Identification and quantification of eight <Emphasis Type="Italic">Listeria monocytogene</Emphasis> serotypes from <Emphasis Type="Italic">Listeria spp.</Emphasis> using a gold nanoparticle-based lateral flow assay

A lateral flow assay for rapid, simple and efficient determination of L. monocytogenes is presented. A monoclonal antibody (mAb) 1C1 against the peptide from P60 protein of L. monocytogenes was prepared and labeled with gold nanoparticles (AuNPs). The mAb 1C1 was paired with the mAb 10E7 against the P60 protein of all the Listeria spp. and used as a capture bioligand in a lateral flow assay. The AuNP-based strip test can detect the supernatant of eight common L. monocytogenes serotypes including 1/2a, 1/2b, and 4b with an equivalent detection limit of 3.7 × 10⁶ CFU?mL^?1 but does not detect four other Listeria spp. (L. ivanovii, L. innocua, L. welshimeri, and L. grayi). There was no cross-reactivity with six other Gram-negative and Gram-positive bacteria. The method was applied to the quantification of L. monocytogenes species in spiked milk samples within 13 h.

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Graphical abstract Monoclonal antibody (mAb) 1C1 was prepared against the peptide from P60 protein of Listeria monocytogenes. The gold nanoparticle (AuNP) based strip sensor detects all the eight tested Listeria monocytogenes serotypes.

     

Epitope mapping of 7S cashew antigen in complex with antibody by solution‐phase H/D exchange monitored by FT‐ICR mass spectrometry

The potential epitope of a recombinant food allergen protein, cashew Ana o 1, reactive to monoclonal antibody, mAb 2G4, has been mapped by solution‐phase amide backbone H/D exchange (HDX) monitored by Fourier transform ion cyclotron resonance mass spectrometry (FT‐ICR MS). Purified mAb 2G4 was incubated with recombinant Ana o 1 (rAna o 1) to form antigen:monoclonal antibody (Ag:mAb) complexes. Complexed and uncomplexed (free) rAna o 1 were then subjected to HDX‐MS analysis. Five regions protected from H/D exchange upon mAb binding are identified as potential conformational epitope‐contributing segments. Copyright © 2015 John Wiley & Sons, Ltd.     

Quantification of an intact monoclonal antibody, rituximab, by (RP)HPLC/DAD in compliance with ICH guidelines

We studied the quantification of an intact therapeutic monoclonal antibody (mAb), rituximab (RTX), using (reverse-phase) high-performance liquid chromatography with diode array detection ((RP)HPLC/DAD). To this end, we developed a chromatographic method and validated it as stability-indicating in accordance with the International Conference on Harmonization guidelines (ICH). A 300-Å C8 column (250 mm?×?4.6 mm, 5 μm) was used to perform the analysis, and the temperature was maintained at 70 °C. Although only one mAb was analyzed, it was necessary to apply a gradient to elute it with a complex organic mixture. Chromatograms were registered at several wavelengths, with λ?=?214 nm employed for quantification purposes. The method was developed to quantify marketed RTX under typical hospital administration conditions. Further dilution was avoided in order to prevent additional mAb modification, and in this way the method was shown to be linear from 60 to 5000 mg/L. The precision of the method (repeatability and intermediate precision, estimated as the relative standard deviation, RSD %), was less than 1.0 %. Accuracy, specificity, robustness, and system suitability were also evaluated as specified in the ICH guidelines. We conducted a comprehensive chromatographic analysis by submitting RTX to several informative stress conditions. These forced degradation studies were conducted for two reasons: to estimate the specificity of the method, and to evaluate the robustness of the mAb formulation against external stress factors when handling it in preparation for administration. Thus, we investigated the effects of acid, base, oxidation, ionic strength, temperature, and UV light. Although a slight modification to the intact mAb could not be distinguished chromatographically in the stress studies we conducted, the procedure proposed here to evaluate peak purity enabled us to detect it with a satisfactory level of confidence. The proposed method could therefore be considered stability-indicating for quantyfying the intact mAb since it is qualified to detect its degradation/modification. Finally, the method was used to evaluate RTX in a long-term stability study performed under hospital conditions of use.     

Preparation of Anti-Glycyrrhetinic Acid Monoclonal Antibody for Application in an Indirect Competitive Enzyme-Linked Immunosorbent Assay

Glycyrrhetinic acid is a major metabolite of glycyrrhizin, which is one of the main components of licorice roots and is considered to be one of the pharmacologically active substances in licorice. A new hybridoma cell line, named G-2A6, was generated by fusing mouse myeloma cells and splenocytes, which were immunized using glycyrrhetinic-acid–keyhole limpet hemocyanin to produce a monoclonal antibody (mAb) against glycyrrhetinic acid. Using the anti-glycyrrhetinic acid mAb, we attempted to develop a simple, rapid, and highly sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA). The developed icELISA had a range from 3.91 to 125?ng/mL with low coefficients of variation (less than 5%) and demonstrated a high recovery rate of glycyrrhetinic acid spiked into licorice powder (average?=?101.76%). In addition, the icELISA could determine the glycyrrhetinic acid concentration in glycyrrhetinic-acid-spiked human serum with simple pretreatment, which suggests that the developed ELISA system using anti-glycyrrhetinic acid mAb would prove to be an effective and useful tool for determining glycyrrhetinic acid in various fields such as the analysis of Glycyrrhiza plants and pharmacokinetic studies of glycyrrhetinic acid during the administration of glycyrrhetinic acid, glycyrrhizin, and/or licorice-based medical agents.     

Binding properties of a monoclonal antibody against the Cry1Ab from <Emphasis Type="Italic">Bacillus Thuringensis</Emphasis> for the development of a capillary electrophoresis competitive immunoassay

Experimental work performed was aimed at the assessment of a competitive capillary electrophoresis immunoassay with laser-induced fluorescence (CEIA-LIF) detection for the determination of the Cry1Ab endotoxin from Bacillus thuringensis. The binding constant of a monoclonal antibody, raised against the insecticide protein Cry1Ab, was determined on a microplate by indirect enzyme-linked immunosorbent assay (ELISA) and compared with that obtained in-capillary under nonequilibrium separation conditions. The two binding constants appear comparable—(5.0 ± 1.2) × 10⁶ M⁻¹ and (9.06 ± 5.7) × 10⁶ M⁻¹—reflecting good preservation of the antibody binding behavior in the capillary electrophoresis format. These results allow use of a calibration curve possible between 0.2 and 150 nM of endotoxin protein, with a limit of detection of 0.5 nM (33 μg L⁻¹). Preliminary recovery experiments on maize extracts spiked with known amounts of Cry1Ab endotoxin also showed promising results in detecting the toxin in complex real matrices.     

The synthesis and cytotoxic evaluation of a series of benzodioxole substituted titanocenes

Using 6‐benzo[1,3]dioxolefulvene ( 1a ), a series of benzodioxole substituted titanocenes was synthesized. The benzyl‐substituted titanocene bis[(benzo[1,3]dioxole)‐5‐methylcyclopentadienyl] titanium (IV) dichloride ( 2a ) was synthesized from the reaction of Super Hydride with 1a . An X‐ray determined crystal structure was obtained for 2a . The ansa‐titanocene {1,2‐di(cyclopentadienyl)‐1,2‐di‐(benzo[1,3]dioxole)‐ethanediyl} titanium(IV) dichloride ( 2b ) was synthesized by reductive dimerisation of 1a with titanium dichloride. The diarylmethyl substituted titanocene bis(di‐(benzo[1,3]dioxole)‐5‐methylcyclopentadienyl) titanium(IV) dichloride ( 2c ) was synthesized by reacting 1a with the para‐lithiated benzodioxole followed by transmetallation with titanium tetrachloride. When titanocenes 2a–c were tested against pig kidney (LLC‐PK) cells inhibitory concentrations (IC₅₀) of 2.8 × 10^?4, 1.6 × 10^?4 and 7.6 × 10^?5 M , respectively, were observed. These values represent improved cytotoxicity against LLC‐PK, when compared with unsubstituted titanocene dichloride, but are not as impressive as values obtained for titanocenes previously synthesized using the above methods. Copyright © 2006 John Wiley & Sons, Ltd.     

Lactic acid production by pellet-formRhizopus oryzae in a submerged system

Monoclonal antibodies (mAbs) of the IgM isotypes were produced from mice immunized with blood forms ofTrypansoma cruzi Y strain. Characterization of the epitope recognized by one of the mAbs, 164C11, as well as the effects of this mAb on complement-mediated lysis and host cell invasion are reported. Immunocytochemical analysis showed that the mAb was reactive with various strains ofT. cruzi (Y, WSL, and Colombiana) as well as other trypanosomatids. The mAb 164C11 demonstrated a high complement-mediated lytic activity against bloodstream trypomastigotes, being more effective than chronic mouse serum. A protein with an apparent molecular weight of 72 kDa was detected by this mAb on all developmental stages ofT. cruzi. Studies using periodate and endoglycosidase treatments suggested that the epitope is not a carbohydrate and seems to be located on the parasite membrane. In addition, preliminary results are presented, suggesting that the72-kDa protein is involved in adhesion/or internalization of bloodstream trypomastigotes.     

Herba Ecliptae Protects against Hydroxyl Radical‐induced Damages to DNA and Mesenchymal Stem Cells via Antioxidant Mechanism

Herba Ecliptae (HE) is a typical Chinese herbal medicine used in China for 1500 years. In the study, HE was extracted by various solvents to prepare five HE extracts. They were observed to possess a protective effect against ×OH‐induced DNA damage, and scavenging effects on ×OH radical, ×O₂^? radical, DPPH×(1,1‐diphenyl‐2‐picrylhydrazyl) radical, and ABTS×⁺ (2,2′‐azino‐bis(3‐ethyl‐benzothiazoline‐6‐sulfonic acid) radical, and reduce Cu²⁺ ion. The contents of total phenolics and wedelolactone in five extracts were determined respectively using Folin‐Ciocalteu method and HPLC method. To identify which chemical component can be responsible for its effects, the correlation graphs between chemical contents and antioxidant abilities (1/IC₅₀ values) were plotted to calculate correlation coefficients (R values). Finally, MTT assay revealed that two HE extracts could effectively protect mesenchymal stem cells (MSCs) against ×OH‐induced damage at 3‐30 μg/mL. On the basis of mechanistic analysis, we concluded that: (i) HE can effectively protect against ×OH‐induced damages to DNA and MSCs, thereby HE may have a therapeutic potential in MSCs transplantation or prevention of many diseases; (ii) the effects can be mainly attributed to total phenolics (R = 0.678) especially wedelolactone (R = 0.618); (iii) they exert antioxidant action via hydrogen atom transfer (HAT) and sequential electron proton transfer (SEPT) mechanisms.     

Transient phosphorylation of tumor associated microtubule associated protein (TMAP)/cytoskeleton associated protein 2 (CKAP2) at Thr-596 during early phases of mitosis

Tumor associated microtubule associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2) is a mitotic spindle-associated protein whose expression is cell cycle-regulated and also frequently deregulated in cancer cells. Two monoclonal antibodies (mAbs) against TMAP/CKAP2 were produced: B-1-13 and D-12-3. Interestingly, the reactivity of mAb D-12-3 to TMAP/CKAP2 was markedly decreased specifically in mitotic cell lysate. The epitope mapping study showed that mAb D-12-3 recognizes the amino acid sequence between 569 and 625 and that phosphorylation at T596 completely abolishes the reactivity of the antibody, suggesting that the differential reactivity originates from the phosphorylation status at T596. Immunofluorescence staining showed that mAb D-12-3 fails to detect TMAP/CKAP2 in mitotic cells between prophase and metaphase, but the staining becomes evident again in anaphase, suggesting that phosphorylation at T596 occurs transiently during early phases of mitosis. These results suggest that the cellular functions of TMAP/CKAP2 might be regulated by timely phosphorylation and dephosphorylation during the course of mitosis.     

Comparison of antibody and albumin catalyzed hydrolysis of steroidalp-nitrophenylcarbonates

A monoclonal antibody (MAb) was produced against thep-nitrophenylphosphate derivative of 3α,5β-lithocholic acid, a transition-state analog for hydrolysis of a steroidalp-nitrophenylcarbonate. The indicated reaction was catalyzed by this Ab with kinetic constants k_cat = 4.0 × 10^-2min and K_m = 3.3 μM at pH 9.0 and 35°C. The Ab also hydrolyzed the isomericp-nitrophenylcarbonate of 3β,5β-lithocholic acid with k_cat = 8.4 × 10^-2/min and K_m = 1.0 μM. Bovine serum albumin (BSA) was found to catalyze the same reactions with similar turnover rates and Michaelis constants of 15 and 14 μM, respectively. Although the BSA-catalyzed reaction was only weakly inhibited by the phosphate ester TSA (IC₅₀ ca. 40 μM), the Ab-catalyzed reaction was completely inhibited at less than 1 μM of the TSA. The relative rates and efficiencies of the MAbcatalyzed and BSA-catalyzed reactions are discussed in the context of the hydrophobic sites and intrinsic reactivity of the protein surfaces, and the induction of groups on the Ab to enhance the enzymatic function.     

Development and characterization of rat monoclonal antibodies for N-acylated homoserine lactones

Quorum sensing (QS) is a communication mechanism between bacteria using diffusible chemical signaling molecules, which are called autoinducers (AI). By detecting the concentration of quorum sensing molecules through binding to a specific receptor protein, bacteria regulate their gene expressions when the concentration of autoinducers and thus the cell density reaches a threshold level. Many Gram-negative bacteria use acylated homoserine lactones (HSLs) as autoinducers. Because of the broad biological functions of HSLs, interest in detection and analysis of HSLs is increasing with a view to their medical, biotechnological, and agricultural applications. In this study, an anti-HSL antibody-based immunochemical detection method has been developed. Four structurally distinct HSL haptens, named HSL1, HSL2, HSL3, and HSL4, have been designed for antibody and assay development. New rat anti-HSL monoclonal antibodies (mAbs) have been produced in-house and characterized with enzyme-linked immunosorbent assays (ELISA), both in the coating antigen and in the enzyme tracer format. Eight mAbs (HSL1-1A5, HSL1-8E1, HSL1/2-2C10, HSL1/2-4H5, HSL4-4C9, HSL4-5E12, HSL4-5H3, and HSL4-6D3) will be presented in this paper. We demonstrate that the anti-HSL mAbs have distinguished sensitivity and selectivity toward HSLs depending upon their chemical structures. The optimized assays are capable of detecting HSLs in the microgram per liter (low micromolar to nanomolar) range. The best IC₅₀ (test midpoint) was 134 ± 30 μg L⁻¹ (n = 54) for N-(3-oxodecanoyl)-l-homoserine lactone (3-oxo-C10-HSL) using mAb HSL1/2-2C10 and HSL1–HRP in the enzyme tracer format. In the coating antigen format, the most selective mAb for N-octanoyl-l-homoserine lactone (C8-HSL) was mAb HSL4-4C9. Additionally, anti-HSL mAbs showed higher sensitivity against hydrolyzed HSLs, namely homoserines. These compounds might also occur under certain biological conditions. This study marks the beginning of new ways for quick and cost-effective HSL detection, requiring small sample amounts (less than 1 mL) and little to no sample preparation.     

A Cerium Hexacyanoferrate (III) Nanoparticle‐modified Carbon Paste Electrode: Voltammetric Characterization and Behavior in the Presence of Dopamine

This study describes a fast and simple methodology for the preparation of Cerium (III) Hexacyanoferrate (II) (CeHCF) nanoparticles (NPs). The NPs were characterized by fourier transform infrared (FTIR), x‐ray diffraction (XRD), scanning electron microscopy (SEM) and cyclic voltammetry (CV). The CeHCF cyclic voltammogram indicate a well‐defined redox pair assigned as Fe²⁺/Fe³⁺ in the presence of cerium (III), with a formal potential of E^θ′=0.29 V (v=100 mV s^?1, KNO₃; 1.0 mol/L, pH 7.0). The carbon paste electrode modified with CeHCF (CeHCF‐CPE) was applied to the catalytic electrooxidation of dopamine applying Differential Pulse Voltammetry (DPV). DPV showed linear response at two concentration ranges, from 9.0×10^?7 to 8.0×10^?6 and 9.0×10^?6 to 1.0×10^?4 mol/L, with an LOD of 1.9×10^?7 and 1.0×10^?5 mol/L, respectively. The CeHCF‐CPE exhibited selectivity against substances commonly found in biological samples, with redox potentials close to that of dopamine, such as urea and ascorbic acid (AA). Subsequently the CeHCF‐CPE was successfully applied to the detection of dopamine in simulated urine samples, with recovery percentages ranging between 99 and 103%.     

Quantitation of bacteria through adsorption of intracellular biomolecules on carbon paste and screen-printed carbon electrodes and voltammetry of redox-active probes

A new electrochemical method for the quantitation of bacteria that is rapid, inexpensive, and amenable to miniaturization is reported. Cyclic voltammetry was used to quantitate M. luteus, C. sporogenes, and E. coli JM105 in exponential and stationary phases, following exposure of screen-printed carbon working electrodes (SPCEs) to lysed culture samples. Ferricyanide was used as a probe. The detection limits (3s) were calculated and the dynamic ranges for E. coli (exponential and stationary phases), M. luteus (exponential and stationary phases), and C. sporogenes (exponential phase) lysed by lysozyme were 3 × 10⁴ to 5 × 10⁶ colony-forming units (CFU) mL⁻¹, 5 × 10⁶ to 2 × 10⁸ CFU mL⁻¹ and 3 × 10³ to 3 × 10⁵ CFU mL⁻¹, respectively. Good overlap was obtained between the calibration curves when the electrochemical signal was plotted against the dry bacterial weight, or between the protein concentration in the bacterial lysate. In contrast, unlysed bacteria did not change the electrochemical signal of ferricyanide. The results indicate that the reduction of the electrochemical signal in the presence of the lysate is mainly due to the fouling of the electrode by proteins. Similar results were obtained with carbon-paste electrodes although detection limits were better with SPCEs. The method described herein was applied to quantitation of bacteria in a cooling tower water sample. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Purification and quantification of recombinant Epstein-Barr viral glycoproteins gp350/220 from Chinese hamster ovary cells.

Truncated Epstein-Barr virus (EBV) membrane antigen gp350/220 (EBV-MA) lacking the membrane anchor was expressed and secreted into the medium of recombinant Chinese hamster ovary cells that had been cultured in Plasmapur hollow-fibre modules using defined serum-free medium. The EBV-MA in the medium was concentrated by 70% (w/v) ammonium sulphate precipitation and subsequently purified by immunoaffinity chromatography using an anti-EBV-MA (EBV.0T6) monoclonal antibody (mAb) column. Adsorbed antigen was eluted with 3 M MgCl2 in phosphate-buffered saline, concentrated by Mono Q anion-exchange chromatography and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, silver staining and Western blotting using EBV-positive serum and anti-EBV-MA specific mAbs. Monospecific polyclonal rabbit antibodies against the purified EBV-MA were raised and purified by protein G affinity chromatography. For the measurement of EBV-MA antigen levels a sandwich enzyme-linked immunosorbent assay using rabbit polyclonal antibodies and a horseradish peroxidase-conjugated anti-MA mAb was developed having a detection level of 10 ng/ml.     

A Solid State Polymer‐Coated Electrode Containing a Chiral Crown Ether Derivative for Enantioselective Detection of Phenylglycine Methyl Ester Isomer

All solid‐state enantioselective electrode (ASESE) based on a newly synthesized chiral crown ether derivative ((R)‐(?)‐(3,3′‐diphenyl‐1,1′‐binaphthyl)‐23‐crown‐6 incorporating 1,4‐dimethoxybenzene) was prepared and characterized by potentiometry. The ASESE clearly showed enantiomer discrimination for methyl esters of alanine, leucine, valine, phenylalanine, and phenylglycine, where the enantioselectivity for phenylglycine methyl ester was the highest (K_R,S=8.5±7.1%). Experimental parameters of ASESE for the analysis of (R)‐(?)‐phenylglycine methyl ester were optimized. The optimized ASESE showed a slope of 55.3±0.2 mV/dec for (R)‐(?)‐phenylglycine methyl ester in the concentration range of 1.0×10^?5–1.0×10^?2 M and the detection limit was 9.0×10^?6 M. The ASESE showed good selectivity for (R)‐(?)‐phenylglycine methyl ester against inorganic cations and various amino acid methyl esters. The concentration of (R)‐(?)‐phenylglycine methyl ester was determined in the mixture of (R)‐(?) and (S)‐(+)‐phenylglycine methyl ester, which ratios varied from 2 : 1 to 1 : 9. The lifespan of the electrode was alleged to be 30 days.     

Fast Epitope Mapping for the Anti‐MUC1 Monoclonal Antibody by Combining a One‐Bead‐One‐Glycopeptide Library and a Microarray Platform

Anti‐MUC1 monoclonal antibodies (mAbs) are powerful tools that can be used to recognize cancer‐related MUC1 molecules, the O‐glycosylation status of which is believed to affect binding affinity. We demonstrate the feasibility of using a rapid screening methodology to elucidate those effects. The approach involves i) “one‐bead‐one‐compound”‐based preparation of bilayer resins carrying glycopeptides on the shell and mass‐tag tripeptides coding O‐glycan patterns in the core, ii) on‐resin screening with an anti‐MUC1 mAb, iii) separating positive resins by utilizing secondary antibody conjugation with magnetic beads, and (iv) decoding the mass‐tag that is detached from the positive resins pool by using mass spectrometric analysis. We tested a small library consisting of 27 MUC1 glycopeptides with different O‐glycosylations against anti‐MUC1 mAb clone VU‐3C6. Qualitative mass‐tag analysis showed that increasing the number of glycans leads to an increase in the binding affinity. Six glycopeptides selected from the library were validated by using a microarray‐based assay. Our screening provides valuable information on O‐glycosylations of epitopes leading to high affinity with mAb.     

Size separation of sodium dodecyl sulfate–proteins by capillary electrophoresis in dilute and ultra-dilute dextran solutions

SDS capillary gel electrophoresis is a widely used in the biopharma and the biomedical fields for rapid size separation of proteins. However, very limited information is available on the use of dilute and ultra-dilute sieving matrices for SDS–protein analysis. Here, background electrolytes (BGEs) containing 1%–0% dextran were used in borate-based BGE to separate a protein sizing ladder (PSL) ≤225 kDa and the intact and subunit forms of a therapeutic monoclonal antibody (mAb). The separation performance for the PSL and mAb components differed significantly with decreasing dextran concentration. Ferguson and reptation plots were used to elucidate the separation mechanism. Highly diluted dextran solutions resulted in linear Ferguson plots for both solute types (cf. Ogston theory) in spite of this model assumes a rigid pore structure, thus cannot describe the separation mechanism in ultra-dilute polymer solutions with no reticulations. The saddle differences between the resolution of the PSL and the intact/subunit mAb forms in ultra-dilute dextran-borate matrices suggested the importance of shape selectivity, manifested by the adequate separation of the SDS covered intact as well as light and heavy chain subunits of the therapeutic mAb even at zero dextran concentration.     

A SERS-based immunoassay for porcine circovirus type 2 using multi-branched gold nanoparticles

We report on a facile immunoassay for porcine circovirus type 2 (PCV2) based on surface enhanced Raman scattering (SERS) using multi-branched gold nanoparticles (mb-AuNPs) as substrates. The mb-AuNPs in the immunosensor act as Raman reporters and were prepared via Tris base-induced reduction and subsequent reaction with p-mercaptobenzoic acid (pMBA). They possess good stability and high SERS activity. Subsequently, the modified mb-AuNPs were covalently conjugated to the monoclonal antibody (McAb) against the PCV2 cap protein to form SERS immuno nanoprobes. These were captured in a microtiterplate via a immunoreaction in the presence of target antigens. The effects of antibody concentration, reaction time and temperature on the sensitivity of the immunoassay were investigated. Under optimized assay conditions, the Raman signal intensity at 1,076 cm^?1 increases logarithmically with the concentrations of PCV2 in the concentration ranging from 8?×?10² to 8?×?10⁶ copies per mL. The limit of detection is 8?×?10² copies per mL. Compared to conventional detecting methods such as those based on PCR, the method presented here is rapid, facile and very sensitive.

Optimized preparation and preliminary evaluation of [64Cu]–DOTA–trastuzumab for targeting ErbB2/Neu expression

Breast cancer radioimmunoscintigraphy targeting HER2/neu expression is a growing field of work in nuclear medicine research. Trastuzumab is a monoclonal antibody that binds with high affinity to HER2/neu, which is over expressed on breast and other tumors. Developing new tracers for the detection of this cancer is of great interest. In this study, trastuzumab was successively labeled with [⁶⁴Cu]CuCl₂ after conjugation with DOTA-NHS-ester. The conjugate was purified by molecular filtration, the average number of DOTA conjugated per mAb was calculated and total concentration was determined by spectrophotometric method. DOTA–trastuzumab was labeled with ⁶⁴Cu produced by ⁶⁸Zn(p,αn)⁶⁴Cu nuclear reaction (30 MeV protons at 180 μA). Radiochemical purity, integrity of protein after radiolabeling and immunoreactivity of radiolabeled mAb trastuzumab with HER2/neu antigen and SkBr3 cell line were performed by RIA. In vitro stability of radiolabeled mAb in human serum was determined by thin layer chromatography. In vitro internalization studies were performed with the SkBr3 cell line and the tissue biodistribution of the ⁶⁴Cu–DOTA–trastuzumab was evaluated in wild-type rat (90 ± 5.5 μCi, 2, 6, 12, 24 h p.i.). The radioimmunoconjugate was prepared with a radiochemical purity of higher than 96 ± 0.5 % (ITLC) and specific activity as high as 5.3 μCi/μg. The average number of chelators per antibody for the conjugate used in this study was 5.8/1. The sample was showed to have similar patterns of migration in the gel electrophoresis. The ⁶⁴Cu–DOTA–trastuzumab showed high immunoreactivity towards HER2/neu antigen and SkBr3 cell line. In vitro stability of the labeled product was found to be more than 94 % in PBS and 82 ± 0.5 % in human serum over 48 h. In vitro internalization studies of the ⁶⁴Cu–DOTA–trastuzumab showed that up to 11.5 % of the radioimmunoconjugate internalized after 10 h. The accumulation of the radiolabeled mAb in liver, skin, intestine, lung, spleen, kidney and other tissues demonstrates a similar pattern to the other radiolabeled anti-HER2 immunoconjugates. ⁶⁴Cu–DOTA–trastuzumab is a potential compound for molecular imaging of PET for diagnosis and treatment studies and follow-up of HER2 expression in oncology.     

Synthesis and In Vivo antitumor and antiviral activities of 2′-deoxyribofuranosyl and arabinofuranosyl nucleosides of certain purine-6-sulfenamides,sulfinamides and sulfonamides

2′-Deoxyribofuranosyl and arabinofuranosyl nucleosides of certain purine-6-sulfenamides, sulfinamides and sulfonamides have been prepared by sequential amination and controlled oxidation of the corresponding 6-thiopurine nucleosides, and evaluated for antiviral and antitumor activities in mice. Amination of 2′-deoxy-6-thioinosine ( 4a ) and 9-β-D-arabinofuranosyl-6-thiopurine ( 4c ) with chloramine solution gave the corresponding 6-sulfenamides 5a and 5c , respectively, which on selective oxidation with 3-chloroperoxybenzoic acid (MCPBA) gave diastereomeric 9-(2-deoxy-β-D-erythro-pentofuranosyl)purine-6-sulfinamide ( 6a ) and 9-β-D-arabinofuranosylpurine-6-sulfinamide ( 6c ), respectively. However, oxidation of 5a and 5c with excess of MCPBA gave the corresponding 6-sulfonamide derivatives 7a and 7c , respectively. Similar amination of 2′-deoxy-6-thioguanosine ( 4b ), ara-6-thioguanine ( 4d ) and α-2′-deoxy-6-thioguanosine ( 8 ) gave the respective 6-sulfenamide derivatives 5b, 5d and 9 . Controlled oxidation of 5b, 5d and 9 gave (R,S)-2-amino-9-(2-deoxy-β-D-erythro-pentofuranosyl)purine-6-sulfinamide ( 6b ), (R,S)-2-amino-9-β-D-arabinofuranosylpurine-6-sulfinamide ( 6d ) and the α-anomer of ( 6b) (10 ), respectively. The diastereomeric mixture of (R,S )-10 was partially resolved and the structure of S -10 was assigned by single-crystal X-ray diffraction analysis. Oxidation of 5b, 5d and 9 with excess of MCPBA afforded the respective 6-sulfonamide derivatives 7b, 7d and 11 . Nucleosides 5c and 7c were significantly active against Friend leukemia virus in mice, whereas 6c was somewhat less active. Of the 20 nucleosides evaluated, 12 exhibited biologically significant anti-L1210 activity in mice. Nucleosides 6b and 7a at 173 mg/kg/day × 1 showed a T/C of 153, whereas 7d at 800 mg/kg/day × 1 showed a T/C of 153 against L1210 leukemia. The α-nucleoside 9 at 480 mg/kg/day × 1 gave a T/C of 172. A single treatment with 6b, 7a, 7d and 9 reduced the body burdens of viable L1210 cells by more than 99.2%. The antileukemic activity of these novel nucleosides tended to parallel solubility.