Multisubstrate-compatible ELISA procedures for rapid and high-sensitivity immunoassays

This protocol describes an improved and optimized approach to develop rapid and high-sensitivity ELISAs by covalently immobilizing antibody on chemically modified polymeric surfaces. The method involves initial surface activation with KOH and an O(2) plasma, and then amine functionalization with 3-aminopropyltriethoxysilane. The second step requires covalent antibody immobilization on the aminated surface, followed by ELISA. The ELISA procedure developed is 16-fold more sensitive than established methods. This protocol could be used generally as a quantitative analytical approach to perform high-sensitivity and rapid assays in clinical situations, and would provide a faster approach to screen phage-displayed libraries in antibody development facilities. The antibody immobilization procedure is of ～3 h duration and facilitates rapid ELISAs. This method can be used to perform assays on a wide range of commercially relevant solid support matrices (including those that are chemically inert) with various biosensor formats.     

Bioluminescent Listeria monocytogenes provide a rapid assay for measuring biocide efficacy

Abstract A recombinant derivative of Listeria monocytogenes 23074, engineered to express the luxAB genes of Vibrio fischeri MJ1, has a bioluminescent phenotype that provides a rapid monitor of microbial viability. The antibacterial activity of phenol and chlorhexidine diacetate (Hibitane) was measured using both bioluminescence and viable counts. Concentration exponents were assessed as 7.3 for phenol and 2.63 for chlorhexidine diacetate using plate counts. The rapidity of bioluminescence measurement constitutes a major advantage in biocide assessment.     

Solid supports in enzyme-linked immunosorbent assay and other solid-phase immunoassays

A very large proportion of modern immunoassays involve the use of synthetic solid phases to immobilize one of the reactants. These solid-phase immunoassays (SPIs) therefore involve ligand-receptor interactions that occur within a reaction volume close to the solution/solid phase interface. As a consequence, the immunochemistry/biochemistry of these ligand-receptor interactions differs from that of their counterparts in solution. Furthermore, the immobilization process can significantly alter the biological activity of the reactant; most adsorbed proteins on polystyrene or silicone are partially or largely denatured. Therefore the use of alternative methods of immobilization is attractive but may result in little increase in the amount of total functional reactant. However, all commonly used solid phases do not have the same properties or the same capacity for reactant immobilization or experience the same level of nonspecific binding. Empiricism plays a major role in SPIs. Derivations of mass law equations for measuring the antigen capture of solid-phase antibodies, for determining the affinity of solid phase for protein adsorption, and for estimating antibody affinity are reviewed.     

Bioluminescent assay as a potential method of rapid susceptibility testing

A method of rapid susceptibility testing by bioluminescent assay was developed. Correlation between the 50% inhibition dose of antimicrobics for bacterial adenosine triphosphate measured by bioluminescent assay and the minimum inhibitory concentration obtained by the broth dilution method was satisfactory. In the bioluminescent assay the incubation time required was only 90 min.     

A multiple homogenizer for rapid sample preparation in immunoassays and electrophoresis

A multiple homogenizer is described for preparing samples of small invertebrates or tissue in a flat-bottom immunoplate. Its efficiency was evaluated by immunoassay of a carboxylesterase (E4), the enzyme conferring insecticide resistance in the peach potato aphid (Myzus persicae). This equipment was shown to release more enzyme, with less variability, than homogenizing individual aphids and its efficiency allows one person to analyze up to 3000 individual insects per day. It is also suitable for preparing samples for electrophoretic analysis. In the present study samples were loaded onto electrophoresis gels rapidly and accurately by using an eight-channel multi-pipette.R. H. ffrench-Constant was supported by an AFRC studentship.     

Bioluminescent immunoassay for alpha-fetoprotein

A bioluminescent immunoassay of alpha-fetoprotein is described. It uses monoclonal antibodies labeled with glucose-6-phosphate dehydrogenase and polyclonal antibodies coimmobilized on Sepharose with bioluminescent enzymes from marine bacteria. The bioluminescent reaction which occurs in the immunosorbent is proportional to the amount of alpha-fetoprotein in the assay. The protocol is simple and rapid, and no separation step is required to remove the excess labeled antibodies. The assay can be performed directly on 25 microliters serum and it is as sensitive as other immunometric assays.     

Novel mass spectrometric immunoassays for the rapid structural characterization of plasma apolipoproteins

Novel mass spectrometric immunoassays (MSIAs) for the isolation and structural characterization of plasma apolipoprotein A-I (apoA-I), apoA-II, and apoE have been developed. The assays combine selective isolation of apolipoprotein species via affinity capture with mass-specific detection using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In application, plasma (from 50 microl of whole blood drawn from individuals, using finger lancet) was addressed with affinity pipette tips derivatized with antibodies toward the specific apolipoprotein. The time required for each assay was approximately 15 min, less if assays on multiple individuals were performed in parallel. In a brief study of five individuals, several recently reported apoA-II variants were identified and observed consistently in all individuals. Additionally, the apoE phenotype of E3/E3 was observed in three of the individuals, and E2/E3 and E3/E4 observed in the remaining two individuals, the latter of whom suffers from Alzheimer's disease. Overall, the MSIA approach offers a rapid, sensitive, and highly accurate means of profiling apolipoproteins from small volumes of plasma.     

Bioluminescent assay of bacterial ATP for rapid detection of bacterial growth in clinical blood cultures

A bioluminescent assay of bacterial ATP for rapid detection of bacterial growth in 512 clinical aerobic blood cultures was evaluated. At the detection limit of bacterial ATP (10^?10 mol/l) in the blood cultures 94.2% of the true positive blood cultures were detected (sensitivity) and the specificity was 85.8%. If the cut-off limit was increased the sensitivity decreased and the specificity increased and at 2 × 10^?9 mol/l ATP the maximum correctly classified blood cultures was reached. At this cut-off limit the sensitivity was 82.9% and the specificity was 99.6%. In 54.3% of the true positive blood cultures bacterial growth was detected more rapidly with the bioluminescent assay than with macroscopic examination and subculture.     

Bioluminescent Salmonella typhimurium provides a rapid assay for measuring the efficacy of freeze-drying suspension media

A strain of Salmonella typhimurium , transformed to a bioluminescent phenotype, was used to compare three freeze-drying suspension media: inositol serum broth with and without added gelatin and sterile skimmed milk. Recovery and growth studies performed by measuring changes in bioluminescence demonstrated that of the three media tested, the routinely used inositol serum broth was the most effective freeze-drying suspension medium.     

Dot-enzyme-linked immunosorbent assay (dot-ELISA) for the rapid diagnosis of human fascioliasis

A dot-enzyme-linked immunosorbent assay (dot-ELISA) was developed as a fast and field applicable antibody detection tool for the diagnosis of human fascioliasis. The assay is performed using partially purified antigens from a species of Fasciola at 180 ng protein/dot (2 microliters) and serum samples at 1:20 dilution (1 microliter). Dot-ELISA results completely agreed with those of micro-ELISA. Antigen-coated nitrocellulose sheets stored for 3 mo at -20 C showed results identical to fresh sheets. Sera from patients with fascioliasis (n = 30) and other parasitic or viral infections (n = 120) were compared with sera from healthy controls (n = 14). Ninety samples can be tested within 90 min. The sensitivity, specificity, and speed of the assay may justify its use in laboratory and field studies.     

Streptavidin-Aequorin Fusion Protein for Bioluminescent Immunoassay

The fusion protein of streptavidin to aequorin (STA-AQ) was highly purified from inclusion bodies in Escherichia coli cells and applied to a bioluminescent sandwich immunoassay. α-Fetoprotein (AFP), which is a serological marker of liver cancer, was used as a model analyte to test STA-AQ in an immunoassay. The measurable range of AFP by the sandwich immunoassay, using the complex of STA-AQ and the biotinylated anti-AFP antibody, was 0.02–200 ng/mL with an average coefficient of variation of 4.9%. The detection sensitivity with the complex of STA-AQ and the biotinylated anti-AFP antibody was similar to that with the complex of biotinylated aequorin, streptavidin and the biotinylated anti-AFP antibody. STA-AQ would be a useful reporter protein for immunoassays.     

Bioluminescent assay for toxicological assessment of nanomaterials

A new method for assessing biotoxicity of nanomaterials, based on the use of soluble bioluminescent coupled enzyme system NAD(P)?H:FMN oxidoreductase and luciferase, is proposed. The results of this study indicate a significant adverse biological effect exerted by nanoparticles at the molecular level. It was found that the most toxic nanoparticles the nanoparticles are based on copper and copper oxide, as well as single-walled carbon nanotubes and multi-walled carbon nanofibers, which are referred to hazard class II.     

Bioluminescent quantum dots

With the goal of using the ciliate Tetrahymena thermophila for biotechnology applications-as a heterologous protein expression system-researchers have identified a copper-inducible and repressible promoter.     

Bioluminescent reporters for identification of gene allelic variants

A method for single nucleotide polymorphism identification was developed, which was based on the primer extension reaction (PEXT) followed by bioluminescent solid-phase microassay. Recombinant Ca²⁺-regulated photoprotein obelin and coelenterazine-dependent Renilla muelleri luciferase were used as reporters. The study was performed as an example of SNP genotyping of the human F5 gene encoding human Factor V Leiden polymorphism 1691 G??A (R506Q). Genomic DNA was amplified by PCR using primers flanking polymorphic site of 140 base pairs. PCR products were used as templates for two PEXT reactions using two primers containing 3??-terminal nucleotides, which were complementary to either normal or mutant alleles. If the template and allele-specific primer were completely complementary, the latter was elongated with DNA polymerase. The resulting extension product contained biotin residue due to the presence of biotinylated deoxyuridine triphosphate (B-dUTP) in the reaction mixture. The products were analyzed using obelin-streptavidin conjugates. The optimal PEXT-reaction conditions were found, which ensured a high reliability of SNP genotyping. A new approach to simultaneously revealing both alleles in one well was developed using two bioluminescent reporters. The efficiency of the proposed approach was shown in the study of clinical DNA samples.     

Bioluminescent Mycobacterium aurum expressing firefly luciferase for rapid and high throughput screening of antimycobacterial drugs in vitro and in infected macrophages

The slow growth and highly infectious nature of Mycobacterium tuberculosis is a limiting factor in its use as test organism in high throughput screening for inhibitory compounds. To overcome these problems, use of surrogate strains and reporter genes have been considered. In this study, we have investigated the application of a fast growing nonpathogenic M. aurum expressing firefly luciferase in rapid screening of antituberculosis compounds in vitro and in infected macrophages using bioluminescence assay. The assay is based on luminescence determination using luciferin as substrate. Inhibition of bioluminescence was obtained with frontline antimycobacterial drugs like streptomycin, rifampicin, isoniazid, ethambutol, ofloxacin, and sparfloxacin at their reported MICs. Inhibition could be observed as early as 2 h in vitro and within 24 h in infected macrophages. The system can reliably be used in high throughput screening.     

Generic liposome reagent for immunoassays

We have derivatized liposomes with antibodies by using avidin to crosslink biotinylated phospholipid molecules in the liposome membranes with biotinylated antibody molecules. A comparison of the biotin binding activity of avidin in solution and avidin associated with liposomes shows that avidin bound to biotinylated phospholipid in liposome membranes retains full binding activity for additional biotin molecules. Changes in the fluorescence spectrum of avidin have been used to characterize the binding capacity of avidin for biotin in solution, and change in intensity of light scattered due to aggregation of liposomes was used to measure the biotin binding activity of avidin associated with liposomes. Relative amounts of the biotinylated phospholipid, avidin, and biotinylated antibody have been optimized to produce stable liposomes which are derivatized with up to 1.7 nmol of antibody/mumol of lipid. These derivatized liposomes are highly reactive to immunospecific aggregation in the presence of multivalent antigen. A linear increase in light scattering was recorded between 1 and 10 pmol of antigen. This work shows that liposomes containing biotinylated phospholipid can be a successful generic reagent for immunoassays.