Mark R. McCoy

Gas chromatographic quantification of fatty acid methyl esters: flame ionization detection vs. electron impact mass spectrometry.

The determination of FAME by GC is among the most commonplace analyses in lipid research. Quantification of FAME by GC with FID has been effectively performed for some time, whereas detection with MS has been used chiefly for qualitative analysis of FAME. Nonetheless, the sensitivity and selectivity of MS methods advocate a quantitative role for GC-MS in FAME analysis—an approach that would be particularly advantageous for FAME determination in complex biological samples, where spectrometric confirmation of analytes is advisable. To assess the utility of GC-MS methods for FAME quantification, a comparative study of GC-FID and GC-MS methods has been conducted. FAME in prepared solutions as well as a biological standard reference material were analyzed by GC-FID and GC-MS methods using both ion trap and quadrupole MS systems. Quantification by MS, based on total ion counts and processing of selected ions, was investigated for FAME ionized by electron impact. Instrument precision, detection limits, calibration behavior, and response factors were investigated for each approach, and quantitative results obtained by each technique were compared. Although there were a number of characteristic differences between the MS methods and FID with respect to FAME analysis, the quantitative performance of GC-MS compared satisfactorily with that of GC-FID. The capacity to combine spectrometric examination and quantitative determination advances GC-MS as a powerful alternative to GC-FID for FAME analysis.

Isolation of alpaca anti-hapten heavy chain single domain antibodies for development of sensitive immunoassay.

Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain (VHH). Although conventional antibodies dominate current assay development, recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. We expressed VHHs from an immunized alpaca and developed a VHH-based immunoassay using 3-phenoxybenzoic acid (3-PBA), a major metabolite of pyrethroid insecticides as a model system. A phage VHH library was constructed, and seven VHH clones were selected by competitive binding with 3-PBA. The best immunoassay developed with one of these VHHs showed an IC(50) of 1.4 ng/mL (limit of detection (LOD) = 0.1 ng/mL). These parameters were further improved by using the phage borne VHH, IC(50) = 0.1 ng/mL and LOD = 0.01 ng/mL. Both assays showed a similar tolerance to methanol and dimethylsulfoxide up to 50% in assay buffer. The assay was highly specific to 3-PBA and its 4-hydroxylated derivative, 4-hydroxy 3-PBA, (150% cross reactivity) with negligible cross reactivity with other tested structural analogues, and the recovery from spiked urine sample ranged from 80 to 112%. In conclusion, a highly specific and sensitive VHH for 3-PBA was developed using sequences from immunized alpaca and phage display technology for antibody selection.

Engineered Antibody Fragments with Infinite Affinity as Reporter Genes for PET Imaging

Reporter gene imaging has great potential for many clinical applications including the tracking of transplanted cells and monitoring of gene therapy. However, currently available reporter gene–reporter probe combinations have significant limitations with the biodistribution of the reporter probe and the specificity and immunogenicity of the reporter gene. The objective of the present study was to evaluate a new approach for reporter gene imaging based on cell surface expression of antibody fragments that can irreversibly bind to radiometal chelates. Methods: We developed a new reporter gene, designated 1,4,7,10-tetraazacyclodocecane-N,N′,N″,N‴-tetraacetic acid (DOTA) antibody reporter 1 (DAbR1), which consists of the single-chain Fv (scFv) fragment of the anti-Y-DOTA antibody 2D12.5/G54C fused to the human T cell CD4 transmembrane domain. The corresponding reporter probe is yttrium-(S)-2-(4-acrylamidobenzyl)-DOTA (*Y-AABD), a DOTA complex that binds irreversibly to a cysteine residue in the 2D12.5/G54C antibody. U-87 glioma cells were stably transfected with a DAbR1 expression vector. Binding of *Y-AABD to transfected and wild-type cells was studied in vitro and in vivo. Results: Flow cytometry revealed high expression of the DAbR1 protein on the cell surface of tumor cells. Uptake of 90Y-AABD in DAbR1-expressing human U-87 glioma xenografts was 6.2 (±1.3) percentage injected dose per gram (%ID/g) at 1 h and 4.9 (±0.62) %ID/g at 24 h after injection. The corresponding tumor-to-plasma ratios were 45:1 and 428:1, respectively. Uptake by U-87 tumors without the DAbR1 gene was 0.16 (±0.02) %ID/g at 1 h and 0.05 (±0.03) %ID/g at 24 h. PET images in mice with 86Y-AABD demonstrated intense uptake in DAbR1-positive tumors and low background activity in the liver. Conclusion: These findings indicate that cell surface expression of radiometal chelate binding antibodies such as 2D12.5/G54C is a promising strategy for reporter gene imaging.

Noncompetitive Phage Anti-Immunocomplex Real-Time Polymerase Chain Reaction for Sensitive Detection of Small Molecules

Immuno polymerase chain reaction (IPCR) is an analytical technology based on the excellent affinity and specificity of antibodies combined with the powerful signal amplification of polymerase chain reaction (PCR), providing superior sensitivity to classical immunoassays. Here we present a novel type of IPCR termed phage anti-immunocomplex assay real-time PCR (PHAIA-PCR) for the detection of small molecules. Our method utilizes a phage anti-immunocomplex assay (PHAIA) technology in which a short peptide loop displayed on the surface of the M13 bacteriophage binds specifically to the antibody-analyte complex, allowing the noncompetitive detection of small analytes. The phagemid DNA encoding this peptide can be amplified by PCR, and thus, this method eliminates hapten functionalization or bioconjugation of a DNA template while providing improved sensitivity. As a proof of concept, two PHAIA-PCRs were developed for the detection of 3-phenoxybenzoic acid, a major urinary metabolite of some pyrethroid insecticides, and molinate, a herbicide implicated in fish kills. Our results demonstrate that phage DNA can be a versatile material for IPCR development, enabling universal amplification when the common element of the phagemid is targeted or specific amplification when the real time PCR probe is designed to anneal the DNA encoding the peptide. The PHAIA-PCRs proved to be 10-fold more sensitive than conventional PHAIA and significantly faster using magnetic beads for rapid separation of reactants. The assay was validated with both agricultural drain water and human urine samples, showing its robustness for rapid monitoring of human exposure or environmental contamination.

Tetramethylenedisulfotetramine Alters Ca2+ Dynamics in Cultured Hippocampal Neurons: Mitigation by NMDA Receptor Blockade and GABAA Receptor-Positive Modulation

Tetramethylenedisulfotetramine (TETS) is a potent convulsant that is considered a chemical threat agent. We characterized TETS as an activator of spontaneous Ca²⁺ oscillations and electrical burst discharges in mouse hippocampal neuronal cultures at 13-17 days in vitro using FLIPR Fluo-4 fluorescence measurements and extracellular microelectrode array recording. Acute exposure to TETS (≥ 2 µM) reversibly altered the pattern of spontaneous neuronal discharges, producing clustered burst firing and an overall increase in discharge frequency. TETS also dramatically affected Ca²⁺ dynamics causing an immediate but transient elevation of neuronal intracellular Ca²⁺ followed by decreased frequency of Ca²⁺ oscillations but greater peak amplitude. The effect on Ca²⁺ dynamics was similar to that elicited by picrotoxin and bicuculline, supporting the view that TETS acts by inhibiting type A gamma-aminobutyric acid (GABA(A)) receptor function. The effect of TETS on Ca²⁺ dynamics requires activation of N-methyl-D-aspartic acid (NMDA) receptors, because the changes induced by TETS were prevented by MK-801 block of NMDA receptors, but not nifedipine block of L-type Ca²⁺ channels. Pretreatment with the GABA(A) receptor-positive modulators diazepam and allopregnanolone partially mitigated TETS-induced changes in Ca²⁺ dynamics. Moreover, low, minimally effective concentrations of diazepam (0.1 µM) and allopregnanolone (0.1 µM), when administered together, were highly effective in suppressing TETS-induced alterations in Ca²⁺ dynamics, suggesting that the combination of positive modulators of synaptic and extrasynaptic GABA(A) receptors may have therapeutic potential. These rapid throughput in vitro assays may assist in the identification of single agents or combinations that have utility in the treatment of TETS intoxication.

Characterization of Seizures Induced by Acute and Repeated Exposure to Tetramethylenedisulfotetramine

Tetramethylenedisulfotetramine (tetramine; TETS) is a potent convulsant poison that is considered to be a chemical threat agent. To provide a basis for the investigation of antidotes for TETS-induced seizures, we characterized the convulsant activity of TETS in mice and rats when administered by the intraperitoneal, intravenous, oral, and intraventricular routes as a single acute dose and with repeated sublethal doses. In mice, parenteral and oral TETS caused immobility, myoclonic body jerks, clonic seizures of the forelimbs and/or hindlimbs, tonic seizures, and death. The CD50 values for clonic and tonic seizures after oral administration were 0.11 and 0.22 mg/kg, respectively. Intraventricular administration of TETS (5–100 μg) in rats also caused clonic-tonic seizures and death. In mice, repeated sublethal doses of TETS at intervals of 2, 24, and 48 h failed to result in the development of persistent enhanced seizure responsivity (“kindling”) as was observed with repeated pentylenetetrazol treatment. In mice, sublethal doses of TETS that produced clonic seizures did not cause observable structural brain damage as assessed with routine histology and Fluoro-Jade B staining 7 days after treatment. However, 1 to 3 days after a single convulsant dose of TETS the expression of glial fibrillary acidic protein, an astrocyte marker, and ionized calcium binding adaptor molecule 1, a microglia marker, were markedly increased in cortex and hippocampus. Although TETS doses that are compatible with survival are not associated with overt evidence of cellular injury or neurodegeneration, there is transient reactive astrocytosis and microglial activation, indicating that brain inflammatory responses are provoked.

Development and Utilization of Camelid VHH Antibodies from Alpaca for 2,2′,4,4′-Tetrabrominated Diphenyl Ether Detection

An antibody-based analytical method for the detection of a chemical flame retardant using antibody fragments isolated from an alpaca has been developed. One specific chemical flame retardant congener, 2,2′,4,4′-tetrabrominated diphenyl ether (BDE-47), is often the major poly-BDE (PBDE) congener present in human and environmental samples and that which is the most frequently detected. An alpaca was immunized with a surrogate of BDE-47 covalently attached to a carrier protein. The resulting mRNA coding for the variable domain of heavy-chain antibodies (VHH) were isolated, transcribed to cDNA, and cloned into a phagemid vector for phage display library construction. Selection of VHHs recognizing BDE-47 was achieved by panning under carefully modified conditions. The assay sensitivity for detecting BDE-47 was down to the part-per-billion (microgram per liter) level. Cross-reactivity analyses confirmed that this method was highly selective for BDE-47 and selected hydroxylated metabolites. When exposed to elevated temperatures, the camelid VHH antibodies retained more reactivity than a polyclonal antibody developed to the same target analyte. The use of this VHH antibody reagent immobilized onto a Au electrode for impedance biosensing demonstrates the increased versatility of VHH antibodies.

Monitoring of Total Type II Pyrethroid Pesticides in Citrus Oils and Water by Converting to a Common Product 3-Phenoxybenzoic Acid

Pyrethroids are a class of insecticides that are becoming increasingly popular in agricultural and home use applications. Sensitive assays for pyrethroid insecticides in complex matrices are difficult with both instrumental and immunochemical methods. Environmental analysis of the pyrethroids by immunoassay requires either knowing which pyrethroids contaminate the source or the use of nonspecific antibodies with cross-reactivities to a class of compounds. We describe an alternative method that converts the type II pyrethroids to a common chemical product, 3-phenoxybenzoic acid (3-PBA), prior to analysis. This method is much more sensitive than detecting the parent compound, and it is much easier to detect a single compound rather than an entire class of compounds. This is useful in screening for pyrethroids as a class or in situations where a single type of pyrethroid is used. We demonstrated this technique in both citrus oils and environmental water samples with conversion rates of the pyrethroid to 3-PBA that range from 45 to 75% and methods that require no extraction steps for either the immunoassay or the liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques. Limits of detection for this technique applied to orange oil are 5 nM, 2 μM, and 0.8 μM when detected by LC-MS/MS, gas chromatography-mass spectrometry, and immunoassay, respectively. The limit of detection for pyrethroids in water when detected by immunoassay was 2 nM.

Behavioral assessment of NIH Swiss mice acutely intoxicated with tetramethylenedisulfotetramine

Tetramethylenedisulfotetramine (TETS) is a potent convulsant poison that is thought to trigger seizures by inhibiting the function of the type A gamma-aminobutyric acid receptor (GABAAR). Acute intoxication with TETS can cause vomiting, convulsions, status epilepticus (SE) and even death. Clinical case reports indicate that individuals who survive poisoning may exhibit long-term neuropsychological issues and cognitive deficits. Therefore, the objective of this research was to determine whether a recently described mouse model of acute TETS intoxication exhibits persistent behavioral deficits. Young adult male NIH Swiss mice received a seizure-inducing dose of TETS (0.15mg/kg, ip) and then were rescued from lethality by administration of diazepam (5mg/kg, ip) approximately 20min post-TETS-exposure. TETS-intoxicated mice typically exhibited 2 clonic seizures prior to administration of diazepam with no subsequent seizures post-diazepam injection as assessed using behavioral criteria. Seizures lasted an average of 72s. Locomotor activity, anxiety-like and depression-relevant behaviors and cognition were assessed at 1week, 1month and 2months post-TETS exposure using open field, elevated-plus maze, light↔dark transitions, tail suspension, forced swim and novel object recognition tasks. Interestingly, preliminary validation tests indicated that NIH Swiss mice do not respond to the shock in fear conditioning tasks. Subsequent evaluation of hot plate and tail flick nociception tasks revealed that this strain exhibits significantly decreased pain sensitivity relative to age- and sex-matched C57BL/6J mice, which displayed normal contextual fear conditioning. NIH Swiss mice acutely intoxicated with TETS exhibited no significant anxiety-related, depression-relevant, learning or memory deficits relative to vehicle controls at any of the time points assessed with the exception of significantly increased locomotor activity at 2months post-TETS intoxication. The general absence of long-term behavioral deficits in TETS-intoxicated mice on these six assays suggests that the neurobehavioral consequences of TETS exposure described in human survivors of acute TETS intoxication are likely due to sustained seizure activity, rather than a direct effect of the chemical itself. Future research efforts are directed toward developing an animal model that better recapitulates the SE and seizure duration reported in humans acutely intoxicated with TETS.

Immunoassays and Biosensors for Monitoring Environmental and Human Exposure to Pyrethroid Insecticides

This paper describes some of the early work on pyrethroid insecticides in the Casida laboratory and briefly reviews the development and application of immunochemical approaches for the detection of pyrethroid insecticides and their metabolites for monitoring environmental and human exposure. Multiple technologies can be combined to enhance the sensitivity and speed of immunochemical analysis. The pyrethroid assays are used to illustrate the use of some of these immunoreagents such as antibodies, competitive mimics, and novel binding agents such as phage-displayed peptides. The paper also illustrates reporters such as fluorescent dyes, chemiluminescent compounds, and luminescent lanthanide nanoparticles, as well as the application of magnetic separation, and automatic instrumental systems, biosensors, and novel immunological technologies. These new technologies alone and in combination result in an improved ability to both determine if effective levels of pyrethroids are being used in the field and evaluate possible contamination.