Bovine Serum Albumin Conjugate Research Articles

Bovine Serum Albumin-Conjugated Ferrimagnetic Iron Oxide Nanoparticles to Enhance the Biocompatibility and Magnetic Hyperthermia Performance.

Magnetic hyperthermia is a fast emerging, non-invasive cancer treatment method which is used synergistically with the existing cancer therapeutics. We have attempted to address the current challenges in clinical magnetic hyperthermia-improved biocompatibility and enhanced heating characteristics, through a single combinatorial approach. Both superparamagnetic iron oxide nanoparticles (SPIONs) of size 10 nm and ferrimagnetic iron oxide nanoparticles (FIONs) of size 30 nm were synthesized by thermal decomposition method for comparison studies. Two different surface modifying agents, viz, Cetyl Trimethyl Ammonium Bromide and 3-Aminopropyltrimethoxysilane, were used to conjugate Bovine Serum Albumin (BSA) over the iron oxide nanoparticles via two different methods—surface charge adsorption and covalent amide bonding, respectively. The preliminary haemolysis and cell viability experiments show that BSA conjugation mitigates the haemolytic effect of the iron oxide nanoparticles on erythrocytes and is non-cytotoxic to the healthy Baby Hamster Kidney cells. It was observed from the results that due to better colloidal stability, the SAR value of the BSA-iron oxide nanoparticles is higher than the iron oxide nanoparticles without BSA, irrespective of the size of the iron oxide nanoparticles and method of conjugation. The BSA-FIONs seem to show improved biocompatibility, as the haemolytic index is less than 2 % and cell viability is up to 120 %, when normalized with the control. The SAR value of BSA-FIONs is 2300 W g−1 when compared to 1700 W g−1 of FIONs without BSA conjugation. Thus, we report here that BSA conjugation over FIONs (with a high saturation magnetization of 87 emu g−1) provide a single combinatorial approach to improve the biocompatibility and enhance the SAR value for magnetic hyperthermia, thus addressing both the current challenges of the same.Graphical

New 3-(heteroaryl)-2-iminocoumarin-based borate complexes: synthesis, photophysical properties, and rational functionalization for biosensing/biolabeling applications.

Members of a series of boron difluoride complexes with 3-(heteroaryl)-2-iminocoumarin ligands bearing both a phenolic hydroxyl group (acting as a fluorogenic center) and an N-aryl substituent (acting as a stabilizing moiety) have been synthesized in good yields by applying a straightforward two-step method. These novel fluorogenic dyes belong to the family of "Boricos" (D. Frath et al., Chem. Commun.- 2013, 49, 4908-4910) and are the first examples of phenol-based fluorophores of which the photophysical properties in the green-yellow spectral range are dramatically improved by N,N-chelation of a boron atom. Modulation of their fluorescence properties through reversible chemical modification of their phenol moieties has been demonstrated by the preparation of the corresponding 2,4-dinitrophenyl (DNP) ethers, which led to a dramatic "OFF-ON" fluorescence response upon reaction with thiols. Additionally, to expand the scope of these "7-hydroxy-Borico" derivatives, particularly in biolabeling, amine or carboxylic acid functionalities amenable to (bio)conjugation have been introduced within their scaffold. Their utility has been demonstrated in the preparation of fluorescent bovine serum albumin (BSA) conjugates and "Borico"-DOTA-like scaffolds in an effort to design novel monomolecular multimodal fluorescence- radioisotope imaging agents.

Initial Growth Kinetics of Luminescent Quantum Clusters of Silver within Albumin Family Protein Templates

We probed the initial growth kinetics of luminescent quantum clusters of silver (AgQCs) within two albumin family proteins, bovine serum albumin (BSA) and ovalbumin (Ova). Shorter time scale (seconds to minutes) growth of AgQCs monitored using real time photoluminescence spectroscopy has shown that, at lower concentrations of Ag+, only unstable QCs were formed. The major role of basic pH in the synthesis was not only to facilitate Ag+-BSA conjugation but also to provide well dispersed medium for controlled nucleation of QCs. Increase in the concentration of NaBH4 affects growth kinetics greatly and leads to increase in the growth rate of AgQCs; but for NaBH4 concentrations higher than the optimum value, growth rate becomes constant. Precise measurements have shown that excitation and emission of AgQCs exhibit linear red-shift with the increasing concentration of NaBH4 whereas protein excitation remains constant. Similar results were observed for both the proteins, Ova and BSA. We believe that various insights provided by this study will be helpful for further improvements in the synthetic methodology and applications of protein protected AgQCs.

Intact reducing glycan promotes the specific immune response to lacto-N-neotetraose-BSA neoglycoconjugates.

The mammalian immune system responds to eukaryotic glycan antigens during infections, cancer, and autoimmune disorders, but the immunological bases for such responses are unclear. Conjugate vaccines containing bacterial polysaccharides linked to carrier proteins (neoglycoconjugates) have proven successful, but these often contain repeating epitopes and the reducing end of the glycan is less important, unlike typical glycan determinants in eukaryotes, which are shorter in length and may include the reducing end. Here, we have compared the effects of two linkage methods, one that opens the ring at the reducing end of the glycan, and one that leaves the reducing end closed, on the glycan specificity of the vaccine response in rabbits and mice. We immunized rabbits and mice with bovine serum albumin (BSA) conjugates of synthetic open- and closed-ring forms (OR versus CR) of a simple tetrasaccharide lacto-N-neotetraose (LNnT, Galβ1-4GlcNAcβ1-3Galβ1-4Glc), and tested reactivity to the immunogens and several related glycans in both OR and CR versions on glycan microarrays. We found that in rabbits the immune response to the CR conjugate was directed toward the glycan, whereas the OR conjugate elicited antibodies to the reducing end of the glycan and linker region but not specifically to the glycan itself. Unexpectedly, mice did not generate a glycan-specific response to the CR conjugate. Our findings indicate that the reducing end of the sugar is crucial for generation of a glycan-specific response to some eukaryotic vaccine epitopes, and that there are species-specific differences in the ability to make a glycan-specific response to some glycoconjugates. These findings warrant further investigation with regard to rational design of glycoconjugate vaccines.

A monoclonal antibody-based enzyme-linked immunosorbent assay for detection of ustiloxin A in rice false smut balls and rice samples

Ustiloxin A, a cyclopeptide mycotoxin, was isolated from the pathogenic fungus Villosiclava virens that causes rice false smut, a worldwide devastating rice disease. A monoclonal antibody (mAb) 2D3G5 was generated with ustiloxin A–bovine serum albumin conjugate. A highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed. It possessed a median inhibition concentration (IC50) of 13.8ng/mL and a working range of 2.8–72ng/mL. The mAb 2D3G5 recognized ustiloxin B with the cross-reactivity as 4%. The average recoveries of ustiloxin A from rice false smut balls and peeled rice samples ranged from 92% to 117% and from 92% to 107%, respectively. Comparison of the concentrations of ustiloxin A in rice false smut balls detected by both icELISA and high performance liquid chromatography–photodiode array detection indicated that the developed icELISA was suitable for detection of ustiloxin A in rice food and feed samples.

Design of iron oxide nanoparticles decorated oleic acid and bovine serum albumin for drug delivery

This study aimed to originally develop a new nanoparticulate drug delivery system of iron oxide nanoparticles (Fe3O4) for biomedical applications. Oleic acid and bovine serum albumin were decorated on the surface of iron oxide nanoparticles in new pattern by conjugation. The decoration was kicked off by the functionalization of arginine on the surface of the iron oxide nanoparticles. It was then followed by the conjugation of oleic acid and bovine serum albumin through the amide bond. Scanning electron microscopy, transmission electron microscopy, powder X-ray diffraction and Fourier transform infrared spectroscopy were used to characterize and determine mechanism of the decorated nanoparticles. Paclitaxel was chosen as the model drug in the study. The nanoparticles demonstrated a potential utility in delivery of anticancer drugs.

Nongenomic effects of estrogen on extracellular signal-regulated kinases through initiating transient calcium flux in endometrial cancer

To study the mechanism on extracellular signal-regulate kinases (ERK) signal transduction by calcium influx initiated by combination of estrogen with calcium channels or estrogen receptor in endometrial cancer cell Ishikawa. Confocal test was used to determine the relative calcium mobilization by stimulation of estrodiol together with and without the inhibition of ICI182780 and nifedipine. Western-blotting was used to detect the protein expression of phosphorylated ERK1/2 (P-ERK1/2) in the same condition. The transient calcium flux initiated by 17β-estrodiol (E2) and a membrane-impermeable conjugate of estrogen and bovine serum albumin (E2-BSA), and the calcium mobilization could be inhibited by ICI182780 and nifedipine in 1 min. In Ishikawa cells, phosphorylation of ERK1/2 was stimulated by E2, and the phosphorylation could not be inhibited by E2 after the combination with ICI182780 in 5 min and in 30 min. The phosphorylation also could not be inhibited by E2-BSA after the combination with nifedipine in 5 min, but in 30 min the phosphorylation was decreased. The phosphorylation of ERK by E2-BSA was decreased by the combination with nifedipine in 30 min. The transient calcium flux initiated by estrogen has an effect on the activation of ERK signal pathway in endometrial carcinoma cells.

Formulation and In-Vitro Evaluation of Piroxicam Loaded BSA Nanospheres by Desolvation

Protein nanoparticles offer improved delivery of drugs particularly targeted delivery to specific cells by ligand attachment, sustained and triggered release. Non steroidal anti-inflammatory drug (NSAID), Piroxicam (PRX) is one of the most widely used drug for treating pain and inflammation. But despite its potent analgesic action it has few draw backs like low absorption rate due to poor water solubility and side effects due to non specific inhibition of Cyclooxygenase enzymes. In this context here, we report a new PRX self-assembled nano sphere delivery system developed from bovine serum albumin (BSA) using a very simple, rapid and reliable method, desolvation. Synthesized nanoparticles were characterized by particle size distribution, zeta potential, polydispersity index, FTIR, scanning electron microscopy and in-vitro release studies. The formulated BSA-PRX nano particles exhibited a uniform spherical shape with an average size of 388.7nm. In vitro release behavior of the drug from BSA conjugate suggests that about 50% of the drug was released during first 3h and 85% after 18h.

Stress-sensitive organs and blood corticosterone after immobilization of active and passive rats immunized with glutamate-bovine serum albumin conjugate.

We studied stress-induced organ and hormonal responses in behaviorally active and passive rats against the background of immunization with glutamate-BSA conjugate. The relative weight of the adrenal glands after immobilization was lower in rats immunized with the conjugate in comparison with non-immunized animals. The weight of the adrenal glands in behaviorally active rats decreased in parallel with the decrease in blood corticosterone. In behaviorally active and passive rats immunized with the conjugate, ulcer formation in the stomach was slightly intensified after immobilization. It was hypothesized that immunization with glutamate-BSA conjugate suppresses activity of the hypothalamic-pituitary-adrenal feedback mechanism underlying the production of glucocorticoid hormones, which is manifested in slightly increased ulceration due to attenuation of the gastroprotective action of corticosterone under stress.

Peroxidase-mediated conjugation of corn fiber gum and bovine serum albumin to improve emulsifying properties.

The emulsifying properties of corn fiber gum (CFG), a naturally occurring polysaccharide-protein complex, was improved by kinetically controlled formation of hetero-covalent linkages with bovine serum albumin (BSA), using horseradish peroxidase (HRP). The formation of hetero-crosslinked CFG-BSA conjugates was confirmed using ultraviolet-visible and Fourier-transform infrared analyses. The optimum CFG-BSA conjugates were prepared at a CFG:BSA weight ratio of 10:1, and peroxidase:BSA weight ratio of 1:4000. Selected CFG-BSA conjugates were used to prepare oil-in-water emulsions; the emulsifying properties were better than those of emulsions stabilized with only CFG or BSA. Measurements of mean droplet sizes and zeta potentials showed that CFG-BSA-conjugate-stabilized emulsions were less susceptible to environmental stresses, such as pH changes, high K ionic strengths, and freeze-thaw treatments than CFG- or BSA-stabilized emulsions. These conjugates have potential applications as novel emulsifiers in food industry.

Synthesis of a pentasaccharide and neoglycoconjugates related to fungal α-(1→3)-glucan and their use in the generation of antibodies to trace Aspergillus fumigatus cell wall.

3-Aminopropyl α-(1→3)-pentaglucoside, a fragment of α-(1→3)-glucan of the cell wall of Aspergillus fumigatus, has been synthesized in a blockwise approach. The application of mono- and disaccharide N-phenyltrifluoroacetimidates bearing a stereodirecting 6-O-benzoyl group was essential for stereoselective α-glucosylations. In the products, p-methoxyphenyl and levulinoyl groups served as orthogonal protecting groups for the anomeric position and 3-OH group, respectively. Their removal from shared blocks led to donors and acceptors that were used for the synthesis of pentasaccharides. Coupling of free α-(1→3)-pentaglucoside with biotin and bovine serum albumin (BSA) gave glycoconjugate tools for mycological studies. Immunization of mice with the BSA conjugate induced the generation of antibodies that recognize α-(1→3)-glucan on A. fumigatus cell wall and distinguish its morphotypes. This discovery represents a first step to the development of a diagnostic test system and a vaccine to detect and fight this life-threatening pathogen.

An ultra-sensitive monoclonal antibody-based competitive enzyme immunoassay for sterigmatocystin in cereal and oil products.

Sterigmatocystin (STG), a biosynthesis precursor of aflatoxin B1, is well known for its toxic and carcinogenic effects in humans and animals. STG derivatives and protein conjugates are needed for generation of monoclonal antibodies (mAbs). This work describes a reliable and fast synthesis of novel STG derivatives, based on which novel STG bovine serum albumin conjugates were prepared. With the novel STG bovine serum albumin conjugates, three sensitive and specific mAbs against STG, named VerA 3, VerA 4, and VerA 6, were prepared by semi-solid hypoxanthine/aminopterin/thymidine (HAT) medium using a modified two-step screening procedure. They exhibited high affinity for STG and no cross-reactivity (CR) with aflatoxins B1, B2, G1, G2, and M1. Based on the most sensitive antibody VerA 3, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for STG in wheat, maize, and peanuts. Assays were performed in the STG-GA-BSA-coated (0.5 µg·mL−1) ELISA format, in which the antibody was diluted to 1∶80,000. Several physicochemical factors influencing assay performance, such as pH, ionic strength, blocking solution, and diluting solution, were optimized. The final results showed that the assays had the detection limits of 0.08 ng·g−1 for wheat, 0.06 ng·g−1 for maize, and 0.1 ng·g−1 for peanuts, inter-assay and intra-assay variations of less than 10%, and recoveries ranging from 83% to 110%. These recoveries were in good agreement with those obtained by using HPLC-MS/MS method (90–104%), indicating the importance of the mAb VerA 3 in the study of STG in crude agricultural products.

Preparation and sonodynamic activities of water-soluble tetra-α-(3-carboxyphenoxyl) zinc(II) phthalocyanine and its bovine serum albumin conjugate

Sonodynamic therapy (SDT) is a new approach for cancer treatment, involving the synergistic effect of ultrasound and certain chemical compounds termed as sonosensitizers. A water-soluble phthalocyanine, namely tetra-α-(3-carboxyphenoxyl) zinc(II) phthalocyanine (ZnPcC4), has been prepared and characterized. The interactions between ZnPcC4 and bovine serum albumin (BSA) were also investigated by absorption and fluorescence spectroscopy. It was found that there were strong interactions between ZnPcC4 and BSA with a binding constant of 6.83×107M−1. A non-covalent BSA conjugate of ZnPcC4 (ZnPcC4–BSA) was prepared. Both ZnPcC4 and ZnPcC4–BSA exhibited efficient sonodynamic activities against HepG2 human hepatocarcinoma cells. Compared with ZnPcC4, conjugate ZnPcC4–BSA showed a higher sonodynamic activity with an IC50 value of 7.5μM. Upon illumination with ultrasound, ZnPcC4–BSA can induce an increase of intracellular reactive oxygen species (ROS) level, resulting in cellular apoptosis. The results suggest that the albumin conjugates of zinc(II) phthalocyanines functionalized with carboxyls can serve as promising sonosensitizers for sonodynamic therapy.

Fabrication and sensing property for conducting polymer nanowire-based biosensor for detection of immunoglobulin G

Conducting polymers are excellent sensing materials in the design of bioanalytical sensors because of their electronic conductivity, low energy optical transitions, biocompatibility, and room temperature operation. Among them, Polypyrrole (Ppy) is one of the most extensively used conducting polymers because of a number of properties such as redox activity, rapid electron transfer, and ability to link a variety of biomolecules to pyrrole groups by chemical treatment. In this study, Ppy nanowires were synthesized by an electrospinning method. The nanowires were prepared from a solution mixture of Ppy and poly(ethylene oxide). The method of detection in such a device is based on the selective binding of antigen onto an antibody that is covalently attached to the nanowires. Thus, anti-IgG was immobilized on Ppy nanowires using an EDC {[N-(3-dimethyl aminopropyl)-N2-ethylcarbodiimide hydrochloride]}-NHS(N-hydrosuccinimide) modified technique. Fluorescence images of BSA–FITC (fluorescein isothiocyanate labeling of bovine serum albumin) conjugation demonstrated that antibody was functionalized on the Ppy nanowires without non-specific binding and facilitated selective detection of antigen. Current–voltage (I–V) characterization was used to monitor the change in the conductivity of nanowires while the specific binding interaction occurred. These results of electrical properties enable Ppy nanowire-based biosensors to detect biomolecules in real-time.

Transdermal delivery of proteins using a combination of iontophoresis and microporation.

This study aimed to investigate transdermal delivery of proteins using combination of microporation and iontophoresis (ITP). Materials & methods & results: Delivery of model protein, Alexa Fluor 555 bovine serum albumin conjugate (AF-BSA) using ITP alone, microneedle (MN) alone, and ITP plus MN combination was assessed using confocal microscopy. Compared to MN alone, combination of MN plus ITP significantly increased skin's penetration depth of AF-BSA (300 vs 110 μm) and achieved lateral distribution of the model protein. Average fluorescence intensity quantified around each microchannel was 23.7-fold (8.2-fold, in vivo) higher for combination treatment compared with MN alone, in vitro. After 1 h in vitro permeation study, the unlabeled BSA amount delivered across skin was found to be 0, 1.4, 0.63 and 14 μg by passive, MN alone, ITP alone and ITP plus MN combination delivery, respectively.

Gold nanocatalyst-based immunosensing strategy accompanying catalytic reduction of 4-nitrophenol for sensitive monitoring of chloramphenicol residue

A new competitive-type immunosensing system based on gold nanoparticles toward catalytic reduction of 4-nitrophenol (4-NP) was developed for sensitive monitoring of antibiotic residue (chloramphenicol, CAP, used in this case) by using ultraviolet–visible (UV–vis) spectrometry. Gold nanoparticle (AuNP) with 16nm in diameter was initially synthesized and functionalized with CAP–bovine serum albumin (CAP–BSA) conjugate, which were used as the competitor on monoclonal anti-CAP antibody-coated polystyrene microtiter plate (MTP). In the presence of target CAP, the labeled CAP–BSA on the AuNP competed with target CAP for the immobilized antibody on the MTP. The conjugated amount of CAP–BSA–AuNP on the MTP decreased with the increase of target CAP in the sample. Upon addition of 4-NP and NaBH4 into the MTP, the carried AuNP could catalytically reduce 4-NP to 4-aminophenol (4-AP), and the as-produced 4-AP could be monitored by using UV–vis absorption spectroscopy. Experimental results indicated that the absorbance at 403nm increased with the increment of target CAP concentration in the sample, and exhibited a dynamic range from 0.1 to 100ngmL−1 with a detection limit (LOD) of 0.03ngmL−1 at the 3sblank level. Intra- and inter-assay coefficients of variation were lower than 5.5% and 8.0%, respectively. In addition, the methodology was evaluated for CAP spiked honey and milk samples, respectively. The recovery was 92–112%.

A Magnetic Nanoparticle Stabilized Gas Containing Emulsion for Multimodal Imaging and Triggered Drug Release

To develop a multimodal imaging guided and triggered drug delivery system based on a novel emulsion formulation composed of iron oxide nanoparticles, nanoscopic bubbles, and oil containing drugs. Iron oxide paramagnetic nanoparticles were synthesized and modified with surface conjugation of polyethylenimide (PEI) or Bovine Serum Albumin (BSA). Both particles were used to disperse and stabilize oil in water emulsions containing coumarin-6 as the model drug. Sulfur hexafluoride was introduced into the oil phase to form nanoscopic bubbles inside the emulsions. The resulted gas containing emulsions were evaluated for their magnetic resonance (MR) and ultrasound (US) imaging properties. The drug release profile triggered by ultrasound was also examined. We have successfully prepared the highly integrated multi-component emulsion system using the surface modified iron oxide nanoparticles to stabilize the interfaces. The resulted structure had distinctive MR and US imaging properties. Upon application of ultrasound waves, the gas containing emulsion would burst and encapsulated drug could be released. The integrated emulsion formulation was multifunctional with paramagnetic, sono-responsive and drug-carrying characteristics, which may have potential applications for disease diagnosis and imaging guided drug release.

A Direct Enzyme Immunoassay to Detect Erythrosine in Foods

As a synthetic food dye, erythrosine is associated with serious toxicity in inducing thyroid tumors, and the use of erythrosine is strictly regulated in most counties including China. In this study, a direct enzyme-linked immunosorbent assay (ELISA) has been developed for analysis of erythrosine in food products. A highly specific monoclonal antibody (MAb) for erythrosine was produced using erythrosine–bovine serum albumin (BSA) conjugate as an artificial antigen, and horseradish peroxidase (HRP)-labeled MAb for erythrosine was utilized as the detection antibody. Under the optimized condition, the UV absorbance in microplate related well with erythrosine concentration in the range of 0.1–10.0 μg/g. The proposed method could be applied to determine erythrosine in beverage and cookie, with good recoveries (80 –103 %) for the three spiked levels (30, 50, and 100 μg/g), and the relative standard deviations of detected amount were <12.3 %.

High specificity detection of Pb2+ ions by p-SCN-Bz-DTPA immunogen and p-NH2-Bn-DTPA coating antigen

Only one bifunctional metal-chelator was used to prepare immunogen and coating antigen in all of the previous researches. However, the antibody-specific recognition to the spacer arm of the bifunctional metalchelator might lower the specificity of heavy metal ions immunoassay. Two different bifunctional metal-chelators were adopted to prepare the immunogen and coating antigen respectively in our study to avoid this problem. The conjugates of keyhole limpet hemocyanin (KLH) and p-SCN-Bz-DTPA-Pb were used as immunogen, whereas the conjugates of bovine serum albumin (BSA) and p-NH2-Bn-DTPA-Pb were used as coating antigen. Polyclonal antibodies specific to DTPA-Pb chelates were obtained from rabbits. Indirect competitive enzyme-linked immunosorbent assay (ELISA) was adopted to detect Pb2+ ion solutions prepared by Pb2+ standard solution and ultrapure water. In the mixing microplate, DTPA and Pb2+ ions formed chelates and combined with specific antibodies. After incubation, the DTPA-Pb and the antibodies complex were added into the wells of the reaction microplate. The reaction microplate was coated by the conjugates of BSA and p-NH2-DTPA-Pb, which competed for the specific antibodies. The result signals presented a good sigmoid curve when the Pb2+ concentration ranges from 0.01 to 100 mg·L−1. The IC50 of the indirect competitive ELISA is 0.23±0.04 mg·L−1 Pb2+ ion. The cross-reaction with Cd2+, Cu2+, Fe2+, Mn2+, Zn2+, and other divalent ions were less than 5%.

A HIGHLY SENSITIVE POLYCLONAL ANTIBODY-BASED ELISA FOR THERAPEUTIC MONITORING AND PHARMACOKINETIC STUDIES OF LENALIDOMIDE

This article describes, for the first time, a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for therapeutic monitoring and pharmacokinetic studies of lenalidomide (LND), the potent drug for treatment of multiple myeloma (MM). The assay employed a polyclonal antibody that specifically recognizes LND with high affinity, and LND conjugate of bovine serum albumin (LND–BSA) immobilized onto microplate wells as a solid phase. The assay involved a competitive binding reaction between LND, in plasma sample, and the immobilized LND–BSA for the binding sites on a limited amount of the anti–LND antibody. The assay limit of detection was 0.05 ng/mL and the effective working range at relative standard deviations (RSD) of ≤5% was 0.1−20 ng/mL. Analytical recovery of LND from spiked plasma was 100.98 ± 3.05. The precisions of the assay were satisfactory; RSD was 2.96−6.67% and 4.46−7.14%, for the intra- and inter-assay precision, respectively. The procedure is convenient, and one can analyze ∼200 samples per working day, facilitating the processing of large-number batch of samples in clinical laboratories. The proposed ELISA has a great value in routine analysis of LND for its therapeutic monitoring and pharmacokinetic studies.