Monoclonal antibodies as a tool for structure-function studies of the MDR1-P-glycoprotein

P-glycoprotein is considered one of the most important member of the rapidly growing superfamily of integral proteins known as the ATP-binding cassette (ABC) which in human also include several other multidrug resistance membrane proteins (i.e., MRP), the product of the cystic fibrosis gene, the TAP-1/TAP2 peptide transporters encoded by the major histocompatibility complex genes and the gene encoding for breast cancer resistance protein (BCRP) also known as MXR1 (mitoxantrone resistance protein). Many monoclonal antibodies (MAbs) reacting with distinct P-glycoprotein domains have been isolated and used to study the molecular organization and cellular functions of this ABC protein. MAbs have been used for multidrug resistance (mdr) gene cloning, delineation of the secondary and tertiary structure of P-glycoprotein and molecular analysis of the mechanisms involved in substrate recognition and transport. The immunodetection of the distinct products of the mdr gene family in normal and malignant cells and tissues has greatly contributed to the understanding of the physiological role of P-glycoprotein and its possible involvement in the refractory of tumors to chemotherapy. The present article deals with the immunological methods used for the structure-function studies of the P-glycoprotein. After introducing the basic structural features of this ABC transporter, the antibody based-approach is discussed with aiming to furnishing methodological perspectives for further investigations of the physiological role of P-glycoprotein and the multidrug resistance phenomenon.     

Assessing multidrug resistance protein 1-mediated function in cancer cell multidrug resistance by scanning electrochemical microscopy and flow cytometry

Cancer cell multidrug resistance is a molecular signature that highly influences the outcome of chemotherapy treatment and for which there is currently no robust method to monitor in vitro its activity. Herein, we demonstrate that ferrocenemethanol (FcCH(2)OH) and its oxidized form ([FcCH(2)OH](+)) affect the redox state of cancer cells. Specifically, the interaction of FcCH(2)OH with the glutathione couple (GSH/GSSG) is shown in human adenocarcinoma cervical cancer cells HeLa and a multidrug resistant variant overexpressing the multidrug resistant associated protein 1 (MRP1) using bioanalytical techniques, such as flow cytometry and fluorescence microscopy. It is further demonstrated that the differential response to FcCH(2)OH in multidrug-resistant cells is in part due to MRP1's unspecific efflux. Scanning electrochemical microscopy confirmed the interaction between FcCH(2)OH and the cells, and the differential response was observed to depend on MRP1 expression. This newly established relation between FcCH(2)OH/[FcCH(2)OH](+), GSH/GSSG and multidrug resistance in human cancer cells enables than the acquisition of scanning electrochemical microscopy images.     

Detection of recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection

Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of recombinant hirudin (r-hirudin), a specific and reproducible analysis method is required. Capillary electrophoresis (CE) is rapidly becoming an important procedure for the analysis of biological molecules. Recently, CE combined with immunoassay has emerged as a new analytical technique. CE-based immunoassay (CEIA) is a sensitive and specific method combining laser-induced fluorescence (LIF) and immunoassay. Therefore, in this study, we specifically investigated fluorescence labeling and determination of r-hirudin by CEIA with a LIF detector using labeled r-hirudin and polyclonal antibody. r-Hirudin was labeled with fluorescein isothiocyanate (FITC). FITC-labeled r-hirudin was purified using high-performance liquid chromatography (HPLC). The method is based on preincubation of r-hirudin and antibody for 20 min, followed by CE analysis using an uncoated capillary. Free and bound r-hirudin were separated within 5 min using CE with high reproducibility. This study demonstrated that the CEIA method could be applied to quantitative analysis of r-hirudin in biological fluids.     

单分子力谱研究活细胞上多药耐药相关蛋白1的表达

采用原子力显微镜的单分子力谱(SMFM)技术研究了多药耐药相关蛋白1(MRP1)与其抗体间的相互作用, 并考察了人舌癌细胞系TCA8113经高剂量平阳霉素(BLM)反复间歇诱导前后细胞表面MRP1的表达差异. 实验结果表明, MRP1与其抗体之间存在特异性相互作用力, 当针尖运动速率为2.5 μm/s时, 作用力大小约为(182±35) pN; 而且药物诱导后MRP1在人舌癌细胞上的表达明显增强. 本工作为了解活细胞水平上MRP1的表达提供了新方法, 有助于肿瘤细胞多药耐药性(MDR)的研究.     

Antitumor efficacy of doxorubicin released from crosslinked nanoparticulate chondroitin sulfate/chitosan polyelectrolyte complexes

It is demonstrated that nanoparticulate PEC with a crosslinked shell sustains DOX release and increases DOX activity against cancer cells. CSMA was synthesized to prepare PEC with chitosan. The double bonds among CSMA were used to form a shell crosslink. The released DOX from DOX-loaded PECs against human cancer KB cells and A549 cells were qualitatively traced by confocal laser scanning microscopy and flow cytometry, and quantitatively measured by capillary electrophoresis. All the results implied the DOX-loaded PEC with a crosslinked shell had the best anti-cancer potency of free DOX and the DOX-loaded PEC prepared from pure chondroitin sulfate and chitosan in both the cell lines.     

Drug Self‐Delivery Systems Based on Hyperbranched Polyprodrugs towards Tumor Therapy

Amphiphilic hyperbranched polyprodrugs (DOX‐S‐S‐PEG) with drug repeat units in hydrophobic core linked by disulfide bonds were developed as drug self‐delivery systems for cancer therapy. The hydroxyl groups and the amine group in doxorubicin (DOX) were linked by 3,3′‐dithiodipropanoic acid as hydrophobic hyperbranched cores, then amino‐terminated polyethylene glycol monomethyl ether (mPEG‐NH₂) as hydrophilic shell was linked to hydrophobic cores to form amphiphilic and glutathione (GSH)‐responsive micelle of hyperbranched polyprodrugs. The amphiphilic micelles can be disrupted under GSH (1 mg mL^?1) circumstance. Cell viability of A549 cells and 293T cells was evaluated by CCK‐8 and Muse Annexin V & Dead Cell Kit. The disrupted polyprodrugs maintained drug activity for killing tumor cells. Meanwhile, the undisrupted polyprodrugs possessed low cytotoxicity to normal cells. The cell uptake experiments showed that the micelles of DOX‐S‐S‐PEG were taken up by A549 cells and distributed to cell nuclei. Thus, the drug self‐delivery systems with drug repeat units in hydrophobic cores linked by disulfide bonds showed significant special advantages: 1) facile one‐pot synthesis; 2) completely without toxic or non‐degradable polymers; 3) DOX itself functions as fluorescent labeled molecule and self‐delivery carrier; 4) drug with inactive form in hyperbranched cores and low cytotoxicity to normal cells. These advantages make them excellent drug self‐delivery systems for potential high efficient cancer therapy.     

Determination of metolcarb in food by capillary electrophoresis immunoassay with a laser-induced fluorescence detector

A capillary electrophoresis immunoassay (CEIA) was developed for the determination of trace metolcarb (MTMC) in food. The method was based on the competitive reactions between fluorescently labeled MTMC tracer and free MTMC with a limited amount of anti-MTMC antibody and the separation and determination by CE with LIF detector. A fluorescent reagent, FITC was labeled on MTMC to construct an immunofluorescent probe. CEIA experimental parameters such as the pH value and concentration of the running buffer and separation voltage as well as incubation time were systematically investigated. Under the optimized conditions, fluorescently labeled antigen and antibody bound could be well separated within 3 min using Na?B?O?/NaH?PO? buffer (20:10 mmol/L, pH 9.0) for background electrolyte, 20 kV for the separation voltage, and 20°C for the column temperature. The linear range of the method was 0.25-50.0 μg/L with LOD 0.07 μg/L. The RSD for relative migration time and relative fluorescence intensity ratio were 2.90% (intraday) and 4.73% (intraday), respectively. The proposed method has been applied to determine the residue of MTMC in food samples with the satisfactory recovery.     

Multidrug resistance and cancer: the role of the human ABC transporter ABCG2

A variety of human cancers become resistant or are intrinsically resistant to treatment with conventional chemotherapy, a phenomenon called multidrug resistance. This broad-based resistance results in large part, but not solely, from overexpression of members of the ATP-binding cassette (ABC) superfamily of membrane transporters, including P-glycoprotein, various members of the multidrug resistance associated proteins (MRPs), and ABCG2, also known as MXR1, BCRP, and ABCP. When overexpressed in cell lines, ABCG2 has the ability to confer high levels of resistance to anthracyclines, mitoxantrone, bisantrene, and the camptothecins topotecan and SN-38. This review focuses on the discovery, the biochemistry and the normal physiology of human ABCG2, a novel ABC half transporter expressed abundantly in placenta, as well as in liver, intestine and stem cells. ABCG2 may serve a protective function by preventing toxins from entering cells as well as potentially playing a role in regulating stem cell differentiation. We also discuss the involvement of ABCG2 in multidrug resistance in cancer, especially with regard to acute myeloid leukemia. The mechanism by which substrates are recognized by ABCG2 and how the energy of ATP hydrolysis is transduced into transport remain elusive. A complete understanding of the mechanism and biological function of ABCG2 will be important for understanding its normal physiology as well as potentially for the development of novel chemotherapeutic treatment strategies.     

Capillary Electrophoresis Based Immunoassay for Monoclonal Antibody with Diode Laser Induced Fluorescence Detection

ABSTRACT

A capillary electrophoresis based immunoassay (CEIA) for monoclonal antibody using diode laser induced fluorescence (LIF) detection was described. A direct assay for monoclonal anti-BSA in mouse serum was used as a model. BSA was labeled with Cy5 and used as the immunoreagent. The 635 nm line of a diode laser was used as the excitation source for LIF detection. The calibration curve for anti-BSA in mouse serum had a linear dynamic range of 4-40 nM. The concentration limit of detection (LOD) was 1.2 nM. Incubation time and CE conditions such as buffer concentration, pH and separation voltage were optimized, and the performances of different lasers as excitation sources were also compared.     

Affinity protection chromatography for efficient labeling of antibodies for use in affinity capillary electrophoresis

Capillary electrophoresis immunoassay (CEIA) is shown to be substantially more sensitive to the antibody (Ab) reagent quality than are immunosorbent methods such as enzyme-linked immunosorbent assays (ELISA). Cyanine 5 (Cy5)-labeled monoclonal anti-ovalbumin (mAb*) was inactive for CEIA of ovalbumin (Ov), yet was functional in ELISA for Ov. ELISA showed the mAb* was at least ten times less active, accounting for the poor CEIA performance. Labeled polyclonal Ab was inactive for a dye to protein ratio greater than 1.6. An affinity protection chromatography procedure (APC) was developed for Ab labeling, which avoided degradation of the Ab binding site. Ov was covalently bound to cyanogen bromide activated cellulose gel in a column, and used to capture the Ab. The coupling efficiency for Ov to the gel was 74-97%, Ab could then be bound with 95-100% efficiency, and Ab* was recovered in 50% yield following labeling on the column. This procedure was performed successfully in three different laboratories, indicating the robustness of the optimized APC synthetic method. No inactive Ab* could be detected in the APC product. The CEIA detection limit for ovalbumin using APC labeled mAb was 173 nM, when [Ab*] was fixed at 163 nM. The association constants of mAb and mAb* were determined by CEIA.     

Micellar electrokinetic chromatographic analysis for in vitro accumulation of anthracyclines enhanced by inhibitors of cell membrane transporter-proteins in cancer cells

Cell membrane transporter-proteins have been partly implicated in lowering the accumulation of drugs in cancer cells, leading to multidrug resistance (MDR). Two cancer cell lines, A549 and RDES, were continuously exposed to subclinical concentration (250 nM) of anthracyclines and micellar electrokinetic chromatography was used to investigate their in vitro accumulation after treatment with inhibitors of membrane transporter-proteins. The four anthracylines [doxorubicin (DOX), epirubicin (EPI), daunorubicin (DNR), and idarubicin (IDA)] were separated within a short analysis time of less than 15 min in borate buffer (80 mM, pH 9.22) containing sodium taurodeoxycholate (35 mM), 2-hydroxypropyl-γ-cyclodextrin (3.5% wt/v), and sodium dodecylsulfate (20 mM). Laser-induced fluorescence was used for detection of the anthracyclines. Three inhibitors, verapamil, cyclosporine A and probenecid, were examined by adding each inhibitor independently or two inhibitors simultaneously to the culture medium. It was found that independent use of each inhibitor leads to more efficient accumulation than combined use of verapamil and probenecid. In addition, the results show that effect of inhibitors on the accumulation of anthracyclines depended on type of cell: in RDES, inhibitors enhanced accumulation of all four anthracyclines, while in A549, inhibitors showed different accumulation behavior for each anthracycline. Generally higher accumulation of anthracyclines was observed in RDES cells than A549, as evidenced by dead cells (7-16%) after 24 h of continuous exposure to subclinical concentration.     

Block copolymer micelles conjugated with anti‐EGFR antibody for targeted delivery of anticancer drug

Antiepidermal growth factor receptor antibody (anti‐EGFR antibody) was conjugated with the block copolymer micelle based on poly(ethylene glycol) (PEG) and poly(ε‐caprolactone) (PCL) for active targeting to EGFR overexpressing cancer cells. Doxorubicin (DOX) was encapsulated in the core of the block copolymer (MePEG‐b‐PCL) micelle (DOX‐micelle). The mean diameters of the DOX‐micelle and the anti‐EGFR‐PEG‐b‐PCL copolymer micelles loaded with DOX (DOX‐anti‐EGFR‐micelle) were about 25 and 31 nm, respectively. The RKO human colorectal cancer cells expressing moderate degree of EGFR were incubated with free DOX, DOX‐micelle, or DOX‐anti‐EGFR‐micelle to study the distribution of DOX in the cells. When cells were incubated with free DOX, moderate degree of DOX fluorescence was observed in the nuclei. In the cells treated with DOX‐micelle, the DOX fluorescence intensity in the cytoplasm was much greater than that in the nuclei. On the other hand, the nuclei of the cells treated with DOX‐anti‐EGFR‐micelle exhibited DOX fluorescence intensity similar to that in the cytoplasm. The cytotoxicity of DOX‐anti‐EGFR‐micelle to induce apoptosis in RKO cells was significantly greater than that of free DOX or DOX‐micelle. These results demonstrated that the presence of anti‐EGFR antibody on the DOX‐micelle surface (DOX‐anti‐EGFR‐micelle) increased the internalization of the DOX‐micelle and nuclear accumulation of DOX, and enhanced the DOX‐induced cell death. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 7321–7331, 2008     

Development of a double-layer microfluidic chip with flow medium for chemotherapy resistance analysis of lung cancer

Integration and miniaturization are main advantages of microchip-based systems. Vertical integration of the multiple operations within a multiple-layer chip is expected to satisfy the urgent demand for high-throughput and large-scale applications. This study aimed at establishing a double-layer chip to integrate the operations including the cell culture, the identification of the protein and the detection of the cell viability onto a platform systematically and supplied with flow fresh medium continuously via a syringe pump to mimic the microenvironment in vivo. With this device, human non-small cell lung cancer cell line (SPCA-1) was cultured well; the expression and the activity of multidrug resistance-associated protein (MRP1) were detected by immunofluorescence assay for the cells pretreated with or without MK-571, a known inhibitor of MRP1; apoptosis percentages were assayed for the cells after being treated by the anticancer drug etoposide (VP-16). The results demonstrated that the function of the MRP1 was inhibited by MK-571, and the percentage of apoptotic for the cells pretreated with MK-571 was higher than that of the control (38.2±2.5% versus 12.3±0.85%, p<0.005). All these indicated that the new device could provide a suitable condition for cell culture and functional analysis in biomedical research, and MK-571 is an effective inhibitor of MRP1 associated with the viability of SPCA-1 cell line treated by VP-16.     

Measurement of intracellular accumulation of anthracyclines in cancerous cells by direct injection of cell lysate in MEKC/LIF detection

Anthracyclines are chemotherapeutic drugs that are broadly used in the treatment of various types of solid cancers and leukemia. Herein, we report on a novel analytical method for intracellular accumulation of anthracyclines using MEKC/LIF detection. An aqueous separation system permitted the injection of cell lysates directly into the capillary. The MEKC migrating solution was made up of borate buffer at pH 9.22 containing sodium taurodeoxycholate, (2‐hydroxypropyl)‐γ‐CD, and SDS. The anthracyclines, Doxorubicin (DOX) and epirubicin (EPI) were detected by LIF using a Nd:YAG laser (532 nm) or an argon ion laser (488 nm) for excitation. Two cell lines, human humerus tumor cells (RDES) and human lung tumor cells (A549), were treated with a mixture of the two anthracyclines for fixed periods of time, and then intracellular concentrations were determined by injecting cell lysates directly. Recovery values of 96.0–100.8% were obtained for DOX and EPI. Reproducibility quantified by RSD was less than 3.9% intraday and 6.7% interday at concentrations ranging between 50 and 500 nM. The uptake of EPI was found to be slightly less than that of DOX for A549, but higher levels of EPI were observed in RDES. Intracellular accumulation of anthracyclines was greater in RDES than in A549, but both types of cells excreted anthracyclines after 12 h. These results indicate that MEKC with an aqueous medium is useful for investigating intracellular uptake and accumulation of drugs, since cell lysates can be used directly with no pretreatment such as deproteination or solvent extraction of analytes.     

Doxorubicin-Conjugated Zinc Oxide Nanoparticles,Biogenically Synthesised Using a Fungus Aspergillus niger,Exhibit High Therapeutic Efficacy against Lung Cancer Cells

This study reports the therapeutic effectiveness of doxorubicin-conjugated zinc oxide nanoparticles against lung cancer cell line. The zinc oxide nanoparticles (ZnONPs) were first synthesised using a fungus, isolated from air with an extraordinary capability to survive in very high concentrations of zinc salt. Molecular analysis based on 18S rRNA gene sequencing led to its identification as Aspergillus niger with the NCBI accession no. {"type":"entrez-nucleotide","attrs":{"text":"OL636020","term_id":"2155690654"}}OL636020. The fungus was found to produce ZnONPs via the reduction of zinc ions from zinc sulphate. The ZnONPs were characterised by various biophysical techniques. ZnONPs were further bioconjugated with the anti-cancer drug doxorubicin (DOX), which was further confirmed by different physical techniques. Furthermore, we examined the cytotoxic efficacy of Doxorubicin-bioconjugated-ZnONPs (DOX-ZnONPs) against lung cancer A549 cells in comparison to ZnONPs and DOX alone. The cytotoxicity caused due to ZnONPs, DOX and DOX-ZnONPs in lung cancer A549 cells was assessed by MTT assay. DOX-ZnONPs strongly inhibited the proliferation of A549 with IC50 value of 0.34 μg/mL, which is lower than IC50 of DOX alone (0.56 μg/mL). Moreover, DOX-ZnONPs treated cells also showed increased nuclear condensation, enhanced ROS generation in cytosol and reduced mitochondrial membrane potential. To investigate the induction of apoptosis, caspase-3 activity was measured in all the treated groups. Conclusively, results of our study have established that DOX-ZnONPs have strong therapeutic efficacy to inhibit the growth of lung cancer cells in comparison to DOX alone. Our study also offers substantial evidence for the biogenically synthesised zinc oxide nanoparticle as a promising candidate for a drug delivery system.     

Loading-free supramolecular organic framework drug delivery systems (sof-DDSs)for doxorubicin: normal plasm and multidrug resistant cancer cell-adaptive delivery and release

Four water-soluble porous supramolecular organic framework drug delivery systems(sof-DDSs)have been used to adsorb doxorubicin(DOX)in water at physiological pH of 7.4,which is driven exclusively by hydrophobicity.The resulting complexes DOX@SOFs are formed instantaneously upon dissolving the components in water.The drug-adsorbed sof-DDSs can undergo plasm circulation with important maintenance of the drug and overcome the multidrug resistance of human breast MCF-7/Adr cancer cells. DOX is released readily in the cancer cells due to the protonation of its amino group in the acidic medium of cancer cells.In vitro and in vivo experiments reveal that the delivery of SOF-a-d remarkably improve the cytotoxicity of DOX for the MCF-7/Adr cells and tumors,leading to 13-19-fold reduction of the IC50 values as compared with that of DOX.This new sof-DDSs strategy omits the indispensable loading process required by most of reported nano-scaled carriers for neutral hydrophobic chemotherapeutic agents,and thus should be highly valuable for future development of low-cost delivery systems.     

Quantitative study of cellular heterogeneity in doxorubicin uptake and its pharmacological effect on cancer cells

Cellular heterogeneity in doxorubicin (DOX) uptake and its relationship with pharmacological effect on cancer cells were quantitatively investigated for the first time. An in vitro experimental model was established by treating human leukemia K562 and breast cancer MCF‐7 cells with different schedules of DOX with or without surface P‐glycoprotein (P‐gp) inhibitor verapamil (VER). The cellular heterogeneity in DOX uptake was quantitatively examined by single‐cell analysis using capillary electrophoresis coupled with laser‐induced fluorescence detection. The corresponding cytotoxic effect was tested by cellular morphology, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium and flow cytometry assays. The expression of cellular membrane surface P‐gp was determined by flow cytometry. Results showed that the cellular heterogeneity exists in DOX uptake. The single‐high DOX schedule leads to lower uptake heterogeneity and higher mean drug uptake. The cellular heterogeneity in DOX uptake was found to be negatively correlated with drug cytotoxicity and surface P‐gp expression, with r = ?0.7680 to ~ ?0.9587. VER reduces the cellular variation in DOX uptake, suggesting that surface P‐gp may be one of the causes of the cellular heterogeneity in DOX uptake. This research demonstrates the importance of quantitative study of cellular heterogeneity in drug uptake and its potential application in drug schedule design, response prediction and therapy modulation. Copyright © 2014 John Wiley & Sons, Ltd.     

Analysis of oxidized multi-walled carbon nanotubes in single K562 cells by capillary electrophoresis with laser-induced fluorescence

Short oxidized multi-walled carbon nanotubes (CNT) were derivatized with fluorescein isothiocyanate (FITC). Capillary electrophoresis coupled with laser-induced fluorescence (CE–LIF) was then used to separate and detect the fluorescently labeled carbon-nanotube probes (CNTP) in multidrug-resistant cells (K562A) and the parent cells (K562S). Greater expression of P-glycoprotein in K562A cells than in K562S cells was confirmed by use of anti-P-glycoprotein antibody and flow-cytometric analysis. Analyses of CNTP in both cell lines using both CE–LIF and flow cytometry showed that CNTP could traverse the cellular membrane without being pumped out by P-glycoprotein. The CNTP distributed in both cell lines was analyzed at the single cell level and the results were compared with those from analysis of ten cells and of the lysate from bulk cells. The results revealed the CE–LIF method could be used for quantitative analysis of CNT in single cells in studies of drug delivery and multidrug resistance.      

Immunoassays using capillary electrophoresis laser induced fluorescence detection for DNA adducts

Human DNA is exposed to a variety of endogenous and environmental agents that may induce a wide range of damage. The critical role of DNA damage in cancer development makes it essential to develop highly sensitive and specific assays for DNA lesions. We describe here ultrasensitive assays for DNA damage, which incorporate immuno-affinity with capillary electrophoresis (CE) separation and laser induced fluorescence (LIF) detection. Both competitive and non-competitive assays using CE/LIF were developed for the determination of DNA adducts of benzo[a]pyrene diol epoxide (BPDE). A fluorescently labeled oligonucleotide containing a single BPDE adduct was synthesized and used as a fluorescent probe for competitive assay. Binding between this synthetic oligonucleotide and a monoclonal antibody (MAb) showed both 1:1 and 1:2 complexes between the MAb and the oligonucleotide. The 1:1 and 1:2 complexes were separated by CE and detected with LIF, revealing binding stoichiometry information consistent with the bidentate nature of the immunoglobulin G antibody. For non-competitive assay, a fluorescently labeled secondary antibody fragment F(ab′)₂ was used as an affinity probe to recognize a primary antibody that was specific for the BPDE-DNA adducts. The ternary complex of BPDE-DNA adducts with the bound antibodies was separated from the unbound antibodies using CE and detected with LIF for quantitation of the DNA adducts. The assay was used for the determination of trace levels of BPDE-DNA adducts in human cells. Analysis of cellular DNA from A549 human lung carcinoma cells that were incubated with low doses of BPDE (32 nM–1 μM) showed a clear dose–response relationship. BPDE is a potent environmental carcinogen, and the ultrasensitive assays for BPDE-DNA adducts are potentially useful for monitoring human exposure to this carcinogen and for studying cellular repair of DNA damage.     

Direct immunoassay of estrone by capillary electrophoresis with laser-induced fluorescence detection

An immunoassay for estrone (E(1)) in women's serum, based on the competitive reaction between fluorescein-labeled complete antigen and E(1) with limited amount of anti-estrone monoclonal antibody is described. A thermally reversible hydrogel, poly-N-isopropylacrylamide (pNIPA), was added to the buffer to improve the reproducibility. With a laser-induced fluorescence (LIF) detector, the capillary electrophoretic immunoassay (CEIA) can be applied to determine E(1) at a concentration lower than 19.6 pg/mL. The E(1) levels in ten normal women's serum were measured at the range of 118.6-222.0 pg/mL.