Rapid detection and identification of Legionella pneumophila by a membrane immunoassay

Legionella pneumophila was detected and identified by an immunoblot assay using a monoclonal antibody specific to serogroups 1 to 8. Samples containing L. pneumophila were plated on buffered charcoal yeast extract agar supplemented with glycine, vancomycin, and polymyxin B. After incubation at 35 degrees C for 3 days, colonies were transferred to nitrocellulose membranes by blotting. Simultaneous detection and identification of L. pneumophila were done by treating the membrane with the monoclonal antibody and a peroxidase conjugate to mouse immunoglobulins. A diffuse cross-reaction was observed with Pseudomonas fluorescens colonies, but this was a low-level reaction that could easily be differentiated from the strong specific reactions to L. pneumophila.     

Rapid detection and identification of Legionella pneumophila by a membrane immunoassay.

Legionella pneumophila was detected and identified by an immunoblot assay using a monoclonal antibody specific to serogroups 1 to 8. Samples containing L. pneumophila were plated on buffered charcoal yeast extract agar supplemented with glycine, vancomycin, and polymyxin B. After incubation at 35 degrees C for 3 days, colonies were transferred to nitrocellulose membranes by blotting. Simultaneous detection and identification of L. pneumophila were done by treating the membrane with the monoclonal antibody and a peroxidase conjugate to mouse immunoglobulins. A diffuse cross-reaction was observed with Pseudomonas fluorescens colonies, but this was a low-level reaction that could easily be differentiated from the strong specific reactions to L. pneumophila.     

Overcoming antibody expression and screening limitations by smart design: applications to PSA immunoassay development

Improving the functional and structural properties of target proteins can often be a challenge for researchers. This paper highlights the importance of antibody construct on screening performance, and ultimately, the clone that is selected. We report the reformatting of phage-selected single chain antibody variable region fragments (scFvs) into single chain antibody fragments (scAbs) for improved screening and binding studies. The generation of a scAb, which had a fused human kappa light chain constant domain (C(k)), was shown to significantly improve expression levels in Escherichia coli. Antibody expression levels were compared between the two antibody constructs (scFv and scAb) by ELISA and a 100-fold improvement was observed. The C(k) domain in the expressed scAb also facilitated high throughput analysis by a Biacore capture assay approach. Individual functional scAbs were ranked on the basis of their remaining binding percentage after 5 min dissociation. Selected antibodies were further characterised by kinetic analysis and a sandwich-based immunoassay developed. The scAb construct enhanced expression levels significantly, facilitating antibody screening and immunoassay development for prostate-specific antigen (PSA), a marker for prostate cancer.     

Quantitation of plasma membrane fatty acid-binding protein by enzyme dilution and monoclonal antibody based immunoassay

Summary A plasma membrane fatty acid-binding protein (h-FABPPm) has been isolated from rat hepatocytes. Analogous proteins have also been identified in adipocytes, jejunal enterocytes and cardiac myocytes, all cells with high transmembrane fluxes of fatty acids. These 43 kDa, highly basic (pl = 9.1) FABP_pm 's appear unrelated to the smaller, cytosolic FABP's (designated FABP's) identified previously in the same tissues. h-FABP_pm appears closely related to the mitochondrial isoform of glutamic-oxaloacetic transaminase (mGOT), and both the purified protein and liver cell plasma membranes (LPM) possess GOT enzymatic activity. From their relative GOT specific activities it is estimated that h-FABP_pm constitutes approximately 2% of LPM protein, or about 0.7 × 10⁷ sites per cell. A monoclonal antibody-based competitive inhibition enzyme immunoassay (CIEIA) for h-FABP_pm is described; it yields an estimate of 3.4 x 10⁷ h-FABP_pm sites per hepatocyte. Quantitated by either method, h-FABPPm appears to be a highly abundant protein constituent of LPM.     

Rapid immunoassay technique for process monitoring of animal cell fermentations.

A calibration and quality control technique suited to process monitoring with immunoassay is demonstrated. The particle concentration fluorescence immunoassay (PC-FIA) is shown to provide a sensitive and rapid method for the quantification of specific biomolecules in cell cultures. Smoothing of linear calibration parameters is performed by forming weighted averages of standard points as the run progresses. These estimates are then used to determine slope and intercept values for improved calibration. The nonuniformity of the fluorescent signal variance is also considered, and a weight model is developed to describe the relationship between signal fluorescence and signal variance for weighted linear curve fitting. Pooling calibration results over the process run improves overall assay performance as determined by using standard control chart analysis. This method is suitable for semicontinuous monitoring of animal cell fermentations and has been used here to measure cell-associated and culture supernatant concentrations of monoclonal antibody (Ab) from hybridoma cells. The cell-associated Ab concentration correlates with cell-specific production rate. Assay times on the order of 10 min for supernatant and 25-30 min for cell-associated Ab concentrations can be achieved, making this procedure suitable for process monitoring and control. Under these conditions the assay has a detection limit of approximately 10 ng/mL, providing a sensitive and specific method for the quantification of cell culture constituents.     

Evaluation of a malaria antibody enzyme immunoassay for use in blood screening

Transfusion-transmitted malaria is rare, but it may produce severe problem in the safety of blood transfusion due to the lack of reliable procedure to evaluate donors potentially exposed to malaria. Here, we evaluated a new enzyme-linked immunosorbent assay malaria antibody test (ELISA malaria antibody test, DiaMed, Switzerland) to detect antibodies to Plasmodium vivax (the indigenous malaria) in the blood samples in the Republic of Korea (ROK). Blood samples of four groups were obtained and analyzed; 100 samples from P.vivax infected patients, 35 from recovery patients, 366 from normal healthy individuals, and 325 from domestic travelers of non-endemic areas residents to risky areas of ROK. P.vivax antibody levels by ELISA were then compared to the results from microscopic examination and polymerase chain reaction (PCR) test. As a result, the ELISA malaria antibody test had a clinical sensitivity of 53.0% and a clinical specificity of 94.0% for P.vivax. Twenty out of 325 domestic travelers (6.2%) were reactive and 28 cases (8.6%) were doubtful. Of the reactive and doubtful cases, only two were confirmed as acute malaria by both microscopy and PCR test. Thus we found that the ELISA malaria antibody test was insufficiently sensitive for blood screening of P.vivax in ROK.     

Rapid screening of membrane topology of secondary transport proteins

Limited experimental data may be very useful to discriminate between membrane topology models of membrane proteins derived from different methods. A membrane topology screening method is proposed by which the cellular disposition of three positions in a membrane protein are determined, the N- and the C-termini and a position in the middle of the protein. The method involves amplification of the encoding genes or gene fragments by PCR, rapid cloning in dedicated vectors by ligation independent cloning, and determination of the cellular disposition of the three sites using conventional techniques. The N-terminus was determined by labeling with a fluorescent probe, the central position and the C-terminus by the reporter fusion technique using alkaline phosphatase (PhoA) and green fluorescence protein (GFP) as reporters. The method was evaluated using 16 transporter proteins of known function from four different structural classes. For 13 proteins a complete set of three localizations was obtained. The experimental data was used to discriminate between membrane topology models predicted by TMHMM, a widely used predictor using the amino acid sequence as input and by MemGen that uses hydropathy profile alignment and known 3D structures or existing models. It follows that in those cases where the models from the two methods were similar, the models were consistent with the experimental data. In those cases where the models differed, the MemGen model agreed with the experimental data. Three more recent predictors, MEMSAT3, OCTOPUS and TOPCONS showed a significantly higher consistency with the experimental data than observed with TMHMM.     

Rapid expression screening of eukaryotic membrane proteins in Pichia pastoris

The overexpression of milligram quantities of protein remains a key bottleneck in membrane protein structural biology. A challenge of particular difficulty has been the overproduction of eukaryotic membrane proteins. In order to cope with the frequently poor expression levels associated with these challenging proteins, it is often necessary to screen a large number of homologues to find a well expressing clone. To facilitate this process using the heterologous, eukaryotic expression host Pichia pastoris, we have developed a simple fluorescent induction plate‐screening assay that allows for the rapid detection of well expressing clones of eukaryotic membrane proteins that have been fused to GFP. Using a eukaryotic membrane protein known to express well in P. pastoris (human aquaporin 4) and homologues of the ER associated membrane protein phosphatidylethanolamine N‐methyltransferase (PEMT), we demonstrate that when a large number of clones are screened, a small number of highly expressing “jackpot” clones can be isolated. A jackpot PEMT clone resulted in 5 mg/L yield after purification. The method allows for the facile simultaneous screening of hundreds of clones providing an alternate to in‐culture screening and will greatly accelerate the search for overexpressing eukaryotic membrane proteins.     

Rapid enumeration of Staphylococcus aureus in foods by direct demonstration of enterotoxigenic colonies on membrane filters by enzyme immunoassay.

Based on enzyme-linked immunosorbent assay, a convenient method has been devised for the direct demonstration of enterotoxin B production by Staphylococcus aureus colonies grown for 24 h on membrane filters. The problem of false-positive reactions due to binding of immunoglobulin G to protein A was turned to advantage by conjugating horseradish peroxidase directly to protein A, which then mediated the labeling of the antitoxin. The test requires 3 h to complete and yields a purple stain at the site of enterotoxin B-producing colonies, thus allowing direct enumeration of confirmed S. aureus in foods within 27 h. The method should be applicable to other enterotoxins of S. aureus.     

Rapid, high-throughput detection of PrPSc by 96-well immunoassay

Prion diseases are emerging infectious disorders that affect several mammalian species including humans. The transmissible agent is comprised of PrP^Sc, a misfolded isoform of the normal host-encoded prion protein PrP^C. Immunodetection of PrP^Sc is often utilized for prion disease diagnosis and tracking spread of the infectious agent through the host. We have developed a rapid, high-throughput 96-well immunoassay, which is specific for the detection of PrP^Sc. This assay has PrP^Sc detection limits similar to western blot and is advantageous because of its comparatively shorter running time, smaller start-up and operation costs and large sample capacity.Key words: prion disease, immunodetection, PrP^Sc     

Fluorescence polarization immunoassay employing immobilized antibody

The use of an antibody immobilized on latex or silver colloid in fluorescence polarization immunoassay (FPI) is assessed. In FPI it is possible to detect antigens of high molecular weight because the molecular weight of the antibody is effectively increased. In the assay for rabbit immunoglobulin G a limit of detection lower by two orders of magnitude and an assay range wider by one order of magnitude can be obtained in comparison with conventional FPI. The detection limit is 10(-10) mol l-1 and the total assay time for one sample is 8 min. This assay combines a low detection limit with a short assay time.     

Rapid chromatographic monitoring of bioprocesses

This paper describes the use of rapid chromatographic separation systems to monitor the level of specific proteins in various bioprocesses such as downstream processing and fermentation. In these monitoring systems, samples of the liquid are continuously extracted from the process and the proteins resolved from one another by a rapid chromatographic separation. The peak on the chromatogram corresponding to the protein of interest is identified and quantified to obtain on-line information on the level of that protein in the bioprocess. There are a number of advantages in using affinity separations as the rapid chromatographic principle. In particular, the use of immobilised monoclonal antibodies potentially allows a chromatographic sensor to be constructed for any protein against which a suitable antibody can be raised. The potential of this technique is illustrated with various examples, including measurement of the levels of monoclonal antibody in tissue culture supernatant using immobilised Protein A as the affinity adsorbent. A discussion of the inherent limitations of this type of protein biosensor is also included.     

Rapid screening for heat tolerance in Phaseolus species using the photoacoustic technique

The potential utility of photoacoustic measurements in vivo for rapid estimation of heat tolerance was investigated in seven Phaseolus genotypes with known and contrasting heat resistance. When small leaf discs of 1 cm diameter were heated at 40.5°C, both relative quantum yield for oxygen evolution (as estimated by the ratio of the amplitude of the oxygen evolution signal (Aox) to the amplitude of the photothermal signal (Apt)) and photochemical energy storage were reduced drastically within a few minutes in the heat sensitive genotypes. In contrast, in the genotypes processing heat tolerance characteristics, these photoacoustic parameters remained constant for at least 20 min. Photochemical energy storage appeared to be more resistant towards heat stress conditions than oxygen evolution, suggesting differential heat sensitivity of PS2 compared to PS1. Within the heat resistant species, we were able to further select for high temperature tolerance when the rate of decrease of Aox/Apt was measured in leaf discs incubated at a higher temperature (42°C). The results suggest that the photoacoutic technique could be used as a rapid and easy screening test for heat tolerance in crop plants.     

Enzyme channeling immunoassay: A new homogeneous enzyme immunoassay technique

A homogeneous enzyme immunoassay has been developed in which an antigen and glucose-6-phosphate dehydrogenase are coimmobilized on agarose beads. Binding of hexokinase-labeled antibody to the bead-bound antigen results in an accelerated conversion of glucose, ATP, and NAD⁺ to 6-phosphogluconolactone, ADP, and NADH. Critical parameters affecting assay response are discussed.     

Protein biomarker enrichment by biomarker antibody complex elution for immunoassay biosensing

It is very challenging to perform sample enrichment for protein biomarkers because proteins can easily change conformation and denature. In this paper we demonstrate protein enrichment suited for high-sensitivity integrated immuno-biosensing. The method enhances the concentration of the biomarkers and simultaneously removes matrix components that could interfere with the immunoassay. Biomarkers are captured using antibody coated magnetic particles and the biomarker antibody complexes are released by enzymatic elution. The eluted complexes are subsequently detected in a sandwich immunoassay biosensor. A scaling study of the enrichment process demonstrates an enrichment factor of 15 in buffer and plasma. We analyze the enrichment factor in terms of the three basic steps of the assay (capture, concentration, elution) and we quantify their respective efficiencies. The process is suited for integration into bio-analytical tools.     

Rapid measurement of blood ethanol concentrations using the alcohol oxidase membrane technique

The alcohol oxidase membrane technique is available for measurement of ethanol in commercial fluids. In this paper we examined its usefulness for cat and human blood in comparison with gas-liquid chromatography (GLC). The membrane method proved to be simple, reproducible, accurate, and inexpensive. Analysis took 1-2 min per sample and required only 25 microL of whole blood for measurement of concentrations between 0.05 and 1.0 mM (0.25-5 mg/dL) and 10 microL of whole blood for measurement of concentrations between 1.0 and 40 mM (5-190 mg/dL). Background concentrations were undetectable in cats after extraneous sources of alcohols were removed. The alcohol oxidase membrane technique is less specific than GLC, but it may be useful when ethanol is administered after background samples have shown an absence of other nonspecific reactants. Its high sensitivity is useful for kinetic studies where blood ethanol concentrations are below or close to those required for maximal hepatic ethanol metabolism.     

Urine sample preparation of tricyclic antidepressants by means of a supported liquid membrane technique for high-performance liquid chromatographic analysis

Supported liquid membrane (SLM) technique for sample work-up and enrichment was used for determination of tricyclic antidepressant drugs in urine by high-performance liquid chromatography (HPLC) with UV detection. The studied antidepressant drugs were amitriptyline, opipramol, noxiptyline and additionally diethazine was used as possible internal standard. Alkaline phosphoric buffer with urine sample, as the donor solution, was passed over the liquid membrane into which investigated substances were extracted. On the other side of the membrane, analyzed compounds were trapped due to creating non-extractable form in acidic acceptor solution. Enriched and cleaned up drugs were then injected into a HPLC system with ultraviolet detection to analyze of their concentration in acceptor solution. Optimum extraction efficiency was determined by changing acceptor and donor solutions pH, application of different flow rates of donor solution and by using different solvents in the membrane. Also, donor solution volume, extraction time and concentration of analytes were varied to check the linearity of extraction process. The highest extraction efficiency: 43% for opipramol, 56% for noxiptyline, 43% for amitriptyline and 42% for diethazine (R.S.D. values were <6% and n=3) was achieved when 0.05 M phosphate buffer pH 4.0 and 9.5 were used as donor and acceptor solutions, respectively, n-undecane with 5% tri-n-octylphosphine oxide (TOPO) was used as liquid membrane. Limit of quantification (LOQ) for tricyclic antidepressants after enrichment of 100ml of urine sample was about 1 ng/ml.     

Therapeutic antibody engineering by high efficiency cell screening

In recent years, several cell-based screening technologies for the isolation of antibodies with prescribed properties emerged. They rely on the multi-copy display of antibodies or antibody fragments on a cell surface in functional form followed by high through put screening and isolation of cell clones that carry an antibody variant with the desired affinity, specificity, and stability. Particularly yeast surface display in combination with high-throughput fluorescence-activated cell sorting has proven successful in the last fifteen years as a very powerful technology that has some advantages over classical generation of monoclonals using the hybridoma technology or bacteriophage-based antibody display and screening. Cell-based screening harbours the benefit of single-cell online and real-time analysis and characterisation of individual library candidates. Moreover, when using eukaryotic expression hosts, intrinsic quality control machineries for proper protein folding and stability exist that allow for co-selection of high-level expression and stability simultaneously to the binding functionality. Recently, promising technologies emerged that directly rely on antibody display on higher eukaryotic cell lines using lentiviral transfection or direct screening on B-cells. The combination of immunisation, B-cell screening and next generation sequencing may open new avenues for the isolation of therapeutic antibodies with prescribed physicochemical and functional characteristics.     

Fast isolation of recombinant baculovirus by antibody screening

We describe a rapid and efficient scheme for the isolation and purification of recombinant baculoviruses. The method is based on the detection of foreign proteins in cellular lysates of baculovirus-infected insect cells by antibody screening. The recombinant virus is purified by repeated serial dilutions. The method allows the identification and purification of recombinant viruses within 2 to 3 wk. This procedure selects for recombinant baculoviruses that highly overproduce the desired protein product.