Fluorogenic hand-held immunoassay for the identification of ricin: rapid analyte measurement platform.

Rapid analyte measurement platform (RAMPtrade mark) fluorogenic hand-held immunoassays (HHAs) were evaluated for inclusivity/sensitivity, exclusivity/specificity, sample matrix effects, ruggedness/stability, and reproducibility in detection of ricin (RCA(60)), a potential biological threat agent. The limit of detection of HHAs for RCA(60) was 14 ng/mL or approximately 140 pg/test. HHAs were inclusive in detection of ricin RCA(60), RCA(120), ricin A chain, and ricin B chain and exclusive in discrimination of RCA(60) from other toxins. None of the sample matrices tested affected assay performance. RAMPtrade mark ratios were tolerant of increases in sample volume, however, a decrease in sample volume of 25% resulted in significantly increased readings. RAMPtrade mark readings were highly reproducible lot-to-lot, cartridge-to-cartridge, day-to-day, and reader-to-reader.

Development of a biotin mimic tagged ScFv antibody against western equine encephalitis virus: bacterial expression and refolding.

Single chain antibodies (ScFvs) are heavy and light chain variable domains connected by an artificial linker. Because of their smaller size, ScFvs show improved tissue penetration in vivo and reduced immunogenicity, making them ideal for therapeutic applications. We have cloned a ScFv against western equine encephalitis (WEE) using rDNA technology. The ScFv was generated from a hybridoma cell line (11D2) specific to the WEE virus E1 glycoprotein and is arranged in the V(L)-V(H) orientation with a (gly(4)ser)(3) linker. This ScFv was engineered successfully with a biotin mimic tag (11 amino acid peptide) and cloned in the pET22b+ expression vector. The ScFv was expressed as a approximately 32kDa protein in Escherichia coli as inclusion bodies, with an estimated yield of 20-40 mg/l. Different refolding protocols were used to solubilise the inclusion bodies. Most of the functional ScFv was generated when the inclusion bodies were solubilized in a detergent, air oxidised in the presence of CuSO(4) and then denatured in urea buffer in comparison to other protocols. The product was renatured finally in Tris arginine buffer (pH 8.0). Refolded protein was dialysed against phosphate buffer saline (PBS) (pH 7.3) to remove the Tris and arginine. Our refolding protocol generated up to a 50% yield of soluble protein, which retained antigen-binding activity with whole inactivated WEE virus as demonstrated by ELISA and Western blot analysis. This 11D2-biotin mimic ScFv complexed with streptavidin horseradish peroxidase (St-HRPO) will be useful as a detector reagent in the ultrasensitive ELISA detection of WEE virus antigen.

Development of a second generation monoclonal single chain variable fragment antibody against Venezuelan equine encephalitis virus: expression and functional analysis.

We have generated a single chain variable fragment (ScFv) antibody from a well-characterized monoclonal antibody (MAb) against Venezuelan equine encephalitis virus (VEE), by cloning variable regions of the heavy (V(H)) and the light (V(L)) chain antibody genes, connected by a DNA linker, in phagemid expression vector pCANTAB 5 E. MAb 1A4A1 was successfully cloned as a ScFv in Escherichia coli strain TG-1 and expressed as a approximately 30 kDa ScFv protein which was functional in recognizing VEE by ELISA. Results were reproduced in Escherichia coli strain HB2151 where the same clone, designated A116, was expressed primarily as soluble periplasmic protein. The 30 kDa A116 antibody displayed weak binding specificity to VEE antigen. Sequence analysis revealed a frame shift in the N-terminal region of the V(L) domain, upstream to the complementarity-determining region 1 (CDR1), as the probable cause of reduced activity. The protein sequence of A116 was highly homologous to published murine ScFv protein sequences except in the region of the identified frame shift.

Bispecific and bifunctional single chain recombinant antibodies.

Bispecific and bifunctional monoclonal antibodies as second generation monoclonals, produced by conventional chemical or somatic methods, have proved useful in the immunodiagnosis and immunotherapy of cancer and other diseases. Recombinant antibodies produced by genetic engineering techniques have also become available for use in preclinical and clinical studies. Furthermore, through genetic engineering, it is possible to remove or add on key protein domains in order to create designer antibody molecules with two or more desired functions. This review summarizes the strategies for development of single chain variable fragment (scFv) bifunctional and bispecific antibodies. The advantages and disadvantages as well as the problems of generating the various bispecific and bifunctional antibody constructs are reported and discussed. Since conventionally prepared bispecific and bifunctional monoclonal antibodies have already shown promise in clinical trials and results from preclinical studies of recombinant bispecific antibodies are encouraging, clinical trials in humans of recombinant bispecific and bifunctional antibodies, as a new generation of biologicals, are likely to be the thrust in the next decade and beyond.

Microsatellite loci are not abundant in all arthropod genomes: analyses in the hard tick, Ixodes scapularis and the yellow fever mosquito, Aedes aegypti.

Plasmid libraries enriched for microsatellites were generated in the tick, Ixodes scapularis and in the mosquito Aedes aegypti. Libraries were enriched for genomic DNA containing (AC)n, (AG)n, (ATG)n, (CAG)n, (TAG)n, (AAT)n, (CTGY)n or (GATA)n motifs. Clones containing each motif were sequenced in both species for PCR primer design. In I. scapularis, most primers amplified a single locus and alleles varied in the number of microsatellite repeats and segregated as codominant markers. In contrast (AC)n, (TAG)n and (GATA)n microsatellite loci extracted from Ae. aegypti appeared to be members of multigene families. A primer pair designed to amplify a particular TAG locus instead amplified many independently segregating loci, some of which did not contain TAG microsatellites. Alleles at the TAG loci segregated as dominant markers and there was limited evidence for length variation among alleles. These results suggest that microsatellite loci are not universally abundant in arthropod genomes nor do alleles always segregate as codominant markers.

SSCP analysis of cDNA markers provides a dense linkage map of the Aedes aegypti genome.

An intensive linkage map of the yellow fever mosquito, Aedes aegypti, was constructed using single-strand conformation polymorphism (SSCP) analysis of cDNA markers to identify single nucleotide polymorphisms (SNPs). A total of 94 A. aegypti cDNAs were downloaded from GenBank and primers were designed to amplify fragments <500 bp in size. These primer pairs amplified 94 loci, 57 (61%) of which segregated in a single F(1) intercross family among 83 F(2) progeny. This allowed us to produce a dense linkage map of one marker every 2 cM distributed over a total length of 134 cM. Many A. aegypti cDNAs were highly similar to genes in the Drosophila melanogaster genome project. Comparative linkage analysis revealed areas of synteny between the two species. SNP polymorphisms are abundant in A. aegypti genes and should prove useful in both population genetics and mapping studies.

Quantitative trait loci that control vector competence for dengue-2 virus in the mosquito Aedes aegypti.

Quantitative trait loci (QTL) affecting the ability of the mosquito Aedes aegypti to become infected with dengue-2 virus were mapped in an F(1) intercross. Dengue-susceptible A. aegypti aegypti were crossed with dengue refractory A. aegypti formosus. F(2) offspring were analyzed for midgut infection and escape barriers. In P(1) and F(1) parents and in 207 F(2) individuals, regions of 14 cDNA loci were analyzed with single-strand conformation polymorphism analysis to identify and orient linkage groups with respect to chromosomes I-III. Genotypes were also scored at 57 RAPD-SSCP loci, 5 (TAG)(n) microsatellite loci, and 6 sequence-tagged RAPD loci. Dengue infection phenotypes were scored in 86 F(2) females. Two QTL for a midgut infection barrier were detected with standard and composite interval mapping on chromosomes II and III that accounted for approximately 30% of the phenotypic variance (sigma(2)(p)) in dengue infection and these accounted for 44 and 56%, respectively, of the overall genetic variance (sigma(2)(g)). QTL of minor effect were detected on chromosomes I and III, but these were not detected with composite interval mapping. Evidence for a QTL for midgut escape barrier was detected with standard interval mapping but not with composite interval mapping on chromosome III.

Construction and characterization of monoclonal antibodies against western equine encephalitis virus.

A repertoire of mouse monoclonal antibodies (MAbs) against western equine encephalitis virus (WEE) was constructed and characterized. Anti-WEE antibodies were expressed from hybridomas and purified by protein G chromatography. Each of the antibodies was functionally assessed by indirect enzyme-linked immunosorbent assays (ELISAs), Western blotting, and immunoprecipitations. All antibodies bound to WEE antigen in ELISAs, whereas only a subgroup of antibodies was found to be active in Western blotting and immunoprecipitations. A subset of antibodies was found to cross-react with other alphaviruses, such as Sindbis virus (SIN), Venezuelan equine encephalitis (VEE), and eastern equine encephalitis (EEE). Because many of the antibodies were highly reactive to WEE antigen in one or more of the assays, these antibodies are excellent candidates for immunodetection and immunotherapy studies.

Development of a functional monoclonal single-chain variable fragment antibody against Venezuelan equine encephalitis virus.

We have generated a single-chain variable fragment (ScFv) antibody, from a previously well-characterized monoclonal antibody (MAb) to Venezuelan equine encephalitis (VEE) virus, 5B4D-6. The variable regions of the heavy (V(H)) and light (V(L)) chain antibody genes, were connected by a DNA linker and cloned in the phagemid vector pCANTAB5E. The ScFv clone in Escherichia coli strain TG-1, 5B4D-6-6, was expressed as a approximately 30 kDa ScFv protein and higher molecular weight fusion products which were functional in recognizing VEE virus by enzyme-linked immunosorbent assay (ELISA). Results were reproduced in Escherichia coli strain HB2151, where clone D66 was expressed mainly as soluble periplasmic protein. The D66 ScFv antibody bound VEE virus strongly as determined by ELISA. Nucleotide sequence analysis of 5B4D-6-6 ScFv indicated that the Vkappa gene belonged to family XVI, subgroup V, while the V(H) gene was unique in its sequence, though its amino acid sequence could be subgrouped as IA. The deduced protein sequence of D66 was highly homologous to published murine ScFv protein sequences. This work demonstrates, for the first time, cloning of a functional ScFv antibody against VEE virus.

A single chain Fv specific against Western equine encephalitis virus.

A recombinant single chain Fv (scFv) specific against Western equine encephalitis virus (WEE) was developed and characterized. The scFv was generated from 11D2 hybridoma producing anti-WEE antibody reactive to E1 component of viral envelope glycoprotein. V(L) and V(H) gene segments of 11D2 scFv were generated and joined together with a (gly4ser)3 linker by polymerase chain reaction (PCR). The resulting scFv was successfully expressed in P. pastoris expression system. Fifteen individual plasmids were tested and six of them were shown to drive scFv expression. DNA sequence analysis from three productive plasmids showed that they all carried the same VL and V(H) gene segments with a few base differences. Comparison of 11D2 scFv DNA sequence to the Kabat database showed that VH of 11D2 antibody belonged to subgroup IIID and subfamily XIV, while VL domain did not belong to any known subgroup or subfamily. Western blot analysis of 11D2 scFv using anti-c-myc antibody for detection showed different band pattern among clones derived from different plasmids. This was thought to be due to the different glycosylation where amino acid substitution occurred. Successful purification of 11D2 scFv could be done by immobilized metal affinity chromatography with an unoptimized yield of 700 microg/L. Functional studies showed that 11D2 scFv could bind to its respective WEE antigen as demonstrated by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). The binding affinity of 11D2 scFv is reasonably good compared to the parental 11D2 bivalent monoclonal antibody (MAb). Thus, 11D2 scFv and its derivatives have a potential use as immunotherapeutic and immunodiagnostic agents of WEE infections.

Rapid immunofiltration assay of Newcastle disease virus using a silicon sensor.

A rapid nonradioactive sandwich immunoassay which utilizes biotin-streptavidin mediated filtration capture of immune complexes in conjunction with a silicon sensor was developed for the detection of virus. Using purified Newcastle disease virus as a model, the lower limits of detection (LOD) were determined for a number of immunoassay configurations employing both monoclonal and polyclonal antibodies. The LODs ranged from 1.3 ng/ml (sample volume of 100 microliter) for an incubation of 60 min to 400 ng/ml for a 1 min incubation. The sandwich immune complexes were formed from one-step incubation of antibody and antigen. No 'hook' effects were observed over a wide range of analyte concentrations. The assays were easy to perform and required a total time equal to the incubation period plus about 5 min. The assay format is suitable for virus, bacteria and protein antigens. New assays can be developed and optimized readily, often within 1 day.

Epitope specificity of monoclonal antibodies against Newcastle disease virus: competitive fluorogenic enzyme immunoassay.

A test to determine the epitope specificity of monoclonal antibodies (MCA) was developed for hybridoma clones producing antibodies against Newcastle disease virus (NDV). The virus was first immobilized on nitrocellulose membranes of Millititer HA plates. Dilutions of MCA were then added, singly, or simultaneously in pairs, and bound antibody was quantitated with alkaline phosphatase-labelled detector antibody and a fluorogenic substrate, 4-methylumbelliferyl phosphate (4-MUP). Fluorescence count was measured fluorometrically. Additivity indices were calculated and plotted against dilutions of paired MCA. Antibodies that recognized identical epitopes displayed non-additivity at saturating antibody dilutions, followed by partial additivity and by total additivity at low, non-saturating dilutions. In contrast, MCA that recognized distinct epitopes exhibited total additivity throughout the curve. MCA that exhibited partial additivity were interpreted as competing for overlapping shared epitopes, or, distinct epitopes in close proximity, resulting in steric hinderance.

Sensitive avidin-biotin amplified fluorogenic enzyme immunoassay using biotinylated monoclonal antibodies for the identification and quantitation of virus.

A highly sensitive amplified fluorogenic enzyme-linked immunosorbent assay (FELISA), which utilizes the high affinity interaction of the vitamin biotin for the multiple binding sites on the glycoprotein avidin, was developed for the detection and identification of a model virus, Newcastle disease virus (NDV). Monoclonal antibodies (MCA) directed against the virus were purified and labelled with biotin. Biotinylated MCA was then used with avidin-labelled enzyme and a fluorogenic substrate to detect NDV adsorbed directly on nitrocellulose membranes. Reagents were standardized and, using purified virus, the theoretical lower limit of test sensitivity of the amplified FELISA was determined to be 1 fg/ml of test sample (50 ag/well). The specificity of the amplified FELISA was evaluated by challenging the assay system with homologous and heterologous strains of NDV, and with other serologically related and unrelated viruses. The test was simple to perform and multiple samples could be conveniently assayed with results obtainable in 3-4 h.

Sensitive fluorogenic enzyme immunoassay on nitrocellulose membranes for quantitation of virus.

A highly sensitive fluorogenic enzyme-linked immunosorbent assay (FELISA), which utilizes nitrocellulose membranes as solid phase support, has been developed for the detection and identification of virus in clinical samples. Reagents were standardized and, using purified Newcastle disease virus (NDV) as a model, the theoretical lower limits of test sensitivity of the FELISA were compared, in both "sandwich" and "indirect" formats, to those of a comparable chromogenic enzyme-linked immunosorbent assay (CELISA). Of the systems evaluated, the "sandwich" FELISA exhibited maximum sensitivity and detected 10 fg of purified virus protein per milliliter of test sample (500 ag per test volume). Specificity of the "sandwich" FELISA was evaluated by challenging the system with heterologous strains of NDV and with other serologically related and unrelated viruses. In a clinical trial in which fecal materials from chickens undergoing vaccination with NDV were assayed directly by FELISA, the virus was detected from the first to approximately the tenth day post-vaccination. The test is simple to perform and results can be obtained in approximately 4 h.

Rapid rate-kinetic turbidometric assay for quantitation of viral complement-fixing antibodies.

A rapid rate-kinetic turbidometric assay for the quantitation of viral complement fixing antibodies has been developed, using adenovirus as a model. The procedure is based on the turbidometric quantitation of intact sheep erythrocytes and measures the rate of hemolysis (change in absorbance at 640 nm/min/), at maximum velocity, occurring in the presence of residual complement not fixed by the antigen-antibody reaction. Reagents were standardized and assays performed using a microprocessor-controlled spectrophotometer with kinetic assay capability and a thermoregulated cell compartment. Eleven sera were assayed for complement fixing antibodies both by the conventional microtiter technique and by the rapid turbidometric method described here. Good correlation (r = 0.89) was obtained between the two procedures. Unlike the conventional complement fixation test, the rate-kinetic turbidometric complement fixation assay was found to be tolerant of variation in complement and antigen concentration, endpoint titers were objectively quantitated and, once reagents had been standardized, results could be obtained within 45-60 min. The technique is potentially adaptable to large-scale automation.

Single radial hemolysis test for quantitation of complement-fixing antibodies to non-hemagglutinating viruses.

A single radial hemolysis test, which overcomes many of the problems of conventional complement fixation tests, was developed for the quantitation of virus complement-fixing antibodies. The test procedure utilized staphylococcal protein A-coated sheep erythrocytes immobilized in agarose into which antigen was incorporated. Undiluted heat-inactivated serum samples were allowed to diffuse radially from wells punched in the agarose. Protein A served to concentrate the subsequent antigen-antibody reaction on the surface of the erythrocytes. Zones of hemolysis were developed by flooding with complement. With adenovirus as a model, basic test parameters were defined, and optimum reagent concentrations and diffusion conditions were determined. A positive linear relationship was found to exist between zone diameter and increasing log concentration of specific antiserum. No correlation was found between zone diameter and total concentration of immunoglobulin G in test sera. Sera rendered anticomplementary by the addition of carrageenan produced hemolytic zones equal to diameter to those observed with untreated sera. Seventy-seven human sera with known complement fixation titers were tested by this method. Good correlation (r = 0.74) between the complement fixation test and single radial hemolysis was observed. This test was highly reproducible and more sensitive than the conventional complement fixation test.

Coronary artery disease in diabetic and nondiabetic patients: a clinical and angiographic comparison.

A comparison of data from 58 diabetic and 58 nondiabetic patients with arteriographic evidence of coronary artery disease showed that diabetic patients had a significantly greater frequency of major stenoses in the intermediate coronary artery segments but no significant differences in the proximal or distal segments. Thus, the diabetic patients did have more severe coronary disease, but the diabetic group did not have "more distal" disease as represented by the number of major or minor lesions in the distal segments. The diabetic patients had a significantly greater frequency of electrocardiographic intraventricular conduction defects and manifestations of left ventricular dysfunction. There was no significant difference in the severity of coronary artery disease between the diabetic patients with manifestations of myocardial decompensation and the diabetic patients without such manifestations, suggesting that the increased frequency of myocardial dysfunction in diabetic patients may be related to factors other than the greater severity of coronary artery disease.