Development of a noncompetitive phage anti-immunocomplex assay for brominated diphenyl ether 47

We present a new application of the noncompetitive phage anti-immunocomplex assay (PHAIA) by converting an existing competitive assay to a versatile noncompetitive sandwich-type format using immunocomplex binding phage-borne peptides to detect the brominated flame retardant, brominated diphenyl ether 47 (BDE 47). Three phage-displayed 9-mer disulfide-constrained peptides that recognize the BDE 47-polyclonal antibody immunocomplex were isolated. The resulting PHAIAs showed variable sensitivities, and the most sensitive peptide had a dose-response curve with an SC₅₀ (concentration of analyte producing 50% saturation of the signal) of 0.7 ng/ml BDE 47 and a linear range of 0.3-2 ng/ml, which was nearly identical to the best heterologous competitive format (IC₅₀ of 1.8 ng/ml, linear range of 0.4-8.5/ml). However, the PHAIA was 1400-fold better than homologous competitive assay. The validation of the PHAIA with extracts of house furniture foam as well as human and calf sera spiked with BDE 47 showed overall recovery of 80-113%. The PHAIA was adapted to a dipstick format (limit of detection of 3.0 ng/ml), and a blind test with six random extracts of local house furniture foams showed that the results of the PHAIA and dipstick assay were consistent, giving the same positive and negative detection.     

Development of an enzyme-linked immunosorbent assay for thiacloprid in soil and agro-products with phage-displayed peptide

A monoclonal antibody (3A5) that can recognize thiacloprid was produced, and a linear 8-residue peptide phage library was constructed. Six phage-displayed peptides were isolated from the linear 8-residue peptide phage library and a cyclic 8-residue peptide phage library. A phage enzyme-linked immunosorbent assay (ELISA) was developed to detect thiacloprid using a phage-displayed peptide. Under the optimal conditions, the half-maximal inhibition concentration (IC₅₀) and the limit of detection (IC₁₀) of the developed phage ELISA were 8.3 and 0.7 μg/L, respectively. Compared with the conventional ELISA, the sensitivity was improved more than 3-fold. The cross-reactivity (CR) was less than 0.08% for the tested structural analogues and was regarded as negligible. The recoveries of thiacloprid ranged from 80.3% to 116.3% in environmental and agricultural samples, which conformed to the requirements for residue detection. The amount of thiacloprid detected by phage ELISA in the samples was significantly correlated with that detected by high-performance liquid chromatography. The current study indicates that isolating phage-displayed peptides from phage display libraries is an alternative method for the development of a sensitive immunoassay and that the developed assay is a potentially useful tool for detecting thiacloprid in environmental and agricultural samples.     

Detection of 3-phenoxybenzoic acid in river water with a colloidal gold-based lateral flow immunoassay

3-Phenoxybenzoic acid (3-PBA) is a general metabolite of synthetic pyrethroids. It could be used as a generic biomarker for multiple pyrethroids exposure for human or pyrethroid residues in the environment. In this study, monoclonal antibodies (mAbs) against 3-PBA were developed by using PBA–bovine serum albumin (BSA) as an immunogen. In the competitive enzyme-linked immunosorbent assay (ELISA) format, the I₅₀ and I₁₀ values of purified mAbs were 0.63 and 0.13 μg/ml, respectively, with a dynamic range between 0.19 and 2.04 μg/ml. Then, the colloidal gold (CG)-based lateral flow immunoassay was established based on the mAbs. The working concentration of coating antigen and CG-labeled antibodies and the blocking effects were investigated to get optimal assay performance. The cutoff value for the assay was 1 μg/ml 3-PBA, and the detection time was within 10 min. A total of 40 river water samples were spiked with 3-PBA at different levels and determined by the lateral flow immunoassay without any sample pretreatments. The negative false rate was 2.5%, and no positive false results were observed at these levels. This lateral flow immunoassay has the potential to be an on-site screening method for monitoring 3-PBA or pyrethroid residues in environmental samples.     

Selection of phage-displayed peptides for the detection of imidacloprid in water and soil

Imidacloprid is the most widely used neonicotinoid insecticide in the world and shows widespread environment and human exposures. A phage clone designated L7-1 that selectively binds to imidacloprid was selected from a commercial phage display library containing linear 7-mer randomized amino acid residues. Using the clone L7-1, a competitive enzyme-linked immunosorbent assay (ELISA) for imidacloprid was developed. The half-maximum signal inhibition concentration (IC₅₀) and the limit of detection (LOD) of the phage ELISA for imidacloprid were 96 and 2.3 ng ml⁻¹, respectively. This phage ELISA showed relatively low cross-reactivity with all of the tested compounds structurally similar to imidacloprid, less than 2% with the exception of 6-chloronicotinic acid, a metabolite of imidacloprid that showed 11.5%. The average recoveries of the phage ELISA for imidacloprid in water and soil samples were in the ranges of 74.6 to 86.3% and 72.5 to 93.6%, respectively. The results of the competitive phage ELISA for imidacloprid in the fortified samples agreed well with those of a high-performance liquid chromatography (HPLC) method. The simple phage-displayed peptide technology has been proven to be a convenient and efficient method for the development of an alternative format of ELISA for small molecules.     

Extension of dynamic range of sensitive nanoparticle-based immunoassays

Nanoparticles have successfully been employed in immunometric assays that require high sensitivity. Certain analytes, however, require dynamic ranges (DRs) around a predetermined cut-off value. Here, we have studied the effects that antibody orientation and addition of free solid-phase and detection antibodies have on assay sensitivity and DR in traditional sandwich-type immunoassays. D-dimer and cardiac troponin I (cTnI), both routinely used in critical care testing, were applied as model analytes. The assays were performed in microtitration wells with preimmobilized solid-phase antibody. Inherently fluorescent nanoparticles coated with second antibody were used to detect the analyte. The selection of antibody orientation and addition of free solid-phase or detection antibody, with nanoparticles and calibrator, desensitized the assays and extended the DR. With D-dimer the upper limit of the DR was improved from 50 to 10,000 ng/ml, and with cTnI from 25 to 1000 ng/ml. Regression analysis with the Stago STA Liatest D-dimer assay yielded a slope (95% confidence interval) of 0.09 (0.07–0.11) and a y-intercept of −7.79 (−17.87–2.29) ng/L (n = 65, r = 0.906). Thus it is concluded that Europium(III)-chelate-doped nanoparticles can also be employed in immunoassays that require wide DRs around a certain cut-off limit.     

Cloning antifungal single chain fragment variable antibodies by phage display and competitive panning elution

Phage display and two competitive panning elution conditions were used to isolate Candida-specific single chain fragment variable (scFv) antibodies. An scFv phage library constructed from splenic lymphocytes of mice immunized by idiotypic vaccination with an HM-1 killer toxin (HM-1)-neutralizing monoclonal antibody (nmAb-KT) was used for panning against Candidaalbicans membrane fraction (CaMF). Key steps were specific elution conditions to separately release the bound phages with original antigen HM-1 + HM-1 peptide 6 and CaMF. The positive phages were screened by using enzyme-linked immunosorbent assay, and after nucleotide sequencing, clone expression, and purification, clone scFv-C1 was selected for detailed characterization. The scFv-C1 showed IC₅₀ values for cell growth against various Candida species and Saccharomyces cerevisiae as 2.40 to 6.40 μM and 2.20 μM, respectively. By using surface plasmon resonance analysis, the scFv-C1 had a K_d value of 3.09 × 10⁻¹¹ M to nmAb-KT, indicating a 260-fold higher affinity than for HM-1. These results showed the generated scFv-C1 mimicking HM-1-binding affinity to nmAb-KT and in vitro antifungal activity. We believe that the effectiveness of the competitive panning elution method and antigen-specific recombinant scFv antibodies obtained in this study are excellent candidates for antimycotic drugs.     

Screening of boldenone and methylboldenone in bovine urine using disposable electrochemical immunosensors

Electrochemical based immunosensors for the detection of boldenone and methylboldenone in bovine urine were described in this paper. The immunosensors were fabricated by immobilizing boldenone-bovine serum albumin conjugate on the surface of screen-printed electrodes (SPEs), and followed by the competition between the free analyte and coating conjugate with corresponding antibodies. The use of anti-species IgG-horseradish peroxidase conjugate determined the degree of competition. The electrochemical technique chosen was chronoamperometry, performed at a potential of +100 mV whereby the product of the catalysis of 3,3′,5,5′-tetramethylbenzidine undergoes reduction produced by the enzyme label. The limits of detection of assay were 30.9 ± 4.3 pg ml⁻¹ for boldenone and 120.2 ± 8.2 pg ml⁻¹ for methylboldenone, respectively. Results of repeated analysis of each androgen carried out using three different batches of electrodes indicate suitable repeatability (EC₅₀ = 1.0 ± 0.3 ng ml⁻¹ (n = 3, N = 3), R² = 0.969, R.S.D. = 9.6% for boldenone and 1.5 ± 0.3 ng ml⁻¹, 0.971, 10.5% for methylboldenone, respectively). Urine samples were determined directly after a single dilution step, omitting extraction and hydrolysis. This method offers the advantage to pick up both boldenone and its major metabolites in an efficient manner due to the high cross-reactivity pattern of α-boldenone with this antibody. The concentration of methylboldenone in urine detected by developed methods does indicate methylboldenone administration to heifers. Gas chromatography coupled to mass spectrometry analysis was performed to quantitate the individual metabolites present in urine samples, and results were validated with both ELISA and immunosensor data.     

Group determination of 14-membered macrolide antibiotics and azithromycin using antibodies against common epitopes

Erythromycin (ERY), clarithromycin (CLA), roxithromycin (ROX), and azithromycin (AZI) are macrolide antibiotics widely used in livestock and human medicine. Therefore, they are frequently found as pollutants in environmental water. A method based on indirect competitive enzyme-linked immunosorbent assay (ELISA) for group determination of these macrolides in foodstuffs, human biofluids, and water was developed. Carboxymethyloxime of clarithromycin (CMO–CLA) was synthesized and conjugated to bovine serum albumin (BSA) and gelatin to prepare immunogen and coating antigen with advantageous presentation of target epitopes, l-cladinose and d-desosamine, common for these analytes. Antibodies generated in rabbits were capable of recognizing ERY, CLA, and ROX as a group (100–150%), and AZI (12%) and did not cross-react with ERY degradants, which lack antibiotic activity. Assay displayed sensitivity of determination of 14-membered macrolides (IC₅₀ = 0.13–0.2 ng/ml) and low limit of detection (LOD) that was achieved at 0.02 to 0.03 ng/ml. It allowed performing analysis of milk, muscle, eggs, bovine serum, water, human serum and urine, and avoiding matrix effect without special pretreatment using simple dilution with assay buffer. For 15-membered macrolide AZI, the corresponding characteristics were IC₅₀ = 1.6 ng/ml and LOD = 0.14 ng/ml. The recoveries of veterinary and human medicine macrolides from corresponding matrices were validated and found to be satisfactory.     

Selection of staphylococcal enterotoxin B (SEB)-binding peptide using phage display technology

In this study, peptides were selected to recognize staphylococcal enterotoxin B (SEB) which cause food intoxication and can be used as a biological war agent. By using commercial M13 phage library, single plaque isolation of 38 phages was done and binding affinities were investigated with phage-ELISA. The specificities of the selected phage clones showing high affinity to SEB were checked by using different protein molecules which can be found in food samples. Furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (SPR) sensors. Sequence analysis was realized for three peptides showing high binding affinity to SEB and WWRPLTPESPPA, MNLHDYHRLFWY, and QHPQINQTLYRM amino acid sequences were obtained. The peptide sequence with highest affinity to SEB was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide-SEB interaction were determined by using isothermal titration calorimetry (ITC) and compared with those of antibody-SEB interaction. The binding constant of the peptide was determined as 4.2 ± 0.7 × 10⁵ M⁻¹ which indicates a strong binding close to that of antibody.     

Peptide sequences mediating tropism to intact blood–brain barrier: An in vivo biodistribution study using phage display

Peptide motifs that demonstrate tropism for the blood brain barrier (BBB) are of real translational value in developing innovative delivery strategies for biological brain targeted therapies. In vivo peptide-phage display affords peptide selection against the full complement of biological markers within the correct cellular macro- and micro-environments. Here a stringent in vivo biopanning protocol was employed in the rat aimed at identifying cyclic 7-mer peptide motifs that mediate tropism to brain microvasculature. Five rounds of biopanning identified 349 unique peptide motifs in the brain tissue gray matter compartment (microvasculature and parenchyma). While in general no consensus was evident linking peptide physico-chemical properties and brain tropism, peptides bearing c-SxTSSTx-c or c-xxxSSTx-c motifs were found to be present in high abundance. Based on amino acid frequency distribution of the 349 unique peptides sequences a theoretical ‘idealized’ peptide pattern, c-PP(S/P)SSST-c, could be derived. For the most abundant experimental peptide sequence found in brain tissue, c-SYTSSTM-c, an in vivo pharmacokinetic and whole body tissue biodistribution study was performed. Based upon tissue exposure data (i.e. tissue AUC_{(0–infinity)}) the sequence c-SYTSSTM-c efficiently retargeted phage virions to the brain providing an approximate 5-fold greater (P < 0.05) accumulation in brain over control phage; in all other organs no significant (P > 0.05) difference in tissue tropism between c-SYTSSTM-c and control phages were evident. This peptide and more generally the peptide motifs, -SxTSSTx- or -xxxSSTx-, warrant further investigation as agents mediating sequence-dependent tropism to brain microvasculature potentially able to deliver biologic cargo to the CNS.     

Design of a PKCδ-specific small peptide as a theragnostic agent for glioblastoma

Glioblastoma is an aggressive malignant brain tumor that starts in the brain or spine and frequently recurs after anticancer treatment. The development of an accurate diagnostic system combined with effective cancer therapy is essential to improve prognosis of glioma patients. Peptides, produced from phage display, are attractive biomolecules for glioma treatment because of their biostability, nontoxicity, and small size. In this study, we employed phage display methodology to screen for peptides that specifically recognize the target PKCδ as a novel biomarker for glioma. The phage library screening yielded four different peptides displayed on phages with a 20- to 200-pM K_d value for the recombinant PKCδ catalytic domain. Among these four phage peptides, we selected one to synthesize and tagged it with fluorescein isothiocyanate (FITC) based on the sequence of the PKCδ-binding phage clone. The synthetic peptide showed a relative binding affinity for antibody and localization in the U373 glioma cell. The kinase activity of PKCδ was inhibited by FITC-labeled peptide with an IC₅₀ of 1.4 μM in vitro. Consequently, the peptide found in this study might be a promising therapeutic agent against malignant brain tumor.     

Generation and application of a fluorescein-specific single chain antibody

A recombinant single chain antibody fragment (designated scDE1) of the murine monoclonal anti-fluorescein antibody B13-DE1 was generated using the original hybridoma cells as source for the variable antibody heavy and light chain (VH and VL) genes. After cloning the variable genes into a phage vector a functional antibody fragment was selected by phage display panning. Recombinant antibody could be expressed as phage antibody and as soluble single chain antibody in Escherichia coli. High yield of scDE1 could also be detected in bacterial culture supernatant. The scDE1 showed the same binding specificity as the parental monoclonal antibody, i.e. it bound fluorescein, fluorescein derivatives and a fluorescein peptide mimotope. Surface plasmon resonance revealed a K(D) of 19 nM for the scDE1 compared to 0.7 nM for the monoclonal antibody. The isolated soluble scDE1 could easily be conjugated to horseradish peroxidase which allowed the use of the conjugate as universal indicator for the detection of fluorescein-labelled proteins in different immunoassays. Detection of hCG in urine was performed as a model system using scDE1. In addition to E. coli the scFv genes could also be transferred and expressed in eukaryotic cells. Finally, we generated HEK293 cells expressing the scDE1 at the cell surface.     

Development of a competitive enzyme immunoassay for 17α-19-nortestosterone

Because 17β-19-nortestosterone and its esters are frequently used anabolic steroids in cattle in Europe, there is a need for an assay that can also detect certain metabolites. The enzyme immunoassay described here involves the detection and quantitation of the major metabolite 17α-19-nortestosterone in urine. The assay is based on the coating of an antibody raised in a rabbit against 17α-19-nortestosterone-3-carboxy-methyloxime—bovine serum albumin (17α-19-NT-3-CMO-BSA), the competitive incubation of 17α-19-NT and the 17α-19-nortestosterone-3-CMO—horseradish peroxidase label, followed by the detection of the blue colour developed by the action of the enzyme on tetramethylbenzidine. The 3-CMO conjugate of 17α-19-nortestosterone was used to produce an antibody with selective affinity for the 17α-epimer. For the optimization of the assay, different coatings and incubation conditions were tested. The standard curve ranged between 0.98 and 4000 pg per well, with a B/B₀ 50% of ± 65 pg per well. Colour was measured with a microtitre plate reader. The method was validated by means of certified blank and spiked cattle urine samples.     

A peptide-based fluorescent ratiometric sensor for quantitative detection of proteins

A ratiometric fluorescent sensor was obtained by solid-phase synthesis of a peptide singly labeled at its N-terminus with a 3-hydroxychromone (3HC) derivative, an environmentally sensitive fluorophore with a two-band emission. The construct contains the binding site recognized by an antibody fragment, scFv1F4_Q34S, with nanomolar (nM) affinity. The dye only marginally affected the kinetic and equilibrium binding parameters of the scFv-peptide interaction, as measured by surface plasmon resonance. On interaction with the antibody fragment, the sensor showed up to 47% change in the ratio of its two emission bands, indicating an enhanced screening of the 3HC fluorophore from bulk water. Competition with two unlabeled peptides of different lengths led to a dynamic displacement of the construct governed by the relative binding constants. Calibration showed that the response is proportional to the ratio of scFv1F4_Q34S to labeled peptide. The detection limit of scFv1F4_Q34S was 15 nM. In a more complex medium (100 μg/ml bovine serum albumin), the scFv could be detected in the 50- to 100-nM range. This work demonstrates that, with the perspective of further improvements of the dye spectroscopic properties, fluorescent ratiometric sensing based on small synthetic peptides represents a promising tool for quantitative target detection.     

Bacteriophage-based biosorbents coupled with bioluminescent ATP assay for rapid concentration and detection of Escherichia coli

Wild type T4 bacteriophage and recombinant T4 bacteriophages displaying biotin binding peptide (BCCP) and cellulose binding module (CBM) on their heads were immobilized on nano-aluminum fiber-based filter (Disruptor™), streptavidin magnetic beads and microcrystalline cellulose, respectively. Infectivity of the immobilized phages was investigated by monitoring the phage-mediated growth inhibition of bioluminescent E. coli B and cell lysis using bioluminescent ATP assay. The results showed that phage immobilization resulted in a partial loss of infectivity as compared with the free phage. Nevertheless, the use of a biosorbent based on T4 bacteriophage immobilized on Disruptor™ filter coupled with a bioluminescent ATP assay allowed simultaneous concentration and detection of as low as 6 × 10³ cfu/mL of E. coli in the sample within 2 h with high accuracy (CV = 1-5% in log scale). Excess of interfering microflora at levels 60-fold greater than the target organism did not affect the results when bacteriophage was immobilized on the filter prior to concentration of bacterial cells.     

A small compound targeting the interaction between nonstructural proteins 2B and 3 inhibits dengue virus replication

The non-structural protein NS2B/NS3 serine-protease complex of the dengue virus (DENV) is required for the maturation of the viral polyprotein. Dissociation of the NS2B cofactor from NS3 diminishes the enzymatic activity of the complex. In this study, we identified a small molecule inhibitor that interferes with the interaction between NS2B and NS3 using structure-based screening and a cell-based viral replication assay. A library containing 661,417 small compounds derived from the Molecular Operating Environment lead-like database was docked to the NS2B/NS3 structural model. Thirty-nine compounds with high scores were tested in a secondary screening using a cell-based viral replication assay. SK-12 was found to inhibit replication of all DENV serotypes (EC₅₀ = 0.74–4.92 μM). In silico studies predicted that SK-12 pre-occupies the NS2B-binding site of NS3. Steady-state kinetics using a fluorogenic short peptide substrate demonstrated that SK-12 is a noncompetitive inhibitor against the NS2B/NS3 protease. Inhibition to Japanese encephalitis virus by SK-12 was relatively weak (EC₅₀ = 29.81 μM), and this lower sensitivity was due to difference in amino acid at position 27 of NS3. SK-12 is the promising small-molecule inhibitor that targets the interaction between NS2B and NS3.     

Development of enzyme-linked immunosorbent assay for determination of polybrominated diphenyl ether BDE-121

Our interests are in the development of immunoassay-based fast scanning methods for persistent organic pollutants. To develop the immunoassay method of polybrominated diphenyl ether (PBDE), a model compound of PBDE, 2,3′,4,5′,6-pentabromodiphenylether (BDE-121), has been chosen to develop its antibody and the competitive indirect enzyme-linked immunosorbent assay (ELISA) is developed. The hapten of BDE-121 containing reactive carboxylic acid was synthesized and conjugated to carrier proteins (bovine serum albumin [BSA] and ovalbumin [OVA]). Anti-BDE-121 polyclonal antibody was then developed in rabbits as a result of immunization with the BDE-121–BSA conjugate. The optimal amount of coating antigen BDE-121–OVA conjugate and the dilution of antiserum needed in the ELISA were determined with the checkerboard method, and the effects of the properties of PBST (phosphate-buffered saline and Tween 20) buffer (pH and salt concentration) and chemical solvent (types and concentrations) on the ELISA were investigated to achieve a rapid robust assay with high sensitivity. Under the optimized conditions, the developed indirect ELISA shows a linear detection range from 1.74 to 84.1 ng/ml, with an IC₅₀ value of 8.07 ng/ml and a detection limit of 0.644 ng/ml. In total, 11 kinds of compounds were tested for calculating the cross-reactivity, which was less than 8% for nearly all of them. Real samples were analyzed by the proposed immunoassay and gas chromatography/mass spectrometry (GC/MS).     

Development of a highly sensitive ELISA for the determination of PBAN and its application to the analysis of hemolymph in Spodoptera littoralis

A highly sensitive enzyme linked immunosorbent assay (ELISA) for the determination of the pheromone biosynthesis activating neuropeptide (PBAN) has been developed. Six antisera have been obtained that recognize the carboxyl terminal side of this peptide. Two immunogens have been rationally designed and synthesized in order to direct antibody specificity, using as haptens PBAN or PBAN(20-33) with a Cys residue attached to their amino-terminal side. The Cys thiol group has been used to covalently bind the peptide to keyhole limpet hemocyanin (KLH) by using N-succinimidyl-4-(maleidimidomethyl) cyclohexane carboxylate (SMCC) as a convenient heterobifunctional cross-linker. Several usable competitive immunoassays have been obtained by synthesizing eight different coating antigens and screening the sera against all of them. The best assay was obtained with antibody 4 using Cys-Hez-PBAN(20-33) coupled to bovine serum albumin (BSA) through the Lys groups by using the homobifunctional cross-linker dimethylpimelidate dihydrochloride (DMP) as the coating antigen. The optimized assay allows to detect PBAN at concentrations as low as 1 fmol/well (l₅₀ = 2.5 fmol/well). An extraction procedure for the hemolymph has been developed that allows to perform PBAN measurements in this tissue even after a tenfold dilution. In these conditions matrix effect is negligible. Preliminary results on the presence of PBAN like immunoreactivity (PBAN-IR) in the hemolymph of Spodoptera littoralis females are reported.© 1995 Wiley-Liss, Inc.     

Determination of uric acid in human urine and serum by capillary electrophoresis with chemiluminescence detection

A simple and sensitive method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of uric acid (UA). The sensitive detection was based on the enhancement effect of UA on the CL reaction between luminol and potassium ferricyanide (K₃[Fe(CN)₆]) in alkaline solution. A laboratory-built reaction flow cell and a photon counter were deployed for the CL detection. Experimental conditions for CL detection were studied in detail to achieve a maximum assay sensitivity. Optimal conditions were found to be 1.0 × 10⁻⁴ M luminol added to the CE running buffer and 1.0 × 10⁻⁴ M K₃[Fe(CN)₆] in 0.2 M NaOH solution introduced postcolumn. The proposed CE-CL assay showed good repeatability (relative standard deviation [RSD] = 3.5%, n = 11) and a detection limit of 3.5 × 10⁻⁷ M UA (signal/noise ratio [S/N] = 3). A linear calibration curve ranging from 6.0 × 10⁻⁷ to 3.0 × 10⁻⁵ M UA was obtained. The method was evaluated by quantifying UA in human urine and serum samples with satisfactory assay results.