Secreted Expression of mRNA-Encoded Truncated ACE2 Variants for SARS-CoV-2 via Lipid-Like Nanoassemblies.

The transfer of foreign synthetic messenger RNA (mRNA) into cells is essential for mRNA-based protein-replacement therapies. Prophylactic mRNA COVID-19 vaccines commonly utilize nanotechnology to deliver mRNA encoding SARS-CoV-2 vaccine antigens, thereby triggering the body's immune response and preventing infections. In this study, a new combinatorial library of symmetric lipid-like compounds is constructed, and among which a lead compound is selected to prepare lipid-like nanoassemblies (LLNs) for intracellular delivery of mRNA. After multiround optimization, the mRNA formulated into core-shell-structured LLNs exhibits more than three orders of magnitude higher resistance to serum than the unprotected mRNA, and leads to sustained and high-level protein expression in mammalian cells. A single intravenous injection of LLNs into mice achieves over 95% mRNA translation in the spleen, without causing significant hematological and histological changes. Delivery of in-vitro-transcribed mRNA that encodes high-affinity truncated ACE2 variants (tACE2v mRNA) through LLNs induces elevated expression and secretion of tACE2v decoys, which is able to effectively block the binding of the receptor-binding domain of the SARS-CoV-2 to the human ACE2 receptor. The robust neutralization activity in vitro suggests that intracellular delivery of mRNA encoding ACE2 receptor mimics via LLNs may represent a potential intervention strategy for COVID-19.

Purification, characterization and specificity of a new GH family 35 galactosidase from Aspergillus awamori.

Galactosidases, ubiquitous in nature, are complex carbohydrate-active enzymes and find extensive applications in food, pharma, and biotechnology industries. The present study deals with the production of galactosidases from fungi by solid-state fermentation. Fifteen fungi were screened and Aspergillus awamori (MTCC 548), exhibited the highest α and ß-galactosidase activities of 75.11±0.29 U/g and 155.34±1.26 U/g, respectively. 30 g of wheat bran substituted with 6% defatted soy flour, at 28°C, pH 5.0 for 120 h, was established as the optimum production conditions by one-factor approach. The enzyme was purified to homogeneity with an apparent mass of 118 ± 2 kDa by ammonium sulfate precipitation (50-80%), ion exchange and hydrophobic interaction chromatography. Specific activities for α and ß-galactosidase were 22 and 74 U/mg, respectively. Optimum temperature and pH ranges for enzyme activities were 55-60 °C, 5.0-5.5, respectively. The thermal inactivation mid-point was 65 °C. The purified enzyme not only exhibited α and ß-galactosidase activities, but also exhibited ß-xylosidase and ß-glucosidase activities, indicating the enzyme has broad substrate specificity. Sequence analysis by in-gel digestion and tandem mass spectrometry (MS/MS) revealed that the enzyme was a probable ß-galactosidase A, belonging to glycoside hydrolase 35 family, and is being reported for the first time.

Trienzymatic Complex System for Isomerization of Agar-Derived d-Galactose into d-Tagatose as a Low-Calorie Sweetener.

d-Tagatose is a rare monosaccharide that is used in products in the food industry as a low-calorie sweetener. To facilitate biological conversion of d-tagatose, the agarolytic enzyme complexes based on the principle of the cellulosome structure were constructed through dockerin-cohesin interaction with the scaffoldin. The construction of agarolytic complexes composed of l-arabinose isomerase caused efficient isomerization activity on the agar-derived sugars. In a trienzymatic complex, the chimeric ß-agarase (cAgaB) and anhydro-galactosidase (cAhgA) from Zobellia galactanivorans could synergistically hydrolyze natural agar substrates and l-arabinose isomerase (LsAraA Doc) from Lactobacillus sakei 23K could convert d-galactose into d-tagatose. The trienzymatic complex increased the concentration of d-tagatose from the agar substrate to 4.2 g/L. Compared with the monomeric enzyme, the multimeric enzyme showed a 1.4-fold increase in tagatose production, good thermostability, and reusability. A residual activity of 75% remained, and 52% of conversion was noted after five recycles. These results indicated that the dockerin-fused chimeric enzymes on the scaffoldin successfully isomerized d-galactose into d-tagatose with synergistic activity. Thus, the results demonstrated the possibility of advancing efficient strategies for utilizing red algae as a biomass source to produce d-tagatose in the industrial food field that uses marine biomass as the feedstock.

Improvements in the production of Aspergillus oryzae ß-galactosidase crosslinked aggregates and their use in repeated-batch synthesis of lactulose.

Aspergillus oryzae ß-galactosidase was immobilized by aggregation and crosslinking, obtaining catalysts (CLAGs) well-endowed for lactulose synthesis. Type and concentration of the precipitating agent were determinants of immobilization yield, specific activity and thermal stability. CLAGs with specific activities of 64,007, 48,374 and 44,560 IUH g-1 were obtained using 50% v/v methanol, ethanol and propanol as precipitating agents respectively, with immobilization yields over 90%. Lactulose synthesis was conducted at 50 °C, pH 4.5, 50% w/w total sugars, 200 IUH g-1 of enzyme and fructose/lactose molar ratio of 8 in batch and repeated-batch operation. Lactulose yields were 0.19 g g-1 and 0.24 g g-1 for fructose to lactose molar ratios of 4 mol mol-1 and 8 mol mol-1 while selectivities were 3.3 mol mol-1 and 6.6 mol mol-1 respectively for CLAGs obtained by ethanol and propanol precipitation. Based on these results, both CLAGs were selected for the synthesis in repeated-batch mode. The cumulative mass of lactulose in repeated-batch was higher with CLAGs produced by ethanol and propanol precipitation than with the free enzyme. 86 and 93 repeated-batches could have been respectively performed with those CLAGs considering a catalyst replacement criterion of 50% of residual activity, as determined by simulation.

Nanoparticles decorated carbon nanotubes as novel matrix: A comparative study of influences of immobilization on the catalytic properties of Lensculinarisß-galactosidase (Lcß-gal).

In the present study, Multiwalled carbon nanotubes (MWCNT) decorated with two different nanoparticles namely tungsten disulfide (WS2) and tin oxide (SnO2), nanocomposites (NCs) were synthesized via hydrothermal method. Spectroscopic studies showed that both synthesized NCs possess nearly same functional groups but MWCNT-SnO2 NCs are rich in O-functional group. Microscopic studies revealed that both NCs have different morphological microstructure. Lens culinaris ß-galactosidase (Lcß-gal) was immobilized using glutaraldehyde cross-linker resulted in immobilization efficiency of 91.5% and 88% with MWCNT-WS2 and MWCNT-SnO2 NCs, respectively. Remarkable increase in rate of hydrolysis of whey lactose has been observed with both NCs i.e. Lcß-gal immobilized MWCNT-WS2 hydrolyzes the 97% whey lactose in 1.5 h while MWCNT-SnO2 showed maximum 92% of whey hydrolysis in 2 h at optimum conditions. Both nanobiocatalyst could serve as a promising candidates for dairy industries and would offer a potential platform for enzyme based biosensor fabrication.

Development of 11-DGA-3-O-Gal-Modified Cantharidin Liposomes for Treatment of Hepatocellular Carcinoma.

BACKGROUND: Liver cancer is a common malignant tumor worldwide, and its morbidity and mortality increase each year. The disease has a short course and high mortality, making it a serious threat to human health. PURPOSE: The objective of this study was to create novel liver-targeting nanoliposomes to encapsulate cantharidin (CTD) as a potential treatment for hepatic carcinoma. METHODS: 3-Galactosidase-30-stearyl deoxyglycyrrhetinic acid (11-DGA-3-O-Gal)-modified liposomes (11-DGA-3-O-Gal-CTD-lip) for the liver-targeted delivery of CTD were prepared via the film-dispersion method and characterized. In vitro analyses of the effects on cellular cytotoxicity, cell migration, cell cycle, and cell apoptosis were carried out and an in vivo pharmacokinetics study and tissue distribution analysis were performed. RESULTS: Compared with unmodified liposomes (CTD-lip), 11-DGA-3-O-Gal-CTD-lip showed higher cytotoxicity and increased the inhibition of HepG2 cell migration, but they did not increase the apoptotic rate of cells. The inhibition mechanism of 11-DGA-3-O-Gal-CTD-lip on hepatocellular carcinoma was partly through cell cycle arrest at the S phase. Analysis of pharmacokinetic parameters indicated that 11-DGA-3-O-Gal-CTD-lip were eliminated more rapidly than CTD-lip. Regarding tissue distribution, the targeting efficiency of 11-DGA-3-O-Gal-CTD-lip to the liver was (41.15 ± 3.28)%, relative targeting efficiency was (1.53 ± 0.31)%, relative uptake rate was( 1.69 ± 0.37)%, and peak concentration ratio was (2.68 ± 0.12)%. CONCLUSION: 11-DGA-3-O-Gal-CTD-lip represent a promising nanocarrier for the liver-targeted delivery of antitumor drugs to treat hepatocellular carcinoma.

Substrate preference of an ABC importer corresponds to selective growth on ß-(1,6)-galactosides in Bifidobacterium animalis subsp. lactis.

Bifidobacteria are exposed to substantial amounts of dietary ß-galactosides. Distinctive preferences for growth on different ß-galactosides are observed within Bifidobacterium members, but the basis of these preferences remains unclear. We previously described the first ß-(1,6)/(1,3)-galactosidase from Bifidobacterium animalis subsp. lactis Bl-04. This enzyme is relatively promiscuous, exhibiting only 5-fold higher efficiency on the preferred ß-(1,6)-galactobiose than the ß-(1,4) isomer. Here, we characterize the solute-binding protein (Bal6GBP) that governs the specificity of the ABC transporter encoded by the same ß-galactoside utilization locus. We observed that although Bal6GBP recognizes both ß-(1,6)- and ß-(1,4)-galactobiose, Bal6GBP has a 1630-fold higher selectivity for the former, reflected in dramatic differences in growth, with several hours lag on less preferred ß-(1,4)- and ß-(1,3)-galactobiose. Experiments performed in the presence of varying proportions of ß-(1,4)/ß-(1,6)-galactobioses indicated that the preferred substrate was preferentially depleted from the culture supernatant. This established that the poor growth on the nonpreferred ß-(1,4) was due to inefficient uptake. We solved the structure of Bal6GBP in complex with ß-(1,6)-galactobiose at 1.39 Å resolution, revealing the structural basis of this strict selectivity. Moreover, we observed a close evolutionary relationship with the human milk disaccharide lacto-N-biose-binding protein from Bifidobacterium longum, indicating that the recognition of the nonreducing galactosyl is essentially conserved, whereas the adjacent position is diversified to fit different glycosidic linkages and monosaccharide residues. These findings indicate that oligosaccharide uptake has a pivotal role in governing selectivity for distinct growth substrates and have uncovered evolutionary trajectories that shape the diversification of sugar uptake proteins within Bifidobacterium.

Mapping the Secretome and Its N-Linked Glycosylation of Pleurotus eryngii and Pleurotus ostreatus Grown on Hemp Stalks.

Our previous research showed that Pleurotus eryngii and Pleurotus ostreatus were effective fungi for pretreatment of industrial hemp stalks to improve enzymatic saccharification. The secretomes of these two fungi were analyzed to search for the effective enzyme cocktails degrading hemp lignin during the pretreatment process. In total, 169 and 155 proteins were identified in Pleurotus eryngii and Pleurotus ostreatus, respectively, and 50% of the proteins involved in lignocellulose degradation were CAZymes. Because most of the extracellular proteins secreted by fungi are glycosylated proteins, the N-linked glycosylation of enzymes could be mapped. In total, 27 and 24 N-glycosylated peptides were detected in Pleurotus eryngii and Pleurotus ostreatus secretomes, respectively. N-Glycosylated peptides of laccase, GH92, exoglucanase, phenol oxidase, α-galactosidase, carboxylic ester hydrolase, and pectin lyase were identified. Deglycosylation could decrease enzymatic saccharification of hemp stalks. The activities of laccase, α-galactosidase, and phenol oxidase and the thermal stability of laccase were reduced after deglycosylation.

Identification and biochemical characterization of a novel cold-adapted 1,3-α-3,6-anhydro-L-galactosidase, Ahg786, from Gayadomonas joobiniege G7.

Agar is a major polysaccharide of red algal cells and is mainly decomposed into neoagarobiose by the co-operative effort of ß-agarases. Neoagarobiose is hydrolyzed into monomers, D-galactose and 3,6-anhydro-L-galactose, via a microbial oxidative process. Therefore, the enzyme, 1,3-α-3,6-anhydro-L-galactosidase (α-neoagarobiose/neoagarooligosaccharide hydrolase) involved in the final step of the agarolytic pathway is crucial for bioindustrial application of agar. A novel cold-adapted α-neoagarooligosaccharide hydrolase, Ahg786, was identified and characterized from an agarolytic marine bacterium Gayadomonas joobiniege G7. Ahg786 comprises 400 amino acid residues (45.3 kDa), including a 25 amino acid signal peptide. Although it was annotated as a hypothetical protein from the genomic sequencing analysis, NCBI BLAST search showed 57, 58, and 59% identities with the characterized α-neoagarooligosaccharide hydrolases from Saccharophagus degradans 2-40, Zobellia galactanivorans, and Bacteroides plebeius, respectively. The signal peptide-deleted recombinant Ahg786 expressed and purified from Escherichia coli showed dimeric forms and hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose into 3,6-anhydro-L-galactose and other compounds by cleaving α-1,3-glycosidic bonds from the non-reducing ends of neoagarooligosaccharides, as confirmed by thin-layer chromatography and mass spectrometry. The optimum pH and temperature for Ahg786 activity were 7.0 and 15 °C, respectively, indicative of its unique cold-adapted features. The enzymatic activity severely inhibited with 0.5 mM ethylenediaminetetraacetic acid was completely restored or remarkably enhanced by Mn2+ in a concentration-dependent manner, suggestive of the dependence of the enzyme on Mn2+ ions. Km and Vmax values for neoagarobiose were 4.5 mM and 1.33 U/mg, respectively.

Carrageenan catabolism is encoded by a complex regulon in marine heterotrophic bacteria.

Macroalgae contribute substantially to primary production in coastal ecosystems. Their biomass, mainly consisting of polysaccharides, is cycled into the environment by marine heterotrophic bacteria using largely uncharacterized mechanisms. Here we describe the complete catabolic pathway for carrageenans, major cell wall polysaccharides of red macroalgae, in the marine heterotrophic bacterium Zobellia galactanivorans. Carrageenan catabolism relies on a multifaceted carrageenan-induced regulon, including a non-canonical polysaccharide utilization locus (PUL) and genes distal to the PUL, including a susCD-like pair. The carrageenan utilization system is well conserved in marine Bacteroidetes but modified in other phyla of marine heterotrophic bacteria. The core system is completed by additional functions that might be assumed by non-orthologous genes in different species. This complex genetic structure may be the result of multiple evolutionary events including gene duplications and horizontal gene transfers. These results allow for an extension on the definition of bacterial PUL-mediated polysaccharide digestion.

Crystal structure of ß1→6-galactosidase from Bifidobacterium bifidum S17: trimeric architecture, molecular determinants of the enzymatic activity and its inhibition by α-galactose.

In a search for better comprehension of ß-galactosidase function and specificity, we solved the crystal structures of the GH42 ß-galactosidase BbgII from Bifidobacterium bifidum S17, a well-adapted probiotic microorganism from the human digestive tract, and its complex with d-α-galactose. BbgII is a three-domain molecule that forms barrel-shaped trimers in solution. BbgII interactions with d-α-galactose, a competitive inhibitor, showed a number of residues that are involved in the coordination of ligands. A combination of site-directed mutagenesis of these amino acid residues with enzymatic activity measurements confirmed that Glu161 and Glu320 are fundamental for catalysis and their substitution by alanines led to catalytically inactive mutants. Mutation Asn160Ala resulted in a two orders of magnitude decrease of the enzyme kcat without significant modification in its Km , whereas mutations Tyr289Phe and His371Phe simultaneously decreased kcat and increased Km values. Enzymatic activity of Glu368Ala mutant was too low to be detected. Our docking and molecular dynamics simulations showed that the enzyme recognizes and tightly binds substrates with ß1→6 and ß1→3 bonds, while binding of the substrates with ß1→4 linkages is less favorable. DATABASE: Structural data are available in the PDB under the accession numbers 4UZS and 4UCF.

Structure of a plant ß-galactosidase C-terminal domain.

Most plant ß-galactosidases, which belong to glycoside hydrolase family 35, have a C-terminal domain homologous to animal galactose and rhamnose-binding lectins. To investigate the structure and function of this domain, the C-terminal domain of the rice (Oryza sativa L.) ß-galactosidase 1 (OsBGal1 Cter) was expressed in Escherichia coli and purified to homogeneity. The free OsBGal1 Cter is monomeric with a native molecular weight of 15kDa. NMR spectroscopy indicated that OsBGal1 Cter comprises five ß-strands and one α-helix. The structure of this domain is similar to lectin domains from animals, but loops A and C of OsBGal1 Cter are longer than the corresponding loops from related animal lectins with known structures. In addition, loop A of OsBGal1 Cter was not well defined, suggesting it is flexible. Although OsBGal1 Cter was predicted to be a galactose/rhamnose-binding domain, binding with rhamnose, galactose, glucose, ß-1,4-d-galactobiose and raffinose could not be observed in NMR experiments.

Enzyme Shielding in an Enzyme-thin and Soft Organosilica Layer.

The fragile nature of most enzymes is a major hindrance to their use in industrial processes. Herein, we describe a synthetic chemical strategy to produce hybrid organic/inorganic nanobiocatalysts; it exploits the self-assembly of silane building blocks at the surface of enzymes to grow an organosilica layer, of controlled thickness, that fully shields the enzyme. Remarkably, the enzyme triggers a rearrangement of this organosilica layer into a significantly soft structure. We demonstrate that this change in stiffness correlates with the biocatalytic turnover rate, and that the organosilica layer shields the enzyme in a soft environment with a markedly enhanced resistance to denaturing stresses.

A mechanism-based inactivator of glycoside hydrolases involving formation of a transient non-classical carbocation.

The design of mechanism-based enzyme inactivators to generate chemical probes for biological research is an important challenge in carbohydrate chemistry. Here we describe the synthesis and biological evaluation of a novel carbocyclic mechanism-based inactivator of galactosidases (glycoside hydrolase families 27 and 36). Upon catalysis of this unnatural substrate, a transient non-classical carbocation forms within the enzyme active site. We show that the inactivation event, which proceeds via a bicyclobutonium ion intermediate, leads to a single alkylation event that occurs on the enzymatic nucleophile, an aspartic acid residue in this case. We also show that the catalytic proficiencies for enzymatic hydrolysis of substrates and inactivation by our bicyclo[4.1.0]heptyl analogue of galactose differ by only a factor of 20. This inactivator has the potential for further development as a useful biological research tool for both basic research and biotechnological applications.

Evaluation of the binding of polyhedral borane anions to representative proteins.

The ability of three polyhedral borane anions, [B20H18](2-), [B20H17SH](4-), and [B20H19](3-), to bind to proteins was evaluated by measuring the total boron content using inductively coupled plasma-atomic emission spectroscopy after purification by gel electrophoresis. Results were correlated to the known chemical reactivity of the compounds as well as the reported murine biodistributions of the liposomally encapsulated sodium salts of each of the polyhedral borane anions. Qualitative reactions were performed with the [B20H18](2-) anion to determine the potential reactivity with simple molecular building blocks.

A thermally-stable enzyme detection assay that amplifies signal autonomously in water without assistance from biological reagents.

This Communication describes a thermally-stable small molecule and a corresponding assay strategy that autonomously amplifies a colorimetric signal when a specific enzyme biomarker is detected.

Role of protons in sugar binding to LacY.

WT lactose permease of Escherichia coli (LacY) reconstituted into proteoliposomes loaded with a pH-sensitive fluorophore exhibits robust uphill H(+) translocation coupled with downhill lactose transport. However, galactoside binding by mutants defective in lactose-induced H(+) translocation is not accompanied by release of an H(+) on the interior of the proteoliposomes. Because the pK(a) value for galactoside binding is ∼10.5, protonation of LacY likely precedes sugar binding at physiological pH. Consistently, purified WT LacY, as well as the mutants, binds substrate at pH 7.5-8.5 in detergent, but no change in ambient pH is observed, demonstrating directly that LacY already is protonated when sugar binds. However, a kinetic isotope effect (KIE) on the rate of binding is observed, indicating that deuterium substitution for protium affects an H(+) transfer reaction within LacY that is associated with sugar binding. At neutral pH or pD, both the rate of sugar dissociation (k(off)) and the forward rate (k(on)) are slower in D(2)O than in H(2)O (KIE is ∼2), and, as a result, no change in affinity (K(d)) is observed. Alkaline conditions enhance the effect of D(2)O on k(off), the KIE increases to 3.6-4.0, and affinity for sugar increases compared with H(2)O. In contrast, LacY mutants that exhibit pH-independent high-affinity binding up to pH 11.0 (e.g., Glu325 → Gln) exhibit the same KIE (1.5-1.8) at neutral or alkaline pH (pD). Proton inventory studies exhibit a linear relationship between k(off) and D(2)O concentration at neutral and alkaline pH, indicating that internal transfer of a single H(+) is involved in the KIE.

Prebiotics and bioactive natural substances induce changes of composition and metabolic activities of the colonic microflora in cancerous rats.

Prebiotics are defined as selectively fermented food ingredients that induce specific changes in the composition and/or activity in the gastrointestinal microbiota beneficial to the host well-being and health. The aim of the presented experiment was to investigate the effect of a prebiotic applied alone or in combination with Hyppocastani extractum siccum, and Lini oleum virginale in rats with dimethylhydrazine induced colon cancer. Wistar albino rats were fed high fat diet supplemented with the prebiotic alone or in combination with Horse chestnut and flaxseed oil. The activity of faecal glycolytic enzymes, lipid parameters, bile acids, short chain fatty acids and counts of coliforms and lactobacilli were determined. Treatment with the prebiotic alone and in combination with selected substances significantly decreased the activity of glycolytic bacterial enzyme ß-glucuronidase (P<0.001) and increased activities of ß-galactosidase and ß-glucosidase. Bile acids concentration was significantly decreased (P<0.01) except for the combination of the prebiotic with Horse chestnut. The prebiotic alone decreased the lipid parameters (P<0.001) and enhanced production of short chain fatty acids. Application of prebiotic and bioactive natural substances significantly reduced number of coliforms (P<0.05). Prebiotic alone significantly increased the count of lactobacilli (P<0.05). These results show that prebiotics have a protective effect and may be the useful for colon cancer prevention and treatment.

Cloning, purification, and characterization of ß-galactosidase from Bacillus licheniformis DSM 13.

The gene encoding homodimeric ß-galactosidase (lacA) from Bacillus licheniformis DSM 13 was cloned and overexpressed in Escherichia coli, and the resulting recombinant enzyme was characterized in detail. The optimum temperature and pH of the enzyme, for both o-nitrophenyl-ß-D: -galactoside (oNPG) and lactose hydrolysis, were 50°C and 6.5, respectively. The recombinant enzyme is stable in the range of pH 5 to 9 at 37°C and over a wide range of temperatures (4-42°C) at pH 6.5 for up to 1 month. The K(m) values of LacA for lactose and oNPG are 169 and 13.7 mM, respectively, and it is strongly inhibited by the hydrolysis products, i.e., glucose and galactose. The monovalent ions Na(+) and K(+) in the concentration range of 1-100 mM as well as the divalent metal cations Mg²(+), Mn²(+), and Ca²(+) at a concentration of 1 mM slightly activate enzyme activity. This enzyme can be beneficial for application in lactose hydrolysis especially at elevated temperatures due to its pronounced temperature stability; however, the transgalactosylation potential of this enzyme for the production of galacto-oligosaccharides (GOS) from lactose was low, with only 12% GOS (w/w) of total sugars obtained when the initial lactose concentration was 200 g/L.

Improved specific biodetection with ion trap mobility spectrometry (ITMS): a 10-min, multiplexed, immunomagnetic ELISA.

Enabling trace chemical detection equipment utilized in the field to transduce a biodetection assay would be advantageous from a logistics, training, and maintenance standpoint. Described herein is an assay design that uses an unmodified, commercial off-the-shelf (COTS) ion trap mobility spectrometer to analyze an immunomagnetic enzyme-linked immunosorbant assay (ELISA). The assay, which uses undetectable enzymatic substrates and ELISA-generated detectable products, was optimized to quantitatively report the amount of target in the sample. Optimization of this ELISA design retained the assay specificity and detection limit (approximately 10(3) E. coli per assay) while decreasing the number of user steps and reducing the assay time to 10 min (>9-fold decrease as compared to past studies). Also discussed are previously undescribed, independent substrate/enzyme/product combinations used in the immunomagnetic ELISA. These discoveries allow for the possibility of a quantitative, multiplexed, 10-min assay that is analyzed by the ion trap mobility spectrometer trace chemical detector.