512SC, Photometrics, AZ, USA) equipped with a Uniblitz mechanical shutter (Vincent Associates, Rochester, NY, USA) with an exposure time of 100 ms. All quantitative analysis of data and image acquisition used MetaMorph 7.1 software (Universal Imaging Co., Downing Town, PA, USA).
3. Results and Discussion
A single-molecule sandwich immunoassay for sensitive detection of AFP by evanescent field-enhanced fluorescence was designed. The sensitivity of the AFP assay could calculate the detection limit of 720 zM using 100 nm gold nano-patterned chips. Size reduction and site-specific labeling of antibodies to create a surface with high functional capacity increases the sensitivity of an immunoassay . Furthermore, the 100 nm gold array chips showed no quenching of fluorescence dyes and had increased sensitivity. Under optimal conditions, the sandwich immunoassay had high sensitivity and a wide dynamic range for monitoring on a single-molecule level.
The increase in relative fluorescence intensity (RFI) was proportional to AFP concentration (Figure 2), and the linear response range was 720 zM to 10 nM (linear regression equation, , ) with a low detection limit of 720 zM and a signal-to-noise ratio () of 3 (Table 1). The AFP-nanoarray chip method showed 40,000 times higher sensitivity than other methods (i.e., surface plasmon resonance, bioluminescent immunoassay, electrochemical immunoassay, and chemiluminescence immunoassay). These results showed that the proposed method was highly sensitive, especially for ultralow levels of AFP.
In addition, the wide quantification range (720 zM to 10 nM) would be useful for healthy human serum, which has unestablished normal ranges for AFP. The normal range of AFP for adults and children is variously reported as under 50 ng/mL , under 10 ng/mL , and under 5 ng/mL . Bader et al., Wang and Xureported that the average concentration of AFP is about 25 ng/mL in healthy human serum [41, 42]. Ju et al. also reported a low average value of 3.4 ng/mL . However, a level above 500 ng/mL of AFP in adults can be indicative of HCC, germ cell tumors, and metastatic liver cancer.
The assay method for quantitative analysis was based on evanescent field-enhanced fluorescence imaging via prism-type TIRFM. First, we selected signal regions and background regions with the same area. The sum of TIRF intensities of occupied pixels per single spot was corrected by background subtraction and RFI was calculated. Figure 3(a) shows the peak of fluorescence intensity of serial diluted AFP standard antigen reacted on chip. The peak width was greater than the spot diameter of 100 nm because of fluorescence imaging. The peak shows a moderate increase with increasing AFP concentration from 14.3 aM to 143 pM.
In this study, samples including standard AFP antigen, normal human serum, and human serum with added standard AFP antigen were evaluated using TIRFM and single-molecule sandwich immunoassay chips. The AFP standard (AFP, positive sample) (Figure 4(a), 1.42 pg/mL = 20 fM), normal (nonpathologic) human serum (Figure 4(b), 0.13 pg/mL = 1.8 fM), and the sample of human serum spiked with AFP (Figure 4(c), 1.16 pg/mL = 16.3 fM; theoretical, 1.1 pg/mL = 15.4 fM) were analyzed using gold nanopatterned chips. The results indicated that the sandwich immunoassay using the gold nanopatterned chips gave the high accuracy and sensitivity required for the quantification of biomarkers in human serum samples.
Specificity is an important factor in practical use of immunoassays. AFP is closely related to albumin, both genetically and structurally. The amino acid sequences of AFP and albumin have extensive homology, and the genes coding for the proteins are localized to the same area of human chromosome 4 (4q11–q13) . Since the 1980s, research on monoclonal antibodies with unique specificity for individual binding sites on antigens has been used to improve the sensitivity and specificity of AFP determination . Ding et al. evaluated the selectivity of an immunosensor with four kinds of potential interferents, including L-glutamic acid, bovine serum albumin, hemoglobin, and D-glucose . Chan et al. showed specificity satisfactory with paired monoclonal antibodies for AFP . The monoclonal antibodies against human AFP from Biodesign International (5H7 and 4A3) that we used for the AFP sandwich assay specifically recognized the human AFP molecule . This allowed us to ignore the negligible effects of interfering antigens in the AFP sandwich immunoassay.
We developed a single-molecule sandwich immunoassay on gold nanopatterned chips that was highly sensitive for AFP detection in human serum. The method has a wide range of quantitation and could be applied for testing healthy human serum with the normal range of AFP that has been reported as 47.6 pM–700 pM. The method could be used to diagnose AFP-negative or AFP-positive human serum from pathological clinical samples.
The linear response range of the assay for AFP concentration was 720 zM to 10 nM with a correlation coefficient of 0.9932. The detection limit of 50 × 10–9 ng/mL (720 zM) with a of 3 was in linear range of the calibration curve, and much lower than LODs from 0.004 ng/mL to 20 ng/mL reported for other methods [28–31, 33–35]. For the AutoDELFIA hAFP immunoassay for the quantitative determination of hAFP, Mannings et al. established that falsely low AFP concentrations occur in 2.8% of samples with AFP concentrations <15 kU/L (13.8 ng/mL = 193 pM) due to immunoassay interference . Our results showed that a sandwich immunoassay chip for quantitative analysis of AFP by evanescent field-enhanced fluorescence imaging was simple and sufficiently sensitive for determination of AFP in human serum samples. The assay had good precision and accuracy at the single-molecule level. The new immunoassay is expected to be widely useful for highly sensitive clinical analysis and other biotechnology applications.
Conflict of Interests
The authors have declared no conflict of interests.
This research was supported by a postdoctoral fellowship Grant from Kyung Hee University in 2011 (KHU-20110212).
D. W. Chan and S. Sell, “Tumor markers,” in Tietz Text Book of Clinical Chemistry, C. A. Burtis and E. R. Ashwood, Eds., pp. 897–927, WB Saunders Company, Philadelphia, Pa, USA, 2nd edition, 1994.View at: Google Scholar
C. M. Sturgeon, “Tumor markers in the laboratory: closing the guideline-practice gap,” Clinical Biochemistry, vol. 34, no. 5, pp. 353–359, 2001.View at: Publisher Site | Google Scholar
M. S. Wilson, “Electrochemical immunosensors for the simultaneous detection of two tumor markers,” Analytical Chemistry, vol. 77, no. 5, pp. 1496–1502, 2005.View at: Publisher Site | Google Scholar
T. Konry, A. Novoa, Y. Shemer-Avni et al., “Optical fiber immunosensor based on a poly(pyrrole-benzophenone) film for the detection of antibodies to viral antigen,” Analytical Chemistry, vol. 77, no. 6, pp. 1771–1779, 2005.View at: Publisher Site | Google Scholar
B. Zuo, S. Li, Z. Guo, J. Zhang, and C. Chen, “Piezoelectric immunosensor for SARS-associated coronavirus in sputum,” Analytical Chemistry, vol. 76, no. 13, pp. 3536–3540, 2004.View at: Publisher Site | Google Scholar
R. Kurita, Y. Yokota, Y. Sato, F. Mizutani, and O. Niwa, “On-chip enzyme immunoassay of a cardiac marker using a microfluidic device combined with a portable surface plasmon resonance system,” Analytical Chemistry, vol. 78, no. 15, pp. 5525–5531, 2006.View at: Publisher Site | Google Scholar
S. Lee, N. P. Cho, J. D. Kim, H. Jung, and S. H. Kang, “An ultra-sensitive nanoarray chip based on single-molecule sandwich immunoassay and TIRFM for protein detection in biologic fluids,” Analyst, vol. 134, no. 5, pp. 933–938, 2009.View at: Publisher Site | Google Scholar
S. Lee, S. Lee, Y. H. Ko et al., “Quantitative analysis of human serum leptin using a nanoarray protein chip based on single-molecule sandwich immunoassay,” Talanta, vol. 78, no. 2, pp. 608–612, 2009.View at: Google Scholar
H. Yoshima, T. Mizuochi, M. Ishii, and A. Kobata, “Structure of the asparagine-linked sugar chains of α-fetoprotein purified from human ascites fluid,” Cancer Research, vol. 40, no. 11, pp. 4276–4281, 1980.View at: Google Scholar
G. J. Mizejewski, “α-fetoprotein as a biologic response modifier: relevance to domain and subdomain structure,” Proceedings of the Society for Experimental Biology and Medicine, vol. 215, no. 4, pp. 333–362, 1997.View at: Google Scholar
E. Alpert, R. Hershberg, P. H. Schur, and K. J. Isselbacher, “Alpha-fetoprotein in human hepatoma: improved detection in serum, and quantitative studies using a new sensitive technique,” Gastroenterology, vol. 61, no. 2, pp. 137–143, 1971.View at: Google Scholar
X. W. Wang and H. Xie, “Alpha—fetoprotein enhances the proliferation of human hepatoma cells in vitro,” Life Sciences, vol. 64, no. 1, pp. 17–23, 1998.View at: Publisher Site | Google Scholar
G. I. Abelev, S. D. Perova, N. I. Khramkova, Z. A. Postnikova, and I. S. Irlin, “Production of embryonal alpha-globulin by transplantable mouse hepatomas,” Transplantation, vol. 1, pp. 174–180, 1963.View at: Google Scholar
M. E. Alpert, J. Uriel, and B. de Nechaud, “Alpha-1 fetoglobulin in the diagnosis of human hepatoma,” The New England Journal of Medicine, vol. 278, no. 18, pp. 984–986, 1968.View at: Google Scholar
L. R. Purves, M. MacNab, E. W. Geddes, and I. Bersohn, “Serum-alpha-foetoprotein and primary hepatic cancer,” The Lancet, vol. 1, no. 7548, pp. 921–922, 1968.View at: Google Scholar
J. B. Smith and D. Todd, “Foetoglobin and primary liver cancer,” The Lancet, vol. 2, no. 7572, p. 833, 1968.View at: Google Scholar
M. C. Kew, “Alpha-fetoprotein,” in Modern Trends in Gastroenterology, A. E. Read, Ed., vol. 5, p. 91, Butterworths, London, UK, 1975.View at: Google Scholar
H. F. Deutsch, “Chemistry and biology of α-fetoprotein,” Advances in Cancer Research, vol. 56, pp. 253–312, 1991.View at: Google Scholar
G. J. Mizejewski, “Alpha-fetoprotein structure and function: relevance to isoforms, epitopes, and conformational variants,” Experimental Biology and Medicine, vol. 226, no. 5, pp. 377–408, 2001.View at: Google Scholar
A. A. Terentiev and N. T. Moldogazieva, “Structural and functional mapping of α-fetoprotein,” Biochemistry, vol. 71, no. 2, pp. 120–132, 2006.View at: Publisher Site | Google Scholar
E. Ruoslahti and M. Seppälä, “Studies of carcino-fetal proteins. 3. Development of a radioimmunoassay for -fetoprotein. Demonstration of -fetoprotein in serum of healthy human adults,” International Journal of Cancer, vol. 8, no. 3, pp. 374–383, 1971.View at: Google Scholar
R. P. Liang, G. H. Yao, L. X. Fan, and J. D. Qiu, “Magnetic Fe3O4@Au composite-enhanced surface plasmon resonance for ultrasensitive detection of magnetic nanoparticle-enriched α-fetoprotein,” Analytica Chimica Acta, vol. 737, no. 6, pp. 22–28, 2012.View at: Google Scholar
S. Inouye and J. Sato, “Purification of histidine-tagged aequorin with a reactive cysteine residue for chemical conjugations and its application for bioluminescent sandwich immunoassays,” Protein Expression and Purification, vol. 83, no. 2, pp. 205–210, 2012.View at: Google Scholar
B. Zhang, D. Tang, B. Liu, H. Chen, Y. Cui, and G. Chen, “GoldMag nanocomposite-functionalized graphene sensing platform for one-step electrochemical immunoassay of alpha-fetoprotein,” Biosensors and Bioelectronics, vol. 28, no. 1, pp. 174–180, 2011.View at: Publisher Site | Google Scholar
X. Huang and J. Ren, “Gold nanoparticles based chemiluminescent resonance energy transfer for immunoassay of alpha fetoprotein cancer marker,” Analytica Chimica Acta, vol. 686, no. 1-2, pp. 115–120, 2011.View at: Publisher Site | Google Scholar
J. Bruix, M. Sherman, J. M. Llovet et al., “Clinical management of hepatocellular carcinoma. Conclusions of the barcelona-2000 EASL conference,” Journal of Hepatology, vol. 35, no. 3, pp. 421–430, 2001.View at: Publisher Site | Google Scholar
J. Ylikotila, L. Välimaa, M. Vehniäinen, H. Takalo, T. Lövgren, and K. Pettersson, “A sensitive TSH assay in spot-coated microwells utilizing recombinant antibody fragments,” Journal of Immunological Methods, vol. 306, no. 1-2, pp. 104–114, 2005.View at: Publisher Site | Google Scholar
S. Inouye and J. I. Sato, “Comparison of luminescent immunoassays using biotinylated proteins of aequorin, alkaline phosphatase and horseradish peroxidase as reporters,” Bioscience, Biotechnology and Biochemistry, vol. 72, no. 12, pp. 3310–3313, 2008.View at: Publisher Site | Google Scholar
S. Inouye and J. I. Sato, “Recombinant aequorin with a reactive cysteine residue for conjugation with maleimide-activated antibody,” Analytical Biochemistry, vol. 378, no. 1, pp. 105–107, 2008.View at: Publisher Site | Google Scholar
B. Y. Wu, H. F. Wang, J. T. Chen, and X. P. Yan, “Fluorescence resonance energy transfer inhibition assay for α-fetoprotein excreted during cancer cell growth using functionalized persistent luminescence nanoparticles,” Journal of the American Chemical Society, vol. 133, no. 4, pp. 686–688, 2011.View at: Publisher Site | Google Scholar
B. Zhang, X. Zhang, H. H. Yan, S. J. Xu, D. H. Tang, and W. L. Fu, “A novel multi-array immunoassay device for tumor markers based on insert-plug model of piezoelectric immunosensor,” Biosensors and Bioelectronics, vol. 23, no. 1, pp. 19–25, 2007.View at: Publisher Site | Google Scholar
C. Ding, F. Zhao, R. Ren, and J. M. Lin, “An electrochemical biosensor for α-fetoprotein based on carbon paste electrode constructed of room temperature ionic liquid and gold nanoparticles,” Talanta, vol. 78, no. 3, pp. 1148–1154, 2009.View at: Publisher Site | Google Scholar
R. P. Liang, Z. X. Wang, L. Zhang, and J. D. Qiu, “A label-free amperometric immunosensor for alpha-fetoprotein determination based on highly ordered porous multi-walled carbon nanotubes/silica nanoparticles array platform,” Sensors and Actuators B, vol. 166-167, pp. 569–575, 2012.View at: Google Scholar
N. Gan, L. Jia, and L. Zheng, “A novel sandwich electrochemical immunosensor based on the DNA-derived magnetic nanochain probes for alpha-fetoprotein,” Journal of Automated Methods and Management in Chemistry, vol. 2011, Article ID 957805, 7 pages, 2011.View at: Publisher Site | Google Scholar
B. Su, J. Tang, J. Huang et al., “Graphene and nanogold-functionalized immunosensing interface with enhanced sensitivity for one-step electrochemical immunoassay of alpha-fetoprotein in human serum,” Electroanalysis, vol. 22, no. 22, pp. 2720–2728, 2010.View at: Publisher Site | Google Scholar
Q. Liu, M. Han, J. Bao, X. Jiang, and Z. Dai, “CdSe quantum dots as labels for sensitive immunoassay of cancer biomarker proteins by electrogenerated chemiluminescence,” Analyst, vol. 136, no. 24, pp. 5197–5203, 2011.View at: Google Scholar
J. Lin, Y. Zhao, Z. Wei, and W. Wang, “Chemiluminescence immunoassay based on dual signal amplification strategy of Au/mesoporous silica and multienzyme functionalized mesoporous silica,” Materials Science and Engineering B, vol. 176, no. 18, pp. 1474–1478, 2011.View at: Google Scholar
Y. S. Kim, S. H. Kim, J. K. Seong, B. S. Lee, H. Y. Jeong, and K. S. Song, “Gastric yolk sac tumor: a case report and review of the literature,” Korean Journal of Internal Medicine, vol. 24, no. 2, pp. 143–146, 2009.View at: Publisher Site | Google Scholar
D. Ball, E. Rose, and E. Alpert, “Alpha-fetoprotein levels in normal adults,” American Journal of the Medical Sciences, vol. 303, no. 3, pp. 157–159, 1992.View at: Google Scholar
P. Sizaret, N. Martel, A. Tuyns, and S. Reynaud, “Mean α fetoprotein values of 1,333 males over 15 years by age groups,” Digestion, vol. 15, no. 2, pp. 97–103, 1977.View at: Google Scholar
D. Bader, A. Riskin, O. Vafsi et al., “Alpha-fetoprotein in the early neonatal period—a large study and review of the literature,” Clinica Chimica Acta, vol. 349, no. 1-2, pp. 15–23, 2004.View at: Publisher Site | Google Scholar
X. W. Wang and B. Xu, “Stimulation of tumor-cell growth by alpha-fetoprotein,” International Journal of Cancer, vol. 75, no. 4, pp. 596–599, 1998.View at: Google Scholar
H. Ju, G. Yan, F. Chen, and H. Chen, “Enzyme-linked immunoassay of α-1-fetoprotein in serum by differential pulse voltammetry,” Electroanalysis, vol. 11, no. 2, pp. 124–128, 1999.View at: Google Scholar
P. P. Minghetti, M. E. Harper, E. Alpert, and A. Dugaiczyk, “Chromosomal structure and localization of the human α-fetoprotein gene,” Annals of the New York Academy of Sciences, vol. 417, pp. 1–12, 1983.View at: Google Scholar
D. H. Bellet, J. R. Wands, K. J. Isselbacher, and C. Bohuon, “Serum α-fetoprotein levels in human disease: perspective from a highly specific monoclonal radioimmunoassay,” Proceedings of the National Academy of Sciences of the United States of America, vol. 81, no. 12, pp. 3869–3873, 1984.View at: Google Scholar
D. W. Chan, M. Kelsten, R. Rock, and D. Bruzek, “Evaluation of a monoclonal immunoenzymometric assay for alpha-fetoprotein,” Clinical Chemistry, vol. 32, no. 7, pp. 1318–1322, 1986.View at: Google Scholar
L. Mannings, S. Trow, J. Newman, B. Nix, and C. Evans, “Interference in the autoDELFIA hAFP immunoassay and effect on second-trimester Down’s syndrome screening,” Annals of Clinical Biochemistry, vol. 48, no. 5, pp. 438–440, 2011.View at: Google Scholar