Detection of flunixin in greyhound urine by a kinetic enzyme-linked immunosorbent assay

Anxiety levels in a sample of 65 long-term cancer survivors were assessed in a study of the effects of a planned discharge from an oncology clinic. Thirty-one percent of patients scored > or = 8, and 12% > or = 11 on the anxiety subscale of the Hospital Anxiety and Depression Scale (HADS), indicating that anxiety rates in patients in long-standing remission do not greatly differ from patients with active disease. Despite the provision of continued support and guaranteed fast-access return to the clinic if necessary, 28% of patients refused to be discharged. Fear that recurrence would not be detected was the reason most frequently cited. Seventy-five percent of these patients were HADS anxiety cases. A second assessment 4-5 months later of the 41 patients who were discharged showed a slight, but non-significant increase in anxiety rates suggesting that anxiety in cancer survivors may be persistent and not related to clinic attendance.     

Detection of Vibrio anguillarum by a sandwich enzyme-linked immunosorbent assay performed with monoclonal antibodies

Monoclonal antibodies (Mabs) against V. anguillarum were produced and characterized by Western blotting analysis, competitive binding assays and cross-reactivity tests. Their ability to detect V. anguillarum in a liquid culture was tested in a sandwich enzyme-linked immunosorbent assay (ELISA) performed with different combinations of these Mabs used as capture or tracer antibodies. One combination was selected as the most suitable for diagnostic applications, showing the highest sensitivity and specificity.     

Application of enzyme-linked immunosorbent assay for epidemiological studies of diseases of livestock in the tropics of Mexico

This study was conducted at the Centre for Research, Teaching and Extension in Tropical Livestock (Centro de Investigación, Ense?anza y Extensión en Ganadería Tropical) of the Faculty of Veterinary Medicine of the National Autonomous University of Mexico. During the latter part of 1986 and throughout 1988 and 1989, the herd of Holstein x zebu cattle at the University was tested for IgG antibodies to twenty-one viral, bacterial, rickettsial and parasitic agents. Antigens prepared from twenty infectious disease agents were used as the solid phase in an enzyme-linked immunosorbent assay, and the agar gel immunodiffusion procedure was used to test for antibodies against bovine leukaemia virus. The prevalence of IgG antibodies was high (> 50%) for bluetongue virus, Anaplasma marginale and Mycoplasma bovis. Antibodies to Brucella abortus were absent and antibodies against bovine virus diarrhoea virus and infectious bovine rhinotracheitis virus showed a very low prevalence (< 5%). Antibodies to fifteen other antigens showed intermediate prevalence (15-46%). Antibodies to Campylobacter fetus, A. marginale, bluetongue virus, bovine leukaemia virus and Haemophilus somnus displayed seasonal variations. Levels of antibody to bovine leukaemia virus, M. bovis and Listeria monocytogenes exhibited increasing secular trends while antibodies to bovine virus diarrhoea virus and C. fetus showed declining trends. Prevalence of antibodies increased with the age of animals tested. No consistent difference in antibody prevalence was found between three genotypic groups examined.     

False-positive results in premature infants with the Platelia Aspergillus sandwich enzyme-linked immunosorbent assay

The sensitivity of a sandwich enzyme-linked immunosorbent assay (ELISA) for detecting Aspergillus galactomannan was tested using 783 serum samples obtained from 247 patients (1-15 sera per patient) with severe underlying diseases (haematological malignancies or intensive care unit stay). We selected 146 serum samples from 50 patients for retesting. Serum samples were frozen after routine testing at -18 degrees C until retesting. All patients charts were checked for signs of Aspergillus infection, such as pneumonia or sinusitis. Adult patients were divided into four groups: proven (5), probable (6), suspected (8) or unlikely (25) Aspergillus infection. The results of Platelia ELISA were 100% in proven, 33% in probable and 50% in suspected Aspergillus infection. Patients with unlikely infection had no positive results with Platelia ELISA. Group 5 consists of six paediatric patients with prolonged ICU stay and a birth weight of 400-1320 g. In five out of six infants we found positive results with Platelia ELISA. All positive results in this group of patients are considered as false positive (83.3%).     

Determination of captopril in human blood by an enzyme-linked immunosorbent assay

An immunoassay for the quantitation of the angiotensin-converting enzyme inhibitor, captopril in human plasma is described. Antisera very specific for captopril were produced by immunization with captopril conjugated to bovine serum albumin or porcine thyroglobulin via the drug's thiol group. The antibodies were used to develop an enzyme-linked immunosorbent assay (ELISA) with a detection limit of 0.3 ng mL-1 and intra- and inter-assay coefficients of variation of 7 and 12%, respectively. Apart from stabilizing captopril by the addition of N-ethyl maleimide, the assay was used to detect the drug in human plasma without further extraction or purification. Our immunoassay provides a very sensitive and rapid (four hours) alternative for the study of captopril pharmacokinetics.     

Monoclonal antibody-based blocking enzyme-linked immunosorbent assay for specific detection and titration of peste-des-petits-ruminants virus antibody in caprine and ovine sera

A blocking enzyme-linked immunosorbent assay (B-ELISA), using two neutralizing monoclonal antibodies (MAbs), was established and compared with the virus neutralization test (VNT) for detecting specific peste-des-petits-ruminants virus (PPRV) antibody in caprine and ovine sera. This technique was developed because VNT, the only available specific serological test for PPRV and the cross-reactive rinderpest virus (RPV), is time-consuming and unaffordable for most laboratories in regions where both peste des petits ruminants and rinderpest occur. The test depends on the blocking of the binding of the MAb to a specific epitope in the presence of positive serum. Test conditions were optimized by using peste-des-petits-ruminants and rinderpest sera that were known to be VNT positive and negative. A blocking format, in which serum is preincubated with a solid-phase PPRV antigen and then incubated with the MAb, yielded levels of sensitivity and specificity superior to those of a competitive format, in which the two reagents are added simultaneously. A threshold value of 45% inhibition, representing the mean for a negative population (n = 277) plus 2.7 standard deviations, was adopted for routine screening. A total of 605 serum samples were screened by B-ELISA and the VNT. The sensitivity and specificity of B-ELISA relative to the VNT were 90.4 and 98.9%, respectively. Of 264 field serum samples tested, 11 (4.2%) could not be assayed by the VNT because of contamination or cytotoxicity; the overall agreement quotient between results of the two tests (n = 253) was 0.91. A high correlation (r>/=0.98) was observed between B-ELISA and the VNT for endpoint titration of sera (n=57). Because B-ELISA proved to be nearlyas sensitive and specific as the VNT while being simpler and more rapid, it would be an adequate substitute for the VNT for assessing herd immune status and for epidemiologic surveillance.     

Bridge-heterologous chemiluminescence enzyme-linked immunosorbent assay of estriol 3-sulfate in pregnancy plasma

A simple capillary zone electrophoresis method is developed for the quantitation of the beta-blocker atenolol and the complementary antihypertensive agents bendroflumethiazide, amiloride, and hydrochlorothiazide in human urine samples. The electrophoretic separation is performed using a 78-cm x 75-micron-i.d. (70-cm effective length) fused-silica capillary. A borate buffer (pH 9) is used as running electrolyte. The sample is hydrostatically introduced for 20 s, and the running voltage is 25 kV at the injector end of the capillary. The analysis of urine samples requires the optimization of solid-phase extraction methods, achieving recoveries > or = 61% for all the drugs and good separation from the urine matrix. The method is successfully applied to the determination of these compounds in pharmaceutical formulations and in urine samples collected after the intake of Neatenol Diu (100 mg atenolol-5 mg bendroflumethiazide) and Kalten (50 mg atenolol-25 mg hydrochlorothiazide-2.5 mg amiloride). The method is validated in terms of reproducibility, linearity, and accuracy.     

Reactivities of feline calicivirus field isolates with monoclonal antibodies detected by enzyme-linked immunosorbent assay

Reactivities of feline calicivirus (FCV) field isolates with monoclonal antibodies (MAbs) were examined by enzyme-linked immunosorbent assay (ELISA). The reactivities of the viruses in ELISA were different from our previous results using the neutralization tests (NT). Many isolates were positive in ELISA with MAbs which recognized neutralizing epitope 3B and/or 4. However, most were negative in NT in our previous study. After absorption of two FCV strains with host cells, the non-infectious virus fluid still reacted with MAb, which recognized epitope 3B and/or 4 in ELISA. These results indicated the possibility that neutralizing epitopes are expressed on non-infectious virus particles or exist as proteinaceous molecules in virus fluid.     

Identification and quantitation of sickle cell hemoglobin with an enzyme-linked immunosorbent assay (ELISA)

The experimental details of ELISA for the identification and quantitation of Hb S are presented; the assay is based upon the passive adsorption of Hb S top a solid phase (polystyrene tubes) and the addition of monospecific rabbit antibodies capable of recognizing the (beta 6 Glu leads to Val) substitution in Hb S. After the addition of alkaline phosphatase-conjugated goat antibody to rabbit IgG and substrate, the yellow color produced by hydrolysis of substrate is measured spectrophotometrically. For the identification and quantitation of Hb S in unknown samples, the hemolysate is added to the Hb S-coated tubes before the addition of antibody to Hb S, thus causing an inhibition of the antigen-antibody reaction as evidenced by an absence or reduction of color formation. With this procedure, there is no cross-reactivity with normal hemoglobins, and the immunoassay has a sensitivity in detecting 50 ng quantities of the abnormal hemoglobin in a 5 microgram hemolysate. The assay can be performed on multiple samples in 1 day and offers many advantages over other techniques currently used for the identification and quantitation of Hb S and other abnormal hemoglobins in the clinical laboratory.     

Development of an enzyme-linked immunosorbent assay for human metallothionein-1 in plasma and urine

The development of a sensitive enzyme-linked immunosorbent assay (ELISA) for human metallothionein-1 is reported. Metallothionein was purified from postmortem human liver and used to raise high-titer antibodies in rabbits. The assay was specific for human metallothionein-1 (MT-1), and there was no significant cross-reaction with human metallothionein-2. The detection limit (sensitivity) of the assay was 5 ng/ml, and the added MT-1 could be fully recovered from plasma and urine. The normal reference range for MT-1 was 32 +/- 16 ng/ml in plasma and 10 +/- 6 ng MT-1 per micromole of creatinine in random samples of urine. No significant differences were found between the values for males and females. The concentration of MT-1 was greatly increased between 24 and 48 hours after surgery, indicating that the protein behaves like an acute phase reactant in human subjects.     

Use of a multiwell assembly for chemotaxis and evaluation by enzyme-linked immunosorbent assay (ELISA)

A multiwell chamber assembly for chemotaxis tests was designed, which integrates the established microtiter system. A microtiter plate is covered with a plastic plate containing up to 96 holes of the diameter of the microtiter wells. Between the plates, a Nucleopore filter sheet (5 micron) and a silicon rubber gasket is placed. As a model system, human monocytes and lymphocyte-derived chemotactic factors were used. As it was observed that monocytes migrate through the membrane and settle on the bottom of the microtiter wells, an ELISA was adapted for quantitation of cells. After washing and incubation with a xenoantiserum against human monocytes, the bound antibody was quantitated using protein-A-conjugated alkaline phosphatase and p-nitrophenyl phosphate as detection system. The plates were read in a multichannel photometer. Cell numbers were determined directly from a calibration curve established before with varying numbers of monocytes. Current experience allows the following conclusions: The chemotaxis test in microtiter plates is simpler, faster and uses less material than conventional Boyden chambers. Evaluation by ELISA is much faster and more accurate than by microscopy.     

PCR enzyme-linked immunosorbent assay for diagnosis of leishmaniasis in human immunodeficiency virus-infected patients

A PCR enzyme-linked immunosorbent assay (ELISA) involving the use of bone marrow aspirates (BMA) and blood samples (BS) for the diagnosis of visceral leishmaniasis (VL) in human immunodeficiency virus-infected patients was developed with primers selected from the sequence of the small-subunit rRNA gene and compared with direct examination and in vitro cultivation. The PCR was optimized for routine diagnosis: processing of samples with lysis of erythrocytes without isolation of leukocytes, enzymatic prevention of contamination, internal control of the reaction, and ELISA testing in a microtitration plate hybridization. Of 79 samples (33 BMA and 46 BS) from 77 patients without VL, all the results were negative. Fifty-three samples (9 BMA and 44 BS) were obtained from 13 patients with VL: 6 samples drawn during anti-Leishmania treatment were negative whatever the technique used, and 47 samples (9 BMA and 38 BS) were positive with at least one technique. The sensitivities were 51% (24 of 47), 81% (38 of 47), and 98% (46 of 47) for direct examination, culture, and PCR, respectively. Thus, PCR ELISA is reliable for diagnosing VL in human immunodeficiency virus-infected patients, and blood sampling should be sufficient for the follow-up.     

Increased urinary excretion of glycerol: metabolic studies on a patient

The ratio of the zinc content of boar spermatozoa obtained from semen cooled to 4 degrees C for 30 min to that of the original room temperature control (20-26 degrees C) was constant at 2-28 +/- 0-16 in 22 samples of fresh whole semen from 12 animals. The same ratio occurred when zinc (0 to 0.6 mM in citrate buffer) was added to semen or washed spermatozoa. The increase is dependent only on the initial sperm zinc content at room temperature.     

Sensitive quantitation of endotoxin by enzyme-linked immunosorbent assay with monoclonal antibody against Limulus peptide C

Limulus peptide C, a 28-amino-acid fragment of coagulogen formed by the reaction of endotoxin with Limulus amebocyte lysate, was synthesized, and a monoclonal antibody against it was raised. A new microassay for endotoxin was developed, using this antibody in an enzyme-linked immunosorbent assay for generated peptide C-like immunoreactivity. A linear relationship between absorbance and endotoxin concentration was obtained. Control standard endotoxin in water could be detected to a level of 0.001 endotoxin unit per ml. The endotoxin levels in plasma samples from normal humans, rabbit, mice, and guinea pigs were generally found to be below the detection limit of 0.01 endotoxin unit per ml of plasma. The color and turbidity of specimens did not interfere with the assay. The consumption of Limulus amebocyte lysate in the assay was less than 5% of that in the gel-clot and chromogenic assays. With raw lysate, which was much more stable in solution than chloroform-treated lysate, the assay was still highly sensitive to endotoxin but was totally unresponsive to natural glucans. The monoclonal antibody cross-reacted with peptide C-like immunoreactivity generated in Tachypleus amebocyte lysate, which gave equal sensitivity in the endotoxin assay.     

Effect of sample storage on quantitation of lipoprotein(a) by an enzyme-linked immunosorbent assay

This study evaluated the effect of storage on the quantitation of lipoprotein (Lp)(a) in 25 serum samples. Aliquots of serum were stored for up to three years at either -20 degrees C or -70 degrees C and Lp(a) subsequently analyzed using an enzyme-linked immunosorbent assay kit. Concentrations of Lp(a) declined during storage, and the temperatures employed elicited significantly different (P < 0.05) values within 12 mon which further diverged during three years of storage. Compared to baseline values, significant decreases (P < 0.05) in Lp(a) levels were evident after six months of storage at -20 degrees C with apparent losses (geometric mean) reaching 36.9% (95% confidence interval: 30.9%, 42.9%) after three years. Similarly, significantly lower (P < 0.05) Lp(a) values were recorded after six months of storage at -70 degrees C and at three years the decrease (geometric mean) was 19.1% (95% confidence interval: 14.3%, 24.0%). The losses, after three years, in terms of the arithmetic mean were 53.5 and 26.2% at -20 and -70 degrees C, respectively. Phenotype analysis suggested that large isoforms are more susceptible to degradation than smaller moieties. This may be related to the observation that apparent losses are reduced in samples containing over 8 mg/dL Lp(a). Nevertheless, Lp(a) levels in stored samples retained a strong correlation with the baseline values. These results must be considered specific for the storage conditions and analytical procedures employed.     

Quantitative analysis of Gly m Bd 28K in soybean products by a sandwich enzyme-linked immunosorbent assay

Normative tables for various MMPI-2 code types, which may be used to enhance the interpretation of the Harris and Lingoes subscales, were developed. It was found that scores on the subscales covaried significantly as a function of code type. Gender and code type definition strategy were considered as moderators of the relationship between code type and subscale scores, but neither accounted for a large enough proportion of variance to justify consideration in the tables.     

The clinical usefulness of urinary pyridinoline and deoxypyridinoline as potential markers of bone metastasis in patients with prostate cancer

The aims of this study were to investigate the effects of paracentesis on uterine and intraovarian haemodynamics by colour Doppler ultrasound and the influences of repeated paracentesis on pregnancy outcome in severe ovarian hyperstimulation syndrome (OHSS). Forty-one abdominal paracenteses were performed on seven pregnant women with tense ascites and eight thoracocenteses were performed on three pregnant women with pleural effusion. Pulsatility index (PI) and maximum peak systolic velocity (MPSV) of uterine and intraovarian arteries were measured before and after each intervention. The mean PI of uterine arteries was decreased significantly after paracentesis, but not after thoracocentesis. Furthermore, uterine PI was decreased in 13 out of 14 (92.9%) paracenteses with <2500 ml ascites removed, compared with eight out of 13 (61.5%) with >2500 ml ascites removed. After paracentesis, there were no significant changes in the intraovarian PI and MPSV in either group. The 24-hour urine output increased significantly in the paracentesis group, but not in the thoracocentesis group. There were no significant changes in haematocrit and electrolytes as a result of paracentesis. However, gradual falls in serum total proteins and albumin concentrations were observed in all patients after repeated paracentesis, necessitating post-paracentesis albumin infusion. There was no significant difference in miscarriage rates between the two groups. We conclude that repeated abdominal paracentesis increases uterine perfusion and has no adverse effects on pregnancy outcome in severe OHSS. Extraction of 2500 ml of ascitic fluid did not impair uterine perfusion.     

Quantitation of Salmonella O-antibodies in human sera by enzyme-linked immunosorbent assay (ELISA)

The enzyme-linked immunosorbent assay (ELISA) has been applied to the detection of antibodies against Salmonella O-antigens in human sera. Phenol-water extracted lipopolysaccharides (LPS) from serogroups A (O-antigens 2, 12), B (4, 5, 12) and D (9, 12) were used as antigens. When compared to the tube agglutination method according to Widal employing sera from patients with verified or suspected typhoid--or paratyphoid fever and from healthy controls it was found that ELISA (i) correlated significantly with the Widal reaction, (ii) was up to 100-fold more sensitive, and (iii) showed a greater reproducibility.     

Use of a high molecular weight Entamoeba histolytica antigen fraction in enzyme-linked immunosorbent assay and thin layer immunoassay tests

A high molecular weight Entamoeba histolytica antigen fraction I was employed for the determination of anti-amoebic antibodies in ELISA and thin layer immunoassay (TIA) tests. A large number of human serum and immunized guinea pig serum samples were tested against this antigen. In comparison, ELISA was found more sensitive than the TIA technique. The potent antigen fraction I used in these tests was lyophilized at an optimum concentration of 1 mg/ml for obtaining uniform results.     

Advances in the diagnosis of some parasitic diseases by monoclonal antibody-based enzyme-linked immunosorbent assays

Advances in diagnostic assays for parasitic diseases include the use of monoclonal antibodies (MAbs) in antigen capture and competitive inhibition enzyme-linked immunosorbent assays (C-ELISA). Antigen capture ELISAs for Anaplasma marginale and Cryptosporidium parvum provide direct detection of these parasites during clinical disease, and the C-ELISA format has been adapted for detection of anti-Babesia equi, anti-A. marginale and anti-bluetongue virus antibodies. False-positive results may occur when antigen preparations in other ELISA formats are contaminated with Escherichia coli, erythrocyte or cell-culture antigens. The C-ELISA format overcomes problems of antigen purity, since the specificity of the C-ELISA depends solely on the MAb used. For this reason, the C-ELISA format is highly suited for use with recombinant antigens. Also, the use of recombinant protein in diagnostic assays precludes the need to infect animals for antigen production when the antigen cannot be produced in cell culture.