Establishment of an enzyme-linked immuno-sorbent assay for urinary trypsin inhibitor by using a monoclonal antibody.

Monoclonal antibodies against inter-alpha-trypsin-inhibitor (ITI) were produced. One clone showing specificity for urinary trypsin inhibitor (UTI), a proteolytic fragment of ITI, which is excreted into urine, was selected for the establishment of an enzyme-linked immuno-sorbent assay (ELISA). The ELISA for the quantification of UTI was shown to work reproducibly in the range between 0.5 and 10 ng UTI/ml urine. Urines of several patients suffering from different lung diseases were screened for UTI using the established ELISA. Highest UTI levels were found in the urine of patients with lung empyema. A more moderate increase was observed in patients suffering from lung tuberculosis and from secondary and primary lung tumors.     

Internalization of urinary trypsin inhibitor in human uterine fibroblasts

 We have characterized the molecular species and internalization of urinary trypsin inhibitor (UTI) in human uterine fibroblasts. Link protein (LP) has previously been identified as one of the cell-associated UTI binding proteins. The truncated forms of UTI were readily detectable in the cells after incubating the cells with purified UTI. Immunoblotting analysis with a panel of domain-specific antibodies revealed that the UTI species lacked the amino-terminal domain of UTI, but contained the carboxyl-terminal domain. We have examined whether LP is involved in the UTI internalization in the cells. Internalization of ¹²⁵I-labelled UTI was blocked by the intact UTI, but not by the carboxyl-terminal domain of UTI. Treatment with a polyclonal antibody to the UTI binding domain of LP partially inhibited UTI binding to the cells, but did not significantly prevent UTI internalization. In addition, preincubation of the cells with hyaluronidase reduced the UTI binding to the cells, but had no effect on the rate with which UTI was internalized. These data allow us to conclude that there are at least two different mechanisms for internalization of UTI. The major one is via unknown UTI receptors in a Ca²⁺, Mg²⁺-sensitive manner and another is via LP. Received: 13 November 1997 / Received after revision and accepted: 27 January 1998     

Sandwich ELISA and immunochromatographic strip of Kunitz trypsin inhibitor using sensitive monoclonal antibodies

Kunitz trypsin inhibitor (KTI) is a predominant anti-nutritional factor in soybean. The objective of this study was to develop a sandwich enzyme-linked immunosorbent assay and immunochromato graphic strip (ICS) method for the semi-quantification of KTI in soybean. The limit of detection was 0.1481?ng/ml, and the linear dynamic range was 0.2–20?ng/ml. KTI recovery was 98.40–110.66% from KTI-spiked milk samples, with coefficients of variation of 4.41–10.62%. Based on ICS results, KTI had cutoff values of 5?ng/ml and 2.5?ng/ml in 0.05% Tween-added phosphate-buffered saline and milk, respectively. ICS has potential applications for the fast detection of KTI.     

Human myometrial cells in culture express specific binding sites for urinary trypsin inhibitor

Urinary trypsin inhibitor (UTI), which is present in amniotic fluid, prevents uterine contractility during pregnancy possibly via specific binding protein mechanisms. To test for the presence of UTI binding sites on the cell surface, we prepared cultured myometrial cells obtained at biopsy from 12 pregnant women and performed binding, competition, and cross-linking experiments using a specific radiolabelled UTI as a ligand. We report for the first time two classes of binding sites of differing affinities. Scatchard analysis at 4 degrees C, using radioiodinated UTI, revealed that UTI binds to 35 000 high affinity binding sites/cell (K(d) = 9.1x10(-9) mol/l) and 450 000 lower affinity binding sites/cell (K(d) = 3.5x10(-7) mol/l) in cultured myometrial cells. It appears to be the low affinity site that is internalized, and this has been identified as a protein of approximately 45 kDa by cross-linking and immunoaffinity labelling studies. Monoclonal antibodies against the NH(2)-terminal fragment of UTI abrogated specific binding of this protein to the cells. Treatment of the cells with hyaluronidase resulted in >80% inhibition of the [(125)I]-labelled UTI binding to the cells. These data show that the UTI binding site, which is hyaluronidase sensitive, is expressed on the surface of human uterine myometrial cells to accumulate the UTI molecule during pregnancy.     

Immunochemical determination of the serum protein reacting with antibody against human urinary trypsin inhibitor by single radial immunodiffusion: use of polyethylene glycol

A single radial immunodiffusion method is described for determining the serum protein which reacts with antibody against the urinary trypsin inhibitor, but does not react with anti-inter-alpha-trypsin inhibitor antibody. Since the amount of this protein could not be determined with an agar plate containing antibody alone, we first prepared an agar plate containing 4% polyethylene glycol 6 000 and 1 mg of anti-urinary trypsin inhibitor gamma-globulin and confirmed that the amount of this protein could be measured accurately from the sizes of precipitin rings after 72 h incubation at room temperature. Levels of this serum protein, alpha 1-antitrypsin, alpha 2-macroglobulin and inter-alpha-trypsin inhibitor were measured in normal human serum by the single radial immunodiffusion method, and it was confirmed that the level of this serum protein did not correlate with the levels of these 3 other proteins.     

Antioxidative role of urinary trypsin inhibitor in acute lung injury induced by lipopolysaccharide

We have previously demonstrated the protective role of urinary trypsin inhibitor (UTI) against acute inflammatory lung injury induced by lipopolysaccharide (LPS) using UTI-deficient (-/-) mice and corresponding wild-type (WT) mice. The protection was mediated, at least partly, through inhibition of the enhanced local expression of proinflammatory cytokines, chemokines, and intercellular adhesion molecule-1. In the present study, we addressed whether UTI regulates oxidative stress generated by LPS challenge in the lung. UTI (-/-) and WT mice were treated intratracheally with vehicle or LPS (125 microg/kg). After LPS challenge in both genotypes of mice, the lung levels of mRNA for inducible nitric oxide synthase and hemo oxygenase-1 were elevated, but to a greater extent in UTI (-/-) mice than in WT mice. Immunohistochemistry showed that the formations of 8-hydroxy-2'-deoxyguanosine and nitrotyrosine in the lung were more intense in UTI (-/-) mice than in WT mice after LPS challenge. These results indicate that endogenous UTI is protective against acute lung injury induced by bacterial endotoxin, at least partly, via the antioxidative properties.     

Uptake of human urinary trypsin inhibitor by the kidney epithelial cell line,LLC-PK1

 We investigated the uptake of human urinary trypsin inhibitor (UTI) by the kidney epithelial cells, LLC-PK₁. Indirect immunogold techniques with an electron microscope demonstrated the localization of UTI within the cells after an incubation during which UTI was added to the apical side. Immunoreactivities were found in endocytic vesicles, vacuoles and lysosomes. Subsequently, we tried to characterize the property of the uptake of UTI using the fluorescein isothiocyanate-labelled UTI (FITC-UTI). FITC-UTI uptake was decreased by an incubation with an excess of unlabelled UTI and showed concentration-dependent saturation. This process was markedly suppressed during the incubation at 4°C. The uptake was significantly lessened with 2,4-dinitrophenol and antimycin A, inhibitors of oxidative phosphorylation, and colchicine, a microtubule-depolymerizing agent. These results indicate that exogenous UTI is internalized by LLC-PK₁ cells through an endocytic pathway. From uptake studies, it is suggested that an adsorptive process is partially involved in the mechanisms of endocytosis. Received: 11 March 1996 / Received after revision and accepted: 8 July 1996     

Development and studies of the anti-R7V neutralizing antibody ELISA test: a new serological test for HIV seropositive patients

A new peptide-based ELISA test developed for the detection of anti-R7V specific antibodies in the sera of HIV seropositive patients is described. HIV virus acquires a cellular antigen at the moment the virus is released by budding, the R7V epitope. Certain patients produce anti-R7V Ab which are described as having the capacity to neutralize in vitro cell infection by HIV. Anti-R7V Ab are also detected in asymptomatic patients who have a lower likelihood of progressing to AIDS. Tested with 449 serum samples, the prevalence of anti-R7V Ab was 53.2% in HIV positive patients and 5.5% in HIV negative subjects. According to the duration of infection, the seroprevalence reaches almost 80% of non-treated long-term infected patients. Other retrospective studies were conducted on 308 HIV positive samples; the presence of anti-R7V Ab was significantly higher for 177 asymptomatic patients (64.4%) compared to the 131 symptomatic patients (35.1%). Regarding their neutralizing ability, anti-R7V antibodies were detected at the highest percentage in asymptomatic HIV-infected patients naive of treatment. Besides conventional biological parameters (CD4 and viral load), the detection of anti-R7V antibodies could be proposed to clinicians as an additional tool to manage treatment initiation and to improve the psychological state of their asymptomatic patients.     

Detection of mouse and rat urinary aeroallergens with an improved ELISA

BACKGROUND: Risk analysis of laboratory animal work presupposes allergen monitoring with sensitive methods. Commercial ELISA kits have recently become available for the detection of mouse (Mus m 1) and rat (Rat n 1) urinary allergen from settled dust samples and air samples with high allergen levels. OBJECTIVE: Our aims were to enhance the sensitivities of the commercial ELISA kits for low aeroallergen levels (less than 1 ng/m(3)) and to test these methods with air samples collected from an animal facility. METHODS: Personal and stationary air samples were collected from an animal facility during various tasks of laboratory animal work and from various premises of the animal facility. RESULTS: The sensitivities of the ELISA assays were improved with a careful choice of analysis parameters and reagents. The detection limits of 0.1 ng/m(3) for Mus m 1 and 0.8 ng/m(3) for Rat n 1 were established. The sensitized assays enabled detection of mouse and rat aeroallergens also from premises in which animals or dirty cages were not present during sampling. CONCLUSION: These sensitive assays will help to perform risk assessment in laboratory animal work. However, there remains a lack of standardized analytic procedures and occupational exposure limits for laboratory animal allergens.     

Development of an ELISA for urea herbicides in foods

Four haptens analogous to phenylurea herbicides were synthesized. Immunogens and coating proteins prepared by coupling these haptens to human serum albumin and ovalbumin, respectively. Antisera raised in rabbits immunized with three of the immunogens bound strongly to microtitration plates coated with homologous hapten, such that competitive assays were developed using heterologous coating proteins. The assay systems were characterized with respect to specificity to 13 related compounds and one selected for application to the determination of monolinuron, diuron, and linuron in foods. Satisfactory recrovery data were obtained by direct analysis of methanol extracts of foods fortified with 40–160 ng/g monolinuron or 0.25–2.0 μg/g diuron. Solvent partitioning and silicic acid column chromatography were only partially successful in removing interferences in the determination of linuron.     

Development of an immunoassay for urinary galactosylhydroxylysine

Galactosylhydroxylysine (GHL) is released during bone resorption and has been shown to be elevated in subjects with metabolic bone loss. GHL is relatively specific for bone, it is not recycled or significantly metabolized during collagen turnover, and the levels are not influenced by diet. Previous measurements of GHL levels in urine have been performed using reverse-phase high performance liquid chromatography following pre-column derivatization. We produced polyclonal antibodies to GHL using GHL purified from sea sponges and developed an immunoassay that can recognize GHL in urine. The antibodies have minimal cross-reactivity with a physiological mixture of amino acids (<1%), galactose (<0.2%), lactose (<0.3%), and glucosylgalactosylhydroxylysine (<1%). This competitive immunoassay requires no dilution or pretreatment of the samples and provides a rapid and easy method for the evaluation of GHL in urine. Analysis of clinical samples from normal individuals, post-menopausal women, osteoporotic patients and individuals with Paget's disease show that the assay can discriminate between groups with differing levels of bone resorption as well as deoxypyridinoline (Dpd).     

Development of an indirect competitive ELISA for the detection of acenaphthene and pyrene

Polycyclic aromatic hydrocarbons (PAHs) have mutagenic and carcinogenic properties. Acenaphthene and pyrene were members of 16 PAHs which were listed as the priority pollutants in water environment by US Environmental Protection Agency. The reported instrumental methods and immunoassays did not meet the need for simple and sensitive detection of acenaphthene and pyrene. In this study, a monoclonal antiobody having high affinities with acenaphthene and pyrene was produced and an indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed for sensitive detection of acenaphthene and pyrene in water sample. The linear range of the assay was between 3.53 and 41.98?ng?mL^?1. The sensitivity was 12.17?ng?mL^?1 which was more sensitive than those of reported ELISAs. The average recovery of acenaphthene and pyrene from three kinds of water samples was 99.08% and 98.45, respectively. The developed ELISA could be used for sensitive detection of acenaphthene and pyrene in water samples.     

Comparison between a commercial ELISA, Rubazyme, and hemolysis-in-gel test for determination of rubella antibodies

A commercial enzyme-linked immunosorbent assay, Rubazyme, was compared with the hemolysis-in-gel (HIG) test for antibodies to rubella in 826 sera. The results were in agreement for 99.4% of the 725 sera tested for immunity. However, the Rubazyme assay was no more efficient that either the hemagglutination-inhibition or HIG test in discriminating between sera with low levels of antibody and negative sera. Thus, it was concluded that the HIG test is the method of choice for immunity testing because of the low cost and simplicity. Rubazyme may be of value to confirm equivocal HIG results.     

Quantitative determination of urinary 8-hydroxydeoxyguanosine (8-OH-dg) by using ELISA

In order to establish a quantitative assay for 8-OHdG concentrations in urine, we examined the precision of a test for the recovery of 8-OHdG in urine by using an ELISA method. The coefficient of variation (CV) for assay with incubation in water or air ranged between 7.0% and 8.4% and between 19.2% and 30.6%, respectively. The data by using incubation in water gave higher accuracy than those in air. The recovery rates of 8-OHdG in urine sample ranged from 95 - 114%. Our results indicated that the excellent sensitivity of this ELISA kit by using incubation in water makes its use possible for the determination in urine with a good reproducibility and recovery of 8-OHdG-spiked samples.     

Development of an ELISA for the detection of autoantibodies to BP230

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease associated with autoantibodies against the transmembrane hemidesmosomal protein BP180/collagen type XVII and the intracellular plaque protein BP230. The aim of the present study was to develop an ELISA system for the detection of circulating autoantibodies to BP230. We generated five overlapping cDNA constructs covering the entire length of BP230 and expressed them in baculovirus-infected Sf21 insect cells. ELISA reactivity against BP230 was found in 63% of 56 BP patients' sera; the specificity of the ELISA was 93%. Epitope mapping studies showed that the fragment representing the C-terminal portion of BP230 was by far the most frequent target within the molecule. This ELISA provides a useful tool for the detection of autoantibodies to BP230 in BP and other diseases associated with an autoimmune response to this protein.     

Development of a rapid competitive indirect ELISA procedure for the determination of deoxynivalenol in cereals

A rapid and sensitive method based on competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed and validated for the detection of DON (deoxynivalenol) in cereals with the confirmation of the reliable ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Within this method, the IC50 (half maximal inhibitory concentration of a substance) of the DON-specific monoclonal antibody (MAb) we produced was 61.10 ng/mL. The limit of detection (LOD) value, measured by IC10, was 6.12 ng/mL, which is below the maximum residue levels (MRLs) established for DON in various cereals. In spiked samples, the recoveries ranged from 79.3% to 115.2% for maize and from 74.3% to 118.3% for wheat. This method was compared with the UPLC-MS/MS method by testing four concentrations. The correlation coefficient for the two methods was 0.9884 in a linear-regression analysis. The results illustrated the reliability of the ciELISA method for the determination of cereal samples.