Enzyme-linked Immunofiltration Assay Research Articles

Expression screen by enzyme-linked immunofiltration assay designed for high-throughput purification of affinity-tagged proteins

High-throughput purification of affinity-tagged fusion proteins is currently one of the fastest developing areas of molecular proteomics. A prerequisite for success in protein purification is sufficient soluble protein expression of the target protein in a heterologous host. Hence, a fast and quantitative evaluation of the soluble-protein levels in an expression system is one of the key steps in the entire process. Here we describe a high-throughput expression screen for affinity-tagged fusion proteins based on an enzyme linked immunofiltration assay (ELIFA). An aliquot of a crude Escherichia coli extract containing the analyte, an affinity-tagged protein, is adsorbed onto the membrane. Subsequent binding of specific antibodies followed by binding of a secondary antibody horseradish peroxidase (HRP) complex then allows quantitative evaluation of the analyte using tetramethylbenzidine as the substrate for HRP. The method is accurate and quantitative, as shown by comparison with results from western blotting and an enzymatic glutathione S-transferase (GST) assay. Furthermore, it is a far more rapid assay and less cumbersome than western blotting, lending itself more readily to high-throughput analysis. It can be used at the expression level (cell lysates) or during the subsequent purification steps to monitor yield of specific protein.

Rapid and Sensitive Screening of Sulfamethazine in Porcine Urine with an Enzyme-Linked Immunosorbent Assay and a Field-Portable Immunofiltration Assay

An enzyme-linked immunosorbent assay (ELISA) and an enzyme-linked immunofiltration assay (ELIFA) were developed for the screening of sulfamethazine (SMZ) in porcine urine. Incurred urine samples were measured by ELISA with a working range of 0 to 10 ng of SMZ per ml. The assay showed good accuracy and precision, with recoveries above 99.8% and intra-and interassay coefficients of variation (CVs) ranging from 2.6 to 5.6% and from 5.9 to 12.7%, respectively. Good agreement was observed when the results of the immunoassay were compared with those of liquid chromatography/tandem mass spectrometry analysis. For the ELIFA, a nylon membrane is placed on top of an absorbent material and covalently coated with rabbit anti-rat immunoglobulins. Free binding sites are blocked, and monoclonal anti-SMZ antibodies, SMZ standard or urine, and SMZ–horse radish peroxidase conjugate are subsequently dropped onto the membrane. During the assay, the reactants are drawn through the membrane because of its close contact with the absorbent pad. Finally, a substrate solution is added for blue color development. The blue spot produced can be visually evaluated or instrumentally measured (numeric ▪ value), and the intensity of its color is inversely proportional to the analyte concentration. When a blue dot appears on the membrane, even if its color is less intense than that of the negative control, the sample is considered “negative,” i.e., it is thought to contain a concentration of SMZ that is below the visual detection limit. If no color appears on the test membrane, the sample is considered “positive,” i.e., it is thought to contain a concentration of SMZ that is equal to or above the visual detection limit. Validation of the assay showed good inter- and intra-assay precision (CV < 10%). Because samples can be analyzed after a simple dilution in <30 min with this assay format, it has strong potential for application in the field.

Enzyme-linked immunofiltration assay used in the screening of solid supports and immunoreagents for the development of an azinphos-methyl flow immunosensor

Azinphos-methyl (AM), O, O-dimethyl S-[(4-oxo-1,2,3-benzotriazin-3(4 H)-yl)methyl] phosphorodithioate, is a dithiophosphorous insecticide extensively used for the control of fruit culture pests. In this work the ELIFA system, initially developed and marketed to substitute conventional ELISA methods, was used for the screening of supports and immunoreagents in the development of a flow immunosensor to AM. The objective was to find the optimal antibody concentration, support quantity and enzymatic tracer concentration to develop a sensitive and reusable immunosensor. The influence of chitosan as protein stabilizing agent was also investigated. We observed that, on the basis of immunosorbent characterization, chitosan-modified silica with immobilized LIB-MFH14 monoclonal antibody (MAb) showed the best sensitivity, with a I 50 value of 6 nM AM. All of the immobilized MAbs either in alkylaminated or chitosan-modified silica showed I 50 values between 10 and 36 nM. Regarding the regeneration capability, the best desorption agent tested was 0.1 M glycine/HCl, pH 2.0, performing in most cases a 100% desorption after just one wash and maintaining the antibody activity even after 20 cycles of regeneration. The chitosan-modified silica seemed to be the best support for this purpose.

Anti-PF4-heparin immunoglobulin G is the major class of heparin-induced thrombocytopenia antibody: findings of an enzyme-linked immunofiltration assay using membrane-bound hPF4-heparin.

An enzyme-linked immunofiltration assay (ELIFA) was developed for detecting anti-human platelet factor 4 (hPF4)-heparin antibody in sera of patients with heparin-induced thrombocytopenia (HIT). The immunofiltration assay was developed to capture HIT antibody by hPF4-heparin complex adsorbed onto a positively charged nylon membrane, as an alternative to plastic bound hPF4. Of 75 sera with a positive serotonin-release assay (SRA), anti-PF4-heparin of the immunoglobulin (Ig)G class was detected in 72 (96%) sera. With SRA-negative sera from thrombocytopenic patients treated with heparin, anti-hPF4-heparin IgG and IgA were detected in 16% (n = 126) and 14% (n = 74) of sera respectively; 6% (n = 71) of SRA-negative sera contained both IgG and IgA anti-hPF4-heparin antibodies. The detection of anti-hPF4-heparin IgG in all HIT sera supports the assay of anti-PF4-heparin IgG as being a sensitive screening test for HIT. Alternatively, the absence of anti-hPF4-heparin IgA cannot be used as a test for excluding HIT, as it was detected in only 48% of SRA-positive HIT sera. However, it may be used to support the diagnosis of HIT, when HIT IgG is weak. This study emphasized the need to use different immobilizing media for the capture of anti-PF4-heparin antibody.

Value of the comparative enzyme-linked immunofiltration assay for early neonatal diagnosis of congenital Toxoplasma infection.

The transplacental transfer of specific maternal IgG antibodies makes the diagnosis of congenital Toxoplasma infection quite difficult in the neonate. The enzyme-linked immunofiltration assay (ELIFA), comparing at delivery the immunologic profile of the mother's antibody response and that of her child, allows discrimination between IgG antibodies of maternal origin and IgGs synthesized by the fetus. To evaluate the diagnostic reliability of the comparative ELIFA for diagnosing congenital Toxoplasma infection as well as the reliability of testing for IgM- and IgA-specific antibodies in cord blood. From November, 1991, to December, 1995, an ELIFA was prospectively performed at delivery on blood samples obtained from 227 women with primary Toxoplasma infection during pregnancy and from their infants. For each child the ELIFA result was evaluated in relation to the serologic follow-up: disappearance of specific anti-Toxoplasma gondii IgG antibodies in the absence of treatment before 12 months of age indicating an uninfected child, as opposed to persistence beyond 12 months of age indicative of a congenital infection. Of 227 children 139 were lost to follow-up. Among the 88 children available for follow up, the ELIFA was negative in 70 infants, 69 of whom were confirmed to be uninfected. Thirteen of these 69 cord blood ELIFA-negative samples were positive for anti-T. gondii IgM and/or IgA detected by means of a conventional immunosorbent agglutination assay. Of the remaining 18 children (representing 75% of all new cases of congenital toxoplasmosis diagnosed during the study period at our institution), the ELIFA was positive in 16, negative in 1 and inconclusive in 1. The ELIFA test is a valuable tool for diagnosing congenital T. gondii infection and in differentiating between true neonatal infection and cord blood contamination. In our experience the diagnostic sensitivity of the ELIFA test was 94.1% and the specificity was 98.6%. The cord blood was contaminated by specific maternal anti-T. gondii IgA and/or IgM in as many as 20% of the cases.

Conversion of native oligomeric to a modified monomeric form of human C-reactive protein

C-reactive protein (CRP) is a pentameric oligoprotein composed of identical 23 kD subunits which can be modified by urea-chelation treatment to a form resembling the free subunit termed modified CRP (mCRP). mCRP has distinct physicochemical, antigenic, and biologic activities compared to CRP. The conditions under which CRP is converted to mCRP, and the molecular forms in the transition, are important to better understand the distinct properties of mCRP and to determine if the subunit form can convert back to the pentameric native CRP form. This study characterized the antigenic and conformational changes associated with the interconversion of CRP and mCRP. The rate of dissociation of CRP protomers into individual subunits by treatment in 8 M urea–10 mM EDTA solution was rapid and complete in 2 min as assayed by an enzyme-linked immunofiltration assay using monoclonal antibodies specific to the mCRP. Attempts to reconstitute pentameric CRP from mCRP under renaturation conditions were unsuccessful, resulting in a protein retaining exclusively mCRP characteristics. Using two-dimensional urea gradient gel electrophoresis, partial rapid unfolding of the pentamer occurred above 3 M urea, a subunit dissociation at 6 M urea, and further subunit unfolding at 6–8 M urea concentrations. The urea gradient electrophoresis results suggest that there are only two predominant conformational states occurring at each urea transition concentration. Using the same urea gradient electrophoresis conditions mCRP migrated as a single molecular form at all urea concentrations showing no evidence for reassociation to pentameric CRP or other aggregate form. The results of this study show a molecular conversion for an oligomeric protein (CRP) to monomeric subunits (mCRP) having rapid forward transition kinetics in 8 M urea plus chelator with negligible reversibility.

Serum IgG antibodies against Wallemia sebi and Fusarium species in Finnish farmers.

Wallemia sebi and Fusarium species are common fungi in agricultural environments. Because Fusarium species are difficult to culture, and W. sebi has only recently been found in an agricultural environment with the use of a new culture medium, immunochemical methods may be important for evaluating exposure to these fungi. Immunoglobulin (Ig) G antibodies against W. sebi and two Fusarium species were measured in the sera of Finnish farmers to estimate exposure to fungi. Serum-specific IgG antibody levels against the fungi were determined in farmers with farmer's lung disease and asthma, as well as in asymptomatic farmers with low and high IgG antibody levels against other agricultural microorganisms, and in control persons (printing and office workers). Both enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunofiltration assay (ELIFA) techniques were used. Significantly higher IgG antibody levels against the fungi studied were observed in farmer's lung patients and asymptomatic farmers with high IgG levels against other agricultural fungi, than in the control group. On the other hand, the development of IgG antibodies to the fungi in asthmatic farmers remained unclear because of a discrepancy between the ELISA and ELIFA results. Cross-reactivity between Fusarium species was detected. The results indicate that Finnish farmers are exposed to W. sebi and Fusarium species in their work environment to a greater extent than has earlier been evaluated with air sampling and microbiologic methods.

Enzyme-linked immunofiltration assay To estimate attachment of thiobacilli to pyrite

An enzyme-linked immunofiltration assay (ELIFA) has been developed in order to estimate directly and specifically Thiobacillus ferrooxidans attachment on sulfide minerals. This method derives from the enzyme-linked immunosorbent assay but is performed on filtration membranes which allow the retention of mineral particles for a subsequent immunoenzymatic reaction in microtiter plates. The polyclonal antiserum used in this study was raised against T. ferrooxidans DSM 583 and recognized cell surface antigens present on bacteria belonging to the genus Thiobacillus. This antiserum and the ELIFA allowed the direct quantification of attached bacteria with high sensitivity (10(4) bacteria were detected per well of the microtiter plate). The mean value of bacterial attachment has been estimated to be about 10(5) bacteria mg-1 of pyrite at a particle size of 56 to 65 &mgr;m. The geometric coverage ratio of pyrite by T. ferrooxidans ranged from 0.25 to 2.25%. This suggests an attachment of T. ferrooxidans on the pyrite surface to well-defined limited sites with specific electrochemical or surface properties. ELIFA was shown to be compatible with the measurement of variable levels of adhesion. Therefore, this method may be used to establish adhesion isotherms of T. ferrooxidans on various sulfide minerals exhibiting different physicochemical properties in order to understand the mechanisms of bacterial interaction with mineral surfaces.

Development of ELISA and enzyme-linked immunofiltration assay (ELIFA) methods for monitoring cyclodextrin glycosyltransferase (CGTase) production and bacterial growth in Bacillus macerans batch cultures

Immunochemical methods were developed for monitoring cyclodextrin (CD) glycosyltransferase (CGTase) production and growth of an industrial CD-producing Bacillus macerans strain. Extracellular concentrations of CGTase released into a non-transparent culture medium during a 44 h long fermentation were detected by an indirect antigen inhibition enzyme-linked immunosorbent assay (ELISA). The ELISA was sensitive (minimal detection level 6 ng ml−1) and highly reproducible (coefficients of variation ≤1.2 and 5.9%, within-runs and between-runs, respectively) compared to assays of CGTase activity (coefficients of variation ≤4.2 and 7.0%, respectively). The ELISA, in combination with enzyme activity measurements, was useful to detect the decrease in the specific CGTase activities after 36 h of incubation, which was clearly indicative of the proteolytic degradation of CGTase. B. macerans cell numbers were estimated using an enzyme-linked immunofilter assay (ELIFA). The assay took less than 1 h and the coefficients of variation within and between-runs (2.9–6.4%) were considerably less than for viable counting (10.6–15.4%). In the exponential phase of growth, ELIFA results correlated more closely with the cell counting based on total protein than with viable counts. Nevertheless, in the phase of cell lysis, the bacterial cell number was systematically underestimated by ELIFA in comparison to both viable cell number and total protein determinations. Thus cell antigens detected with immunological procedures might be lost during the transition from vegetative cells to spores. On the other hand, the ELIFA procedure was specific for B. macerans cells and was a better indicator of the onset of the different growth phases than the cell numbers calculated from the protein assay.

Prenatal diagnosis of congenital toxoplasmosis in 261 pregnancies.

Two hundred and sixty-one pregnant women underwent prenatal screening by cordocentesis and/or amniocentesis between 1987 and 1994. The following tests were used: (i) detection of anti-Toxoplasma gondii IgM, IgA, and IgE antibodies by immunocapture and the comparative immunological profile method based on enzyme-linked immunofiltration assay of fetal blood and (ii) direct detection of the parasite in cell culture and by mouse inoculation with fetal blood (FB) and/or amniotic fluid (AF). Of the 31 cases of congenital toxoplasmosis, 24 (77 per cent) were detected prenatally. Overall, the FB and AF inoculation methods were the most effective (50 per cent sensitivity with FB inoculation to mice and/or cell culture and 74 per cent with AF). However, antibody detection in FB was the only positive test in three cases. Of 18 surviving children diagnosed prenatally, only one developed chorioretinitis (9 months of age). Seven newborns (23 per cent) with negative prenatal tests were diagnosed by postnatal laboratory monitoring, but none of these children developed clinical toxoplasmosis. There may have been more false negatives, as only 48 per cent of unaffected children were followed up for at least 12 months. All the tests had a specificity of 100 per cent. Fetal blood sampling has considerable value but also carries some risks and is currently being abandoned in favour of amniocentesis alone with gene amplification and mouse inoculation.

A comparative study by the enzyme-linked immunofiltration asssay of solid phases used in the development of flow immunosensors

The application of an inert membrane-based, enzyme-linked immunofiltration assay (ELIFA) to the characterization of immunosorbents suitable for flow immunosensor development is described. For direct assays, eight monoclonal antibodies (MAb) raised against the insecticide carbaryl were immobilized on three sorbents, namely, controlled pore glass (CPG), hydrazide derivatized agarose beads and a hydrophilic polymer with immobilized Protein A/G. The interaction between immobilized antibodies and antigen was directly detected using a carbaryl hapten conjugated to horseradish peroxidase. Immunosorbent characterization was based on both sensitivity and re-usability. Optimal immunosorbent regeneration was achieved using 0.1 M glycine/HCl, pH 2.0 as the desorbent solution. The best covalent immunosorbent was obtained by immobilizing LIB-CNA36 MAb on hydrazide derivatized agarose beads. The best immunosorbent obtained by reversible immobilization was LIB-CNH45 MAb on Protein A/G. Using this support the eventual irreversible denaturation of covalently immobilized MAbs was overcome. For indirect assays, N-hydroxisuccinimide derivatized agarose beads and glutaraldehyde-activated CPG were used as sorbents for hapten immobilization via the amino groups of a carrier protein. In this format, antigen–MAb interactions were detected using a peroxidase-conjugated rabbit anti-mouse immunoglobulin. The highest sensitivity was achieved by LIB-CNH45 MAb in combination with derivatized agarose beads. All these results demonstrated the suitability of ELIFA as a fast, precise and easy-to-use technique for immunosorbent selection.

Accelerated Detection of DNA on Membranes by Automated Enzyme-Linked Immunofiltration Assay

Methods used to detect DNA after transfer to nitrocellulose or nylon membranes are all based on slow incubation with agitation. We describe an application of the ELIFA technique (enzyme-linked immunofiltration assay) for rapid detection of DNA immobilized on a membrane by active filtration of the reagents across the membrane. The different steps (saturation, hybridization to a nonisotopically labeled probe, washing, and immunoenzymatic revelation) are automated and controlled by a microcomputer that determines the direction of flow and flow rates of the solutions through the membrane. We applied this method to the detection ofToxoplasma gondiiDNA in 108 samples of amniotic fluid during antenatal tests for toxoplasmosis and compared the results with those obtained by the conventional method. In addition to a major time saving (2 h against almost 15 h), automation improves reproducibility and avoids manipulation of the membranes between the different steps, while keeping the same sensitivity and specificity as the standard method.

DEVELOPMENT AND EVALUATION OF A RAPID ENZYME‐LINKED IMMUNOFILTRATION ASSAY (ELIFA) AND AN ENZYME‐LINKED IMMUNOSORBENT ASSAY (ELISA) FOR THE DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN B

An enzyme‐linked immunofiltration assay (ELIFA) and a microtitre plate enzyme‐linked immunosorbent assay (ELISA) were developed and compared for their ability to detect staphylococcal enterotoxin B (SEB). The double antibody capture format was used for both assays. Factors which improved the sensitivity of the ELIFA system were (1) addition of casein and thimerosal to the antigen dilution buffer; (2) addition of polyethylene glycol (MW 6000) to the detection and conjugate antibody dilution buffers; and (3) washing with diethanolamine buffer prior to addition of the substrate/chromogen. The ELIFA system had a turnaround time of approximately 1 h and a detection limit of 1 ng/mL of purified SEB. The ELISA had a total turnaround time of 21 h, or 3 h using plates pre‐coated overnight with the capture antibody. The detection limit of the ELISA for purified SEB was 0.05 ng/mL. The detection limit of SEB in cheese samples spiked with purified enterotoxin and subjected to a simple extraction procedure was 1 ng/mL and 0.1 ng/mL of extract, with the ELIFA and the ELISA, respectively.

Two formats of enzyme immunoassay for the detection of saxitoxin and other paralytic shellfish poisoning toxins.

A competitive direct enzyme-linked immunofiltration assay for the detection of saxitoxin was developed, using polyclonal antibodies against saxitoxin and a saxitoxin-horseradish peroxidase conjugate. The test was performed in an eight-well plastic test device, in which antibody-coated nylon membranes were pressed tightly to an absorbent cellulose layer. Saxitoxin standard or sample extract solution, saxitoxin-conjugate, and enzyme substrate/chromogen solution were sequentially added on to the membrane. The test was evaluated visually by comparing the intensity of the resulting coloured (blue) dot with that of a negative control. The detection limits for saxitoxin in buffer solution and in shellfish tissue were 4 ng/ml and 80 ng/g respectively, with an assay time of less than 15 min. Under the conditions of the immunofiltration assay, decarbamoyl-saxitoxin, gonyautoxin 2/3, and neosaxitoxin standards (in buffer) gave a positive response at concentrations of about 10 ng/ml, 40 ng/ml, and 80 ng/ml, respectively. The relative cross-reactivity of the antibody to these PSPs was similar when determined using both direct and indirect (using a saxitoxin-bovine serum albumin conjugate) competitive enzyme immunoassays in microtitre plate format. In competitive direct microtitre plate assays, the 50% binding values found for saxitoxin, decarbamoyl-saxitoxin, gonyautoxin 2/3 and neosaxitoxin were 15 pg/ml, 47.5 pg/ml, 163.5 pg/ml, and 510 pg/ml respectively. In competitive indirect microtitre assay, the respective values were 138 pg/ml, 404 pg/ml, 1582 pg/ml, and 6982 pg/ml.

Evaluation of risk and diagnostic value of quantitative assays for anti-Toxoplasma gondii immunoglobulin A (IgA), IgE, and IgM and analytical study of specific IgG in immunodeficient patients

To determine their prognostic and diagnostic values for toxoplasmosis in immunodepressed subjects, we assayed immunoglobulin A (IgA) and IgE antibodies by means of immunocapture (IC) tests, with revelation done by using a suspension of T. gondii (ICT). We also carried out a simultaneous analytical study of IgG antibodies on cellulose acetate membranes by using the comparative immunological profile method and an enzyme-linked immunofiltration assay (ELIFA). A total of 1,238 samples (serum, cerebrospinal fluid, and aqueous humor from 318 patients) were tested. IgA and IgE antibodies were detected in all heart, kidney, and liver transplant recipients with clinical manifestations of toxoplasmosis; IgA was detected in the aqueous humor of a patient with chorioretinitis. In patients with AIDS-related toxoplasmosis, including the cerebral form, IgA and IgE antibodies or a significant modification of ELIFA IgG values were observed in 38, 19, and 25% of patients, respectively. IgM was detected by ICT only in 12% of patients and aided the diagnosis in 1 of 71 patients. IC tests for specific IgA and IgE alone and combined with ELIFA were positive in 39 and 46% of patients who developed clinical toxoplasmosis, respectively. In a serial study of 16 patients in whom at least one of these three tests was positive, a significant immunological signal sometimes preceded clinical onset by 1, 6, and even 17 months. Similarly, in a group of human immunodeficiency virus-infected patients with evidence of previous exposure to T. gondii but no clinical manifestations, IgA, IgE, and IgA and/or IgE antibodies were detected in only 11, 4, and 12% of patients, respectively. These two situations point to peripheral T. gondii reactivation. IgA and IgE emerged as interesting markers of the risk of toxoplasmosis in immunodepressed patients. They may also provide valuable assistance in the diagnosis of toxoplasmosis, especially because tests for specific IgM are disappointing. However, at least one in two patients with toxoplasmosis showed no detectable immunological reaction, suggesting that this polyisotypic approach should be combined with other noninvasive methods such as gene amplification.

Rapid detection of toxoplasmic nucleic acid by enzyme-linked immunofiltration-assay after membrane transfer.

We report the use of enzyme-linked immunofiltration-assay (ELIFA) for rapid detection of Toxoplasma gondii DNA after transfer to nitrocellulose or nylon membranes. Saturation, specific labeling with a nonradioactive oligonucleotide probe and washing steps are included in an active filtration process, which is controled by microcomputer regulating reagent flow. This method provides excellent specificity, reproducibility and repeatability. Recycling in a closed circuit or by reversing the direction of flow provides the advantage of reducing the required volume of costly reagents (e.g. probes). ELIFA does not require lengthy membrane manipulation, and hybridization as well as stringency and revelation steps can be automated. The procedure can be carried out in less than 2 h, which is a major time saving compared to conventional solid-phase molecular hybridization techniques.

Direct identification of Staphylococcus aureus in blood cultures by detection of the gene encoding the thermostable nuclease or the gene product.

This study compares methods for direct identification of S. aureus in blood cultures by detection of the thermonuclease (TNase) of this bacterium or the nuc gene encoding it. The protein was detected by an enzyme diffusion test in o-toluidine blue DNA agar with a test time of at least 4 h, by a monoclonal antibody (MAb)-based sandwich enzyme-linked immunosorbent assay (sELISA) with a test time of approximately 4 h, and by a MAb-based sandwich enzyme-linked immunofiltration assay (sELIFA) with a test time of 25-30 min (sample preparation included). The nuc gene was amplified by a polymerase chain reaction (PCR) with a test time (amplification plus detection) of approximately 3.5 h. The tests were optimized for direct examination of blood-containing cultures. All tests were positive with 67/67 blood cultures which grew S. aureus, negative with 35/35 cultures which grew coagulase-negative staphylococci, and negative with 37/37 cultures with various other bacteria. These results showed positive agreement with those of the commercial AccuProbe test but not with the StaphAurex agglutination kit. With an artificially seeded blood culture, minimum total times required (incubation plus testing) were as follows: nuc-PCR, 9.5 h; sELIFA, 12.5 h; enzymatic test, 16-36 h; AccuProbe, 14 h. Direct examination of both the nuc gene and the mecA gene encoding methicillin resistance demonstrated the mecA gene in all the coagulase-negative staphylococci (48.6%) which showed oxacillin resistance. The sELIFA had the particular advantage of its short test time, the PCR its high sensitivity and the possibility of simultaneous detection of the species-specific nuc gene and genes encoding other clinically important characters of the bacteria. These tests offer the prospect of direct application to a variety of clinical specimens for rapid diagnosis of S. aureus infection.

Properties of the Scrapie Prion Protein: Quantitative Analysis of Protease Resistance

The disease-specific isoform of the prion protein (PrPSc) is an essential part of the infectious particle which causes spongiform degeneration in various mammalian species. PrPSc differs from PrP of normal animals (PrPc) by its relative protease resistance. The physical nature of this difference is still unknown. We analyzed the protease resistance of PrPSc quantitatively using an enzyme-linked immunofiltration assay. PrPSc was rendered completely protease-sensitive at alkaline pH or in > 1.5 M guanidinium thiocyanate (GdnSCN). Denaturation in 4 M GdnSCN completely abolished the protease resistance of PrPSc within 15 min, while denaturation in 7.2 M urea showed a slower time course. In the presence of ethanol, PrPSc was protected from denaturation by GdnSCN or alkaline pH. Denaturation curves were used to calculate the free energy (delta GD) as a function of different denaturant concentrations. Linear regression of delta GD values was used to extrapolate the free energy in the absence of denaturants (delta GH2O), yielding similar values (delta GH2O,GdnSCN = -2.3 kcal/mol; delta GH2O,urea = -3.1 kcal/mol). The linear relationship between delta GD and the denaturant concentration is suggestive of a two-state model involving the conformational change of a single protein domain. This is also reflected in the small number of side chains (11.6) additionally exposed to the solvent upon conversion of PrPSc to its protease-sensitive isoform. Our results suggest that only minor rearrangements of the structure of PrP are needed to abolish the protease resistance of PrPSc.

Rapid isotyping of murine monoclonal antibodies by enzyme-linked immunofiltration assay

As a part of the initial characterization of monoclonal antibodies, the isotype is routinely identified by enzyme-linked immunosorbent assay (ELISA). In the present study, we describe an isotyping methodology that uses the technique referred to as enzyme-linked immunofiltration assay (ELIFA) for greatly accelerating the assay compared with ELISA. In the ELIFA method, solutions are filtered through a nitrocellulose membrane using a controlled flow rate to bind proteins. By using this technology, the time required to isotype a monoclonal antibody was reduced from a minimum of 4 to 8 hr using a standard ELISA assay to 30 min with ELIFA.

Secretory leucocyte proteinase inhibitor is produced by human articular cartilage chondrocytes and intervertebral disc fibrochondrocytes.

The objective of this study was to examine the expression of the secretory leucocyte proteinase inhibitor (SLPI) gene by human articular cartilage chondrocytes and intervertebral disc fibrochondrocytes. RNA was extracted from human articular cartilage chondrocytes, synovial fibroblasts and fibrochondrocytes of the annulus fibrosus. Expression was analysed by Northern blotting and hybridisation to a SLPI RNA probe. SLPI was isolated from chondrocyte culture supernatant by gel-permeation and cation-exchange chromatography and examined by a solid-phase enzyme-linked immunofiltration assay for SLPI and by SDS/PAGE using Western blotting with polyclonal IgG samples against bronchial mucus leucocyte proteinase inhibitor and SLPI. This identified biochemically and immunologically the major serine proteinase inhibitory protein synthesised by chondrocytes as SLPI. Secretory leucocyte proteinase inhibitor mRNA was detected in RNA preparations from articular cartilage chondrocytes and annulus fibrosus fibrochondrocytes. The results presented here allowed us to deduce that human articular cartilage chondrocytes and intervertebral disc fibrochondrocytes synthesized SLPI whereas human synovial fibroblasts did not.