Quantification of human tissue transglutaminase by a luminescence sandwich enzyme-linked immunosorbent assay

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes crosslinking of peptidic glutamine residues with primary amines via isopeptide bonds and hydrolysis of ATP or GTP. The enzyme exerts a variety of functions at the cellular and tissue levels that may be disturbed in disease. Its role in pathoprocesses is poorly understood. For investigation of the involvement of tTG in disease, sensitive and specific assays should be available. We have developed the first sandwich enzyme-linked immunosorbent assay (ELISA) based on two monoclonal antibodies (mabs) against human tTG. tTG is captured by mab 3C10 and detected by biotinylated mab 10F3. After incubation with peroxidase-conjugated streptavidin, bound tTG is visualized by peroxidase reaction applying a luminescence substrate. The detection limit was 40 pg/ml. The assay was highly reproducible. Recovery of spiked tTG in crude samples was greater than 92%. The enzyme could be detected in cellular lysates and tissue homogenates of humans. The effect of typical effectors (retinoic acid and interferon-γ) on tTG expression could be demonstrated. A low signal was also obtained in mice samples, suggesting cross-reactivity of the mabs with murine tTG. The new sandwich ELISA may be successfully applied for investigation of physiological functions of tTG and of disorders associated with inadequate tTG expression.     

Quantification of apolipoproteins in rat serum and in cultured rat hepatocytes by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) has been developed to measure apolipoproteins in rat serum. Nondelipidated whole serum was heat-treated at 52 degrees C for 3 h in phosphate-buffered saline containing 0.1% Tween-20 before assay. Monospecific rabbit anti-rat apolipoprotein antibodies were added to 96-well polystyrene microtiter plates which had been coated with purified rat serum apolipoproteins or unknown samples. After incubation and washing, goat anti-rabbit serum antibodies conjugated with horseradish peroxidase were added to the plates and incubated. The bound peroxidase activity was assayed after further washing. Serum apolipoprotein concentrations were calculated by comparison against purified standards that were assayed simultaneously with the unknown samples. The intraassay coefficients of variation for apolipoprotein AI, E, and AIV (Apo AI, E, and AIV) were 2.3, 4.4, and 5.3%, and interassay coefficients of variation were 6.1, 5.5, and 7.9%, respectively. The ELISA assay is sensitive to nanogram quantities of rat serum apolipoproteins and the results agree well with those measured by densitometry. The serum concentrations of Apo AI, E, and AIV of a normal fed rat were found to be 504 +/- 8, 413 +/- 20, and 262 +/- 20 micrograms/ml, respectively. When cultured as monolayers in Waymouth's medium for 1 day, rat hepatocytes secreted Apo AI, E, and AIV at rates of 2.51, 61.8, and 48.9 ng protein/mg cell protein/h.     

Quantification of pralidoxime methylsulfate (Contrathion) in human urine by capillary zone electrophoresis

Pralidoxime methylsulfate (Contrathion) is widely used to treat organophosphate poisoning. For the first time, we developed a specific assay for urinary pralidoxime using capillary zone electrophoresis (CZE) in the following conditions: fused-silica capillary (length: 47 cm, internal diameter: 75 microm), electrolyte solution: 25 mM sodium borate (pH 9.1), voltage: 15 kV, temperature: 25 degrees C, injection time: 1 or 2s, on-line UV detection: 280 nm. Sample preparation did not require a deproteinization step (1:5 dilution in water). The method was linear between 0.125 and 2 mg mL-1 of pralidoxime (quantification limit: 0.10 mg mL-1). Coefficients of variation for intra- and inter-assay precision were below 10% for all three control levels (0.15-1.15 mg mL-1). This assay was successfully applied to urine specimens from organophosphate poisoned patients treated by Contrathion (n=10). This CZE method allows the measure of pralidoxime in urine within 15 min with excellent precision, selectivity, and sensitivity. It is simple (no pretreatment) and convenient, thus suitable for the monitoring of Contrathion therapy in organophosphate poisoned patients.     

Quantification of two cytochrome P-450 isoenzymes by an enzyme-linked immunosorbent assay (ELISA)

An enzyme linked immunosorbent assay (ELISA) using monoclonal and polyclonal antibodies has been developed to quantify individual cytochrome P-450 isoenzymes in microsomal preparations, namely UT-A and PB-B. This very sensitive method can be used for the rapid processing of large quantities of determinations and requires only limited amounts of antibodies.     

A monoclonal antibody-based enzyme-linked immunosorbent assay of ursodeoxycholic acid 3-sulfates in human urine

Sulfation of the 3-hydroxy group is assumed to be a major metabolic route of ursodeoxycholic acid (UDCA) which is used for treating various hepatobiliary diseases. We have developed a sensitive enzyme-linked immunosorbent assay (ELISA) for determining the total amount of nonamidated, glycine- and taurine-amidated ursodeoxycholic acid 3-sulfates (UDCA 3-Suls) using a newly established monoclonal antibody. In this study, 12 kinds of antibody-secreting hybridoma clones were generated by a fusion experiment between P3/NS1/1-Ag4-1 myeloma cells and the spleen cells from a BALB/c or an A/J mouse which had been immunized with a conjugate of nonamidated UDCA 3-Sul and bovine serum albumin. One of the monoclonal antibodies, Ba-10 (γ2a, κ), had suitable binding properties for clinical application, which was group-specific to the UDCA 3-Suls, and showed negligible cross-reactivities with various related bile acids including potentially interfering compounds, namely, the unconjugated UDCA, UDCA 7-N-acetylglucosaminide, the 3-sulfates of cholic acid, chenodeoxycholic acid and deoxycholic acid. The antibody Ba-10 allowed us to develop a sensitive competitive ELISA system whose measurable range was approximately 2–200 pg per assay. A serial dilution study indicated that the ELISA enables the direct measurement of the UDCA 3-Suls in human urine before and after the administration of exogenous UDCA. The daily urinary excretion rate of UDCA 3-Suls from healthy male volunteers (n = 5) was determined to be a mean of 131 ± 61.2 (SD) μg as the nonamidated UDCA 3-Sul equivalent.     

Preferential detection of pro-carboxypeptidase R by enzyme-linked immunosorbent assay

We generated two monoclonal antibodies (mAbs), 2A16 and 10G1, against pro-carboxypeptidase R (proCPR), also known as thrombin activatable fibrinolysis inhibitor (TAFI). By use of these mAbs, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) system to detect proCPR. Since the amount of the antigen detectable by the ELISA was essentially the same in fresh plasma and serum incubated at 37 C for 1 hr, we concluded that the ELISA system detected not only proCPR, but also inactivated CPR generated from proCPR. However, an appreciable amount of proCPR remained unactivated in serum. For extensive activation of proCPR in plasma, thrombin and thrombomodulin complexes (TTM) can be used together with CaCl2. Following extensive conversion of proCPR to CPR by T-TM and CaCl2, converting plasma to serum (T-TM serum), antigenicity became undetectable by ELISA. Further analysis revealed that 2A16 reacts only with proCPR although 10G1 reacts with proCPR, active CPR and inactivated CPR. Therefore, we concluded that the ELISA system preferentially detects proCPR and not CPR. Our sandwich ELISA system utilizing 2A16 and 10G1 provides a suitable method for detecting proCPR and can be used to determine levels of proCPR in plasma samples from patients.     

Determination of opiates in urine by capillary electrophoresis

A method for the separation of a mixture of opiates comprising pholcodine, 6-monoacetylmorphine, morphine, heroin, codeine and dihydrocodeine by capillary electrophoresis using a running buffer of 100 mM disodium hydrogenphosphate at pH 6 is described. The characteristics of an analytical method based on this separation for the determination of these drugs following extraction from urine and using levallorphan as internal standard are reported. Detection limits in the region of 10 ng cm⁻³ are achieved when using electrokinetic injection. A comparison is made of the sensitivity and reproducibility of electrokinetic and hydrodynamic injection for these drugs. Data are presented to show the results obtained when the proposed method is applied to urine spiked with all the above opiates and also to urine from a subject following consumption of dihydrocodeine and pholcodine. The concentrations found are compared with those obtained by LC.     

An enzyme-linked immunosorbent assay to quantitate the elastin crosslink desmosine in tissue and urine samples

An enzyme-linked immunosorbent assay method has been developed for the determination of desmosine. The method is based on an inhibition immunoassay (under nonequilibrium conditions) and uses rabbit antisera directed against a desmosine-bovine serum albumin conjugate and microtiter plates coated with desmosine-gelatin conjugate. The assay quantitates desmosine in the range 2.5-50 pmol in tissue and urine samples. Important applications of this rapid and sensitive assay are in studying elastin metabolism and in screening for monoclonal antibodies against desmosine. Methods are described for obtaining a constant level of substitution of desmosine per molecule of bovine serum albumin and for preparing a desmosine-gelatin coating antigen. Five different antibody preparations directed against desmosine exhibit 15-20% cross-reactivity toward pyridinoline (3-hydroxypyridinium), a nonreducible collagen crosslinking compound also present in urine and many tissue samples.     

Quantification of daunorubicin and daunorubicinol in plasma by capillary electrophoresis

Capillary electrophoresis (CE) with laser-induced fluorescence detection was applied to quantify daunorubicin and daunorubicinol in plasma. Separation was carried out in a 47 cm×50 μm I.D. fused-silica capillary, with a running buffer, pH 5 containing 60 μM spermine and 70% acetonitrile. Sample preparation was done either by protein precipitation with acetonitrile or by liquid–liquid extraction. The assay can be applied in a concentration range from 40 mg/l down to 2 μg/l for daunorubicin and from 1 mg/l to 2 μg/l for daunorubicinol. Precision and accuracy were between 2.9 and 14.5% (n=6) on 1 day and between 1.0 and 14.7% from day to day (n=6) for both analytes. Thus, the CE method enables precise and accurate quantification of daunorubicin and daunorubicinol in small sample volumes over a wide concentration range.     

Quantitative analysis of mouse tyrosinase by enzyme-linked immunosorbent assay

A sensitive, specific, competitive enzyme-linked immunosorbent assay (ELISA) was developed for quantitative analysis of tyrosinase. Binding sites of anti-tyrosinase antibodies were competed for by purified tyrosinase adsorbed onto microtiter plates and a known (standard) or unknown (sample) amount of tyrosinase in solution. Adsorbed antibodies were detected by goat anti-rabbit IgG F(ab')2 labeled with peroxidase. A sensitivity range of 2.1 to 14 ng (30-200 fmol)/well was obtained. SDS was found to be the most suitable detergent for solubilizing the enzyme. Tyrosinase was extracted from B16 mouse melanoma and assayed by the ELISA. The tyrosinase content per mg melanoma protein was 505 +/- 106 (S.D.) ng. This assay is not only useful for measuring the content of normal tyrosinase in crude extracts but also is possibly applicable to detecting the unprocessed tyrosinases.     

Serotyping of dengue viruses by an enzyme-linked immunosorbent assay

We have developed a sandwich enzyme-linked immunosorbent assay for serotyping dengue viruses. In this assay, we used antibody from dengue hemorrhagic fever patients for detection of flavivirus common antigens to confirm virus isolation in C6/36 cells and that from hyperimmune mouse ascitic fluids for serotyping. The anti-dengue antibody was immobilized on microplate wells for capturing of dengue antigens, which were then sandwiched with the same biotinylated antibody. Then the biotin in the solid phase was detected with peroxidase-conjugated streptavidin. We found that all the dengue strains tested were unequivocally identified by this method.     

Detection of H-Y in the enzyme-linked immunosorbent assay

Summary We have developed a new enzyme-linked immunosorbent assay for determination of H-Y phenotype in the human. This assay, which measures the inhibition of the reaction of a monoclonal anti-H-Y antibody and a mouse testis extract as a source of H-Y antigen, was applied to the supernatant of lymphocytes from ten normal male and ten normal female subjects. Introduction of supernatant from male cells gave reading of 69%–78% of those obtained with testis supernatant alone; female-cell supernatant did not inhibit the reaction (89%–102%).     

Measurement of rabies-specific antibodies in carnivores by an enzyme-linked immunosorbent assay

We describe an indirect enzyme-linked immunosorbent assay (ELISA) that utilizes anticanine immunoglobulin for the measurement of rabies-specific antibody in the sera of the major domestic and wildlife reservoirs of rabies in North America. Sufficient cross-reactivity was found to exist between anticanine IgG and serum antibody from all carnivores tested, including dogs, cats, foxes (Vulpes vulpes), skunks (Mephitis sp.) and raccoons (Procyon lotor). With sera of most species, good correlation was observed between results obtained with the ELISA and with the fluorescence inhibition microtest (FIMT). Some wildlife specimens, particularly of skunk and raccoon origin, were cytotoxic in the FIMT, resulting in possible false-positive reactions. In view of this, and since the ELISA is rapid, economical and reproducible (coefficient of variation less than 13%), we consider it to be a favorable alternative to the fluorescence inhibition test for assay of wildlife sera.     

Immunodiagnosis of bovine fascioliasis by enzyme-linked immunosorbent assay and immunoprecipitation methods

Calves were infected with 1,000 Fasciola hepatica metacercariae and the antibodies which developed were monitored by gel diffusion, counterelectrophoresis (CEP) and the enzyme-linked immunosorbent assay (ELISA) using crude or tegument antigen preparations of the adult fluke. Serologic diagnosis was positive as early as 2-4 wk of infection by gel diffusion, by 4 wk by CEP, and by 6-8 wk by ELISA, whereas parasitologic diagnosis via the detection of fluke eggs in the feces was possible only after 8-10 wk of infection. In general, both gel diffusion and ELISA were superior to CEP in detecting active infection.     

Quantitation of serum retinol-binding protein by an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent technique for human serum retinol-binding protein (RBP) was developed. The assay detects RBP via a double-antibody (rabbit anti-human RBP) sandwich technique. The antibody is immobilized by passive adsorption to a polystyrene tube; the assay is then carried out by successive additions containing known and unknown amounts of RBP (antigen), alkaline phosphatase linked to the same antibody, and p-nitrophenyl phosphate (substrate). Colorimetric analysis of the hydrolysis of the substrate by the enzyme (indirectly) attached to the antigen is used for RBP quantitation. The intra- and interassay coefficients of variation ranged between 4 and 7 and 9 and 12%, respectively. The assay can be performed in less than 7 h and has a sensitivity in the nanogram range (3–48 ng/ml). RBP content was analyzed in serum and urine samples of 20 healthy donors and 17 patients with renal failure and in 20 serum specimens of patients with liver cirrhosis. Renal patients had higher serum (mean 150, range 50–398 μg/ml) and urine RBP levels (mean 14, range 1–80 μg/ml) than normal donors (mean serum 43, range 30–60 μg/ml; mean urine RBP 0.06, range 0.04 – 0.13 μg/ml). Liver disease patients had lower than normal serum RBP values (mean 22, range 10–43 μg/ml).     

尿样中三种蛋白质的毛细管电泳分离检测方法研究

目的:建立毛细管电泳分离测定人尿样中转铁蛋白、白蛋白和血红蛋白的新方法.方法:通过选择运行缓冲溶液种类及浓度、pH、表面活性荆种类及浓度、分离电压、进样时间对蛋白质分离效果的影响,优化了毛细管电泳法分离转铁蛋白、白蛋白和血红蛋白的条件.结果:利用此方法测定三种蛋白质的含量,浓度在0.01到1.00 g L-1范围内与峰电流呈良好的线性关系,检出限均为10-4 g L-1.结论:所建立的方法用于人尿样中转铁蛋白、白蛋白和血红蛋白的测定,结果满意.     

Analysis of creatine and creatinine in urine by capillary electrophoresis

Creatine is found in the urine of subjects ingesting creatine monohydrate as an ergogenic aid. Creatinine, the catabolic breakdown product of creatine, is a major constituent of normal urine. It is of interest to follow the excretion of creatine and creatinine in urine as a function of time after creatine ingestion. In this study, creatine and creatinine were analyzed in urine by capillary electrophoresis. The optimization of the method was discussed, with the best results being obtained using a 30 mM phosphate–150 mM sodium dodecyl sulfate buffer at pH 6, with the detector set at 214 nm and an applied voltage of 15 kV across a 45 cm capillary. Verification of the method was provided by HPLC analysis and spiking. The application of the method was demonstrated by analysis of creatine and creatinine in urine samples collected in a 24-h period following creatine ingestion.     

Determination of quassin in picogram quantities by an enzyme-linked immunosorbent assay

A double-antibody, enzyme-linked immunosorbent assay has been developed for the detection of quassin, neoquassin and 18-hydroxyquassin, the bitter natural products of Quassia amara and related species. Antiserum was raised in rabbits using 18-hydroxyquassin-bovine serum albumin as immunogen. The assay described is able to detect these closely related seco-triterpenes at concentrations as low as 5 pg per 0.1 ml sample, and the antiserum shows little cross-reactivity with other quassinoids. The distribution of quassin within small plants of Q. amara, Q. indica and Picrasma quassioides is described.     

Determination of retinoid activity by an enzyme-linked immunosorbent assay.

In normal human keratinocytes, retinoic acid suppresses the expression of the plasma membrane associated enzyme transglutaminase (TGm) at the pretranslational level. This finding led us to develop an enzyme-linked immunosorbent assay (ELISA) for the evaluation of the biological activity of retinoids, i.e., natural and synthetic derivatives of vitamin A. In this assay, keratinocytes are cultured in a 96-well cluster in the presence of different retinoid concentrations. The expression of TGm is then quantified, without any extraction or purification step, using a TGm-specific monoclonal antibody and a peroxidase-conjugated secondary antibody. The dose-response curves obtained show this ELISA to be a sensitive and reproducible assay to determine the potency of retinoids.