Fetal fibronectin in cervical and vaginal secretions as a predictor of preterm delivery

BACKGROUND. Preterm delivery is the leading cause of neonatal mortality in the United States, but efforts to address the problem are hampered by the inability to predict accurately which pregnancies are at risk. We postulated that damage to the fetal membranes may release fetal fibronectin into the cervix and vagina, giving rise to a biochemical marker for preterm delivery. METHODS. We measured fetal-fibronectin concentrations in cervical and vaginal secretions, amniotic fluid, and maternal plasma with a sensitive immunoassay using the monoclonal antibody FDC-6. Immunohistochemical studies were used to determine the distribution of fetal fibronectin in the placenta and amniochorionic membranes and to ascertain its cell of origin. RESULTS. Women with uncomplicated pregnancies (n = 163) who delivered at term rarely had cervicovaginal fetal-fibronectin concentrations above 0.05 micrograms per milliliter between 21 and 37 weeks of gestation (11 of 267 cervical samples [4 percent] and 9 of 267 vaginal samples [3 percent]. High levels of fetal fibronectin were detected in amniotic fluid and in the cervical or vaginal secretions of 93.8 percent of the women with preterm rupture of membranes (n = 65). Cervical or vaginal fetal fibronectin was also present in 50.4 percent of the women with preterm uterine contractions and intact membranes (n = 117), and its presence identified the women who delivered before term (n = 60) with a sensitivity of 81.7 percent and a specificity of 82.5 percent. In the placenta and membranes, fetal fibronectin was found at points of contact with the uterine wall. CONCLUSIONS. The presence of cervicovaginal fetal fibronectin in the second and third trimesters of pregnancy identifies a subgroup of women who are at high risk for preterm delivery. This phenomenon may reflect the separation of the chorion from the decidual layer of the uterus, with the release of intact or degraded chorionic components of the extracellular matrix into the cervical and vaginal secretions.     

A competition enzyme-linked immunosorbent assay (ELISA) for the measurement of pancreatic GP-2 glycoprotein

A competition enzyme-linked immunosorbent assay has been developed for the quantitative detection of soluble and membrane-bound GP-2, a glycoprotein which is confined to the exocrine pancreas. Zymogen granule membranes fixed to microtiter plates with poly-L-lysine were used as the source of antigen. Detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS] was added to the assay in order to reveal all the antigens, more particularly in membranous samples. Presence of detergent at concentrations as high as 0.5% did not interfere with any particular steps of the ELISA. This competition ELISA can detect 10 ng of GP-2 and will be useful for measuring soluble as well as membrane GP-2 in order to elucidate its role in the secretory process of the pancreas as well as in certain pathologies such as cystic fibrosis.     

An enzyme-linked immunosorbent assay for human cathepsin X, a potential new inflammatory marker

The human lysosomal cysteine-type carboxypeptidase cathepsin X is mainly present in monocytes and macrophages and may be released into the circulation due to constitutive and/or regulated secretion by (activated) immune cells. To define its potential diagnostic value as an inflammatory marker, we have developed a highly sensitive and specific sandwich-type immunoassay (ELISA) for cathepsin X permitting both intra- and extracellular detection and quantification. The dynamic range of the cathepsin X ELISA was determined to be 100 (detection limit) to 8000 pg/ml. Reproducibility of both within and between runs yielded coefficients of variation (CVs) of 2.7-3.5% and 6.3-7.3%, respectively. Cross-reactivity with other members (cathepsin B, L) of the thiol-dependent cathepsin family was not observed. The ELISA was used to quantify cathepsin X in leukocytes as well as in plasma of healthy volunteers and patients with multiple trauma. During the first 72 h after trauma, plasma levels of cathepsin X increased significantly, particularly in patients who died during the posttraumatic period. In comparison to the well-known inflammation marker neutrophil elastase, cathepsin X levels predicted survival with a higher significance in the later posttraumatic phase. In conclusion, this report provides the first evidence of cathepsin X immunoreactivity not only in cell lysates but also in plasma samples. We suggest that the newly developed highly reproducible ELISA will be of great value for further evaluation of this protease as a diagnostic and/or prognostic marker in inflammatory diseases.     

Enzyme-linked immunosorbent assay (ELISA) for HIV antibody by a glass slide technique

An enzyme-linked immunosorbent assay (ELISA) technique is described which utilizes a commercially available glass microscope slide coated with hydrophobic teflon in such a pattern as to give 30 small circular wells, each of which has a glass bottom. Each well serves as a solid phase, analogous to a microtiter well for adsorption of purified human immunodeficiency virus (HIV) antigens. Since only 5-10 microliter volumes of reagents are used and rinsing processing is simple, the cost per test is much less than most other ELISA technologies. HIV antigen is stable for over 1 year at 37 degrees C when dried on the glass slides. The sensitivity and specificity of the micro slide immunoenzymatic assay (Micro-SIA) was studied by testing randomly selected, known HIV-seropositive and seronegative plasma. Results compare well with microtiter and Western blot assays. A simple vertical-beam colorimeter is described (useful in the Micro-SIA) which can be easily assembled by the user from commonly available components.     

The development of an enzyme-linked immunosorbent assay (ELISA) for cephalexin.

Cephalexin was structurally modified by the attachment of a spacer at the carboxylic acid through which it was subsequently covalently attached to BSA. This method permitted the molecule to be attached without cleavage of the beta-lactam ring giving a conjugate distinct from previously described immunogenic preparations of penicillins and cephalosporins. This approach required the development of a novel spacer molecule, and its synthesis and characterisation are reported. Rabbits were used to raise antisera and the antibodies produced were characterised with respect to their reactivity with cephalexin and various analogues, other cephalosporins, and a number of penicillins.     

Improved enzyme immunosorbent assay for mouse prolactin using penicillinase as label.

Enzyme immunosorbent assay (EIA) for mouse prolactin was established by modifying a method originally developed for human prolactin by Shrivastav et al. This simple, sensitive, rapid, and reproducible assay utilizes penicillinase as the labeling enzyme, rabbit anti-mouse prolactin antibody (Ab) and goat anti-rabbit Ig Ab as the first and second antibodies. Prolactin reference preparations and enzyme-conjugated prolactin were mixed with the first Ab and incubated for 0.5 h at 4 degrees C (24-48 h for serum samples). Then, the sample mixture was transferred to the wells of microtiter plate coated with the second Ab. After being kept at room temperature for 2 h, the plate was washed and filled with substrate solution (penicillin V). Absorbance at 620 nm was measured with an ELISA reader to quantitate the amount of conjugated prolactin bound to the second Ab. The prolactin levels obtained by this assay exhibited good correlation with those measured by radioimmunoassay (RIA) (y = 0.95x + 9.14, r = 0.943), and the sensitivity of EIA was equivalent to that of RIA (1.7 ng/ml). The CVs of intra-assay and inter-assay by EIA for mouse serum samples ranged comparably to those by RIA.     

Evaluation of an enzyme-linked immunosorbent assay for the detection of ectromelia (mousepox) antibody.

Ectromelia virus, an orthopoxvirus that can cause extensive morbidity and mortality (mousepox) in colonized mice, has been epizootically responsible for serious disruption of biomedical research since 1930. The lack of a sensitive and specific serological assay for infection with this virus became apparent during outbreaks of mousepox at the National Institutes of Health, Bethesda, Md., and other biomedical research institutions in 1979 and 1980. To fill this need, we evaluated an enzyme-linked immunosorbent assay. Sucrose gradient-purified ectromelia and vaccinia viruses were compared as antigens in tests on approximately 1,000 mouse sera from experimentally infected mice and conventional colonies of uninfected mice. A statistical analysis based on the frequency distribution of the absorbance values for 152 mouse sera (free of ectromelia antibody) gave 0.22 as a value to differentiate ectromelia-positive sera from ectromelia-negative sera. When enzyme-linked immunosorbent assay results were compared with those obtained by an indirect immunofluorescence assay, the former was found to be at least 10-fold more sensitive. With the procedures employed, including the use of purified vaccinia virions as antigen, the enzyme-linked immunosorbent assay proved to be highly sensitive and specific for detecting antibodies to ectromelia and vaccinia viruses. False-positive results were not encountered. False-negative results were observed in 3% of 108 separate tests of a known positive serum. Although data indicated that ectromelia antibody can be differentiated from vaccinia antibody with homologous and heterologous antigen, this procedure probably cannot be generally used because of unavailability of ectromelia antigens.     

Enzyme-linked immunosorbent assay (ELISA) using antibody class capture for the detection of antitoxoplasma IgM.

Sera from 180 patients with suspected toxoplasmic lymphadenopathy were examined for antitoxoplasma IgM by an enzyme-linked immunosorbent assay (ELISA), using antibody class capture (ACCA). Of 82 positive ACCA results, 78 were confirmed by testing the IgM fractions of the sera, obtained by sucrose density gradient centrifugation (SDGC). The four positive results which could not be confirmed were all from patients with at least a year''s history of lymphadenopathy. Sera from 10 patients with low Sabin Feldman dye test (DT) titers gave positive ACCA results and subsequent specimens from them showed a rise in antibody concentration, confirming the diagnosis of acute toxoplasmosis. The antitoxoplasma IgM immunofluorescent antibody test (IgM-IFA) on whole serum was relatively insensitive and gave false-positive results with sera containing rheumatoid factor (RF) and antinuclear factor (ANF). There were no false-positive ACCA results with such sera, probably because the conjugates were prepared from F(ab'')2 fragments of antitoxoplasma serum. The ACCA proved to be sensitive, specific and easily automated enabling examination of large numbers of specimens.     

A multidot immunobinding assay for the serodiagnosis of tuberculosis. Comparison with an enzyme-linked immunosorbent assay

A simple dot immunobinding (dot blot) assay procedure has been developed for the detection of antibodies directed against a soluble mycobacterial antigen preparation. This technique was compared with the widely used ELISA, in a study of samples from tuberculous patients. Dot blots were read on a densitometer. The correlation between both assays was excellent (r = 0.91; P less than 0.001); 90% of sera from tuberculous patients were detected using both techniques and a serial two-fold dilution method. Assessments of the end-points of titration curves by reflectometry and simple visual interpretation gave similar results. The dot blot assay is easier to perform and appears to be a practical alternative to ELISA for the detection of anti-mycobacterial antibodies in tuberculous patients.     

Bifidobacterial lipoglycan as a new cause for false-positive platelia Aspergillus enzyme-linked immunosorbent assay reactivity

We previously hypothesized that a lipoglycan of Bifidobacterium bifidum subsp. pennsylvanicum cross-reacts with the Platelia Aspergillus (PA) enzyme-linked immunosorbent assay (ELISA) based on the presence of galactofuranosyl epitopes in the cell wall (M. A. S. H. Mennink-Kersten, R. R. Klont, A. Warris, H. J. M. Op den Camp, and P. E. Verweij, Lancet 363:325-327, 2004). We tested this hypothesis by testing bacterial suspensions of different bifidobacterial species and other gram-positive and -negative bacteria with the PA ELISA, which is used to detect circulating galactomannan for the serodiagnosis of invasive aspergillosis. Furthermore, neonatal fecal samples were enumerated for bifidobacteria by fluorescence in situ hybridization (FISH) and tested for PA ELISA reactivity. All bifidobacteria, except B. infantis and B. adolescentis, showed reactivity 6- to 600-fold higher compared to the controls (i.e., Micrococcus luteus and Propionibacterium freudenreichii, which contain a cell wall lipomannan). Eggerthella lenta showed a 25-fold-higher reactivity. ELISA reactivity was clearly shown to be associated with bacterial lipoglycans containing a beta-1,5-galactofuranosyl chain. All neonatal feces showed PA ELISA reactivity and associated numbers of bifidobacteria. Since high concentrations of bifidobacteria are present in the human gut, these bacteria or excreted lipoglycan may cause false serum PA ELISA reactivity in selected patient groups, especially neonates.     

Validation of an enzyme-linked immunosorbent assay for the quantification of citrullinated histone H3 as a marker for neutrophil extracellular traps in human plasma

There is an emerging interest in the diverse functions of neutrophil extracellular traps (NETs) in a variety of disease settings. However, data on circulating NETs rely largely upon surrogate NET markers such as cell-free DNA, nucleosomes, and NET-associated enzymes. Citrullination of histone H3 by peptidyl arginine deiminase 4 (PAD4) is central for NET formation, and citrullinated histone H3 (H3Cit) is considered a NET-specific biomarker. We therefore aimed to optimize and validate a new enzyme-linked immunosorbent assay (ELISA) to quantify the levels of H3Cit in human plasma. A standard curve made of in vitro PAD4-citrullinated histones H3 allows for the quantification of H3Cit in plasma using an anti-histone antibody as capture antibody and an anti-histone H3 citrulline antibody for detection. The assay was evaluated for linearity, stability, specificity, and precision on plasma samples obtained from a human model of inflammation before and after lipopolysaccharide injection. The results revealed linearity and high specificity demonstrated by the inability of detecting non-citrullinated histone H3. Coefficients of variation for intra- and inter-assay variability ranged from 2.1 to 5.1% and from 5.8 to 13.5%, respectively, allowing for a high precision. Furthermore, our results support an inflammatory induction of a systemic NET burden by showing, for the first time, clear intra-individual elevations of plasma H3Cit in a human model of lipopolysaccharide-induced inflammation. Taken together, our work demonstrates the development of a new method for the quantification of H3Cit by ELISA that can reliably be used for the detection of NETs in human plasma.     

Enzyme-linked immunosorbent assay for the phytotoxin cleistanthin A.

Enzyme-linked immunosorbent assay is reported for the estimation of cleistanthin A, a major constituent of the toxic plant Cleistanthus collinus. Rabbit antibodies were obtained by immunisation with cleistanthin A hemisuccinate-BSA conjugate and the ELISA developed thereupon could detect cleistanthin A at as low a concentration as 3 ng/ml. Cross-reactivity studies with structural analogs as well as with other phytotoxins and drugs of common occurrence established the suitability of the ELISA to specifically monitor the C. collinus marker molecules in emergency clinical and forensic cases. The simplicity and specificity make the ELISA superior to the other available techniques.     

Enzyme-linked immunosorbent assay for determination of antibodies to the envelope glycoprotein of rabies virus.

The envelope glycoprotein G of rabies virus induces neutralizing antibodies, which are important in protection against rabies. This protein was solubilized from purified virus and isolated by differential and sucrose density gradient centrifugation followed by high-performance liquid chromatography. Conditions for solubilization and purification of G were optimized by using immunoblotting and enzyme-linked immunosorbent assay techniques. The reaction with conventional antisera and monoclonal antibodies indicated that purified G protein was essentially devoid of internal viral proteins. Microdilution plates were coated with purified G protein, and sera from humans vaccinated against rabies were tested for the presence of antibodies. Results were compared with those of the rapid fluorescent focus inhibition assay, which is the standard neutralization assay for antibodies to rabies virus. The results of this comparison indicate that the enzyme-linked immunosorbent assay for G is a reliable and simple alternative to the neutralization test.     

A new enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus: II. Application

The liquid-phase blocking sandwich ELISA has been evaluated for the serological study of antibodies against foot-and-mouth disease virus (FMDV). The titres recorded for sera from a population of more than 300 British uninfected, unvaccinated cattle which were examined against each of the seven immunologically distinct FMDV types were less than 1 in 40. alternative to the existing VN test for the quantification of antibodies against FMD virus.     

Development of an equine herpesvirus type 4-specific enzyme-linked immunosorbent assay using a B-cell epitope as an antigen

The equine herpesvirus type 4 (EHV-4)-specific region of glycoprotein G has served as an antigen for serodiagnosis and seroepizootic studies of EHV-4 infection (B. S. Crabb and M. J. Studdert, J. Virol. 67:6332-6338, 1993; S. Yasunaga, K. Maeda, T. Matsumura, K. Kai, H. Iwata, and T. Inoue, J. Vet. Med. Sci. 60:1133-137, 1998; S. Yasunaga, K. Maeda, T. Matsumura, T. Kondo, and K. Kai, J. Vet. Med. Sci. 62:687-691, 2000). Here we identified a major B-cell epitope in the type-specific region of EHV-4 and applied it as an antigen in enzyme-linked immunosorbent assays (ELISAs). A 24-amino-acid repeat sequence expressed as a glutathione S-transferase fusion protein specifically reacted as well as the type-specific region with sera from foals infected with EHV-4. Five synthetic peptides (12-mer peptides) in the repeat sequence were included as ELISA antigens. The results indicated that the 12-mer peptide MKNNPIYSEGSL contained a major B-cell epitope specific for EHV-4 infection. Inclusion of this 12-mer peptide in ELISAs for an epidemiological study specifically detected EHV-4 infection in the field. These results indicated that the 12-mer epitope was responsible for the type-specific antibody response and therefore is useful for seroepizootic studies and serodiagnosis of EHV-4 infection.     

Characterization of a monoclonal antibody against neopterin using an enzyme-linked immunosorbent assay with penicillinase as label

An active ester derivative of neopterin was prepared using 4-(N-maleimidomethyl) cyclohexan 4-carboxilic acid N-hydroxy succinimide ester (MCH-NHS), conjugated to bovine serum albumin (BSA) and injected for antibody production (for both monoclonal and polyclonal antibodies). High titer antibody producing spleen cells were removed and fused with myeloma cells of Sp2/0 origin. Neopterin was conjugated to the enzyme penicillinase by a one-step glutaraldehyde method, which was used as tracer. A novel enzyme immunoassay was developed using this conjugate to screen and characterize the monoclonal antibody (MAb) produced in these experiments. After limiting dilutions, it was found that antibody produced by one clone with a Ka value of 7.6 x 10-7 mol/L was specific for a number of structurally related molecules. This clone was found to be of IgG class and IgG2a subclass. The standard curvewas constructed with a sensitivity of 10 pg/well (100 pg/mL) covering up to 1 ng/mL.     

Development of an enzyme‐linked immunosorbent assay (ELISA) for the detection of porcine pepsin as a milk clotting enzyme

An enzyme‐linked immunosorbent assay (ELISA) for the detection of porcine pepsin in milk‐clotting enzyme preparations has been developed. The assay is capable of detecting porcine pepsin in the range 1 μgto 1 mg ml^‐1 without enhancement or modification. The specificity of the technique was studied by inhibition assay. Slight cross‐reactions with bovine rennet and Mucor miehei rennet occurred at high concentrations (1.0 mg ml^‐1). The ELISA used in this investigation appears to provide a quick, sensitive and specific method for the detection of porcine pepsin and has potential applications in the dairy industry.     

Polycarbonate-coated microsticks as solid-phase carriers in an enzyme-linked immunosorbent assay for rubella antibody.

We evaluated the use of microsticks as solid-phase carriers in an enzyme-linked immunosorbent assay for rubella antibody. The microstick enzyme-linked immunosorbent assay was found to be equal in sensitivity to plate and disk enzyme-linked immunosorbent assays and presumably more sensitive than hemagglutination and immunofluorescence assays. The microstick as a solid-phase carrier offers advantages over both plate and bead carriers.     

Rapid enzyme-linked immunosorbent assay (ELISA) for Aspergillus fumigatus antibodies.

A rapid enzyme-linked immunosorbent assay (ELISA) where component incubation periods were shortened to one hour, was compared with agar gel double diffusion (AGDD) and a standard ELISA procedure for detecting antibodies to Aspergillus fumigatus in 28 asthmatic patients with suspected allergic aspergillosis. Using two A fumigatus antigens the rapid ELISA compared well with AGDD and the standard ELISA method. Eleven sera that reacted with both antigens in AGDD were all positive against antigen 1 in both forms of ELISA, but two failed to react with antigen 2 in the standard ELISA and three failed to react with this antigen in the rapid method. Thirteen AGDD-negative sera were also negative in both forms of ELISA. The rapid ELISA provides a sensitive and reproducible test for routine serological investigation of allergic aspergillosis.