Immunoglobulin A-specific capture enzyme-linked immunosorbent assay for diagnosis of dengue fever

Dengue fever (DF) is usually diagnosed by testing for dengue virus immunoglobulin M (IgM) by a capture enzyme-linked immunosorbent assay (ELISA) (MAC-ELISA). However, IgM can last for months, and its presence might reflect a previous infection. We have tested the use of anti-dengue virus IgA capture ELISA (AAC-ELISA) for the diagnosis of DF by comparing the results of MAC-ELISAs and AAC-ELISAs for 178 serum samples taken from patients with confirmed cases of DF. IgM appears more rapidly (mean delay of positivity, 3.8 days after the onset of DF) than IgA (4.6 days) but lasts longer; the peak IgA titer is obtained on day 8. The specificity and the positive predictive value of AAC-ELISA are 100%; its sensitivity and negative predictive value (NPV) are also 100% between days 6 and 25 after the onset of DF, but they decrease drastically when data for tests conducted with specimens from the first days of infection are included, because the IgA titers, like the IgM titers, have not yet risen. AAC-ELISA is a simple method that can be performed together with MAC-ELISA and that can help in interpreting DF serology.     

Evaluation of enzyme-linked immunosorbent assay for antinuclear antibodies

Antinuclear antibodies (ANAs) are clinically important indicators of collagen diseases. As corresponding antigens for ANAs vary considerably, patients with collagen diseases usually demonstrate several ANAs coincidentally, making difficult to detect the full spectrum of ANAs in each patient's serum. To design an efficient system for measuring ANAs, an enzyme-linked immunosorbent assay (ELISA) which adsorbs eight kinds of recombinant or purified antigens in each well of a multiwell plate was used and results were compared to those obtained with conventional assays by the fluorescent antinuclear antibodies (FANA), and double immunodiffusion (DID) methods. The positivity rates of 106 sera from patients with collagen diseases and 286 sera from healthy subjects were 92.5% and 5.5%, respectively. Sixty-one of 65 positive sera (93.8%) in the corresponding ANAs positive sera by DID or other conventional assay methods were positive by ELISA. Anti-SSA/Ro antibody could be detected with higher sensitivity by this assay method than with the FANA and DID method, but the sensitivities for anti-Scl-70 antibody and anti-centromere antibody were lower. Application of this ELISA method for measuring ANAs along with the FANA test may be beneficial for diagnosis of collagen diseases.     

Comparison between enzyme-linked immunosorbent assay and cytotoxic cross-match procedures for detecting IgG anti-donor antibodies

BACKGROUND: Disadvantages inherent to complement-dependent cytotoxicity cross-match (CDC XM) methods are the requirements for complement and viable target cells, detection of antibodies (Abs) against non-HLA antigens, and subjective scoring. Cross-Stat (SangStat Medical Corp., Menlo Park, CA), a recently developed enzyme-linked immunosorbent assay XM procedure for the detection of IgG anti-donor HLA Abs, is theoretically devoid of these flaws. METHODS: We compared results of Cross-Stat and our standard anti-human globulin (AHG)-enhanced CDC XM procedure on 524 sera from 230 transplant candidates, which were evaluated against 51 cadaveric donors. RESULTS: There was a significant correlation between AHG-CDC IgG XM and Cross-Stat results (P<0.001). For false negative sera, repeat AHG-CDC IgG XMs were still positive after platelet absorption, indicating that the Abs present were either non-HLA Abs or anti-HLA class II. Flow cytometry testing of false positive sera usually (42/62) substantiated Cross-Stat results, indicating that the discrepancy with AHG-CDC IgG XM is caused by greater sensitivity of Cross-Stat. Relative to the AHG-CDC XM, the sensitivity of Cross-Stat was 100%, the specificity was 93%, the positive predictive value was 73%, and the negative predictive value was 100%. A technical shortcoming of the Cross-Stat assay is that the frequency of indeterminate samples in the assays was 15%. Among 49 Cross-Stat negative vs. 13 Cross-Stat positive primary cadaveric renal allograft recipients (all AHG-CDC IgG-XM negative), there was no statistical difference in overall graft survival. CONCLUSION: Given the important theoretical advantages of enzyme-linked immunosorbent assay-based XM methods over the CDC XM, however, further testing of the clinical relevance of the Cross-Stat is warranted.     

Moniliformin from Fusarium fujikuroi culture material and deoxynivalenol from naturally contaminated wheat incorporated into diets of broiler chicks

The effects of feeding diets containing 100 mg moniliformin (M)/kg of feed from culture material and 16 mg deoxynivalenol (DON)/kg of feed from naturally contaminated wheat were evaluated in growing broiler chicks from 1 day to 21 days of age. Body weight (BW), body-weight gain, and feed consumption were decreased by feeding M and M plus DON diets. Relative heart weight was increased by the M diet, whereas relative weights of proventriculus, gizzard, and heart were increased by the M plus DON diet. The M diet increased alanine transferase and aspartate transaminase activities and creatinine concentration and decreased mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration (MCHC). The M and DON diet decreased glucose, hemoglobin, and MCHC. Histopathological lesions from the M diet were limited to the kidney and consisted of extensive renal tubular epithelial degeneration plus luminal mineralization. A moderation of the severity of lesions was seen in the tissues of the M plus DON-fed chicks, consisting of generally mild tubular epithelial degeneration. None of the parameters measured were affected by the DON diet. Results indicate additive or less-than-additive toxicity for most parameters when chicks were fed diets containing 100 mg M plus 16 mg DON/kg of feed. Although the concentration of M in this study was high compared with that reported for feedstuffs, additional information on the occurrence and toxicity of M will need to be collected in order to assess the importance of M to the poultry industry.     

Peptide-based OspC enzyme-linked immunosorbent assay for serodiagnosis of Lyme borreliosis

Sera from 210 patients with Lyme borreliosis (LB) were studied by an enzyme-linked immunosorbent assay (ELISA) based on a synthetic peptide (pepC10) comprising the C-terminal 10-amino-acid residues of OspC of Borrelia burgdorferi. We found that 36.3 and 45.0% of the serum samples from patients with erythema migrans (EM) and neuroborreliosis (NB), respectively, displayed immunoglobulin M (IgM) anti-pepC10 reactivities, while these samples rarely (相似文献   

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.     

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.     

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.     

Immunoglobulin M-capture biotin-streptavidin enzyme-linked immunosorbent assay for detection of antibodies to dengue viruses

Defensins and other antimicrobial peptides act in the innate host defense of epithelial surfaces. Human beta defensin 1 (hBD-1) has recently been shown to be expressed in airway epithelial cells and so has been implicated as a primary component of antibacterial activity in human lung. We attempted to purify these molecules from bronchoalveolar lavage fluid (BALF). Extraction of BALF on SepPak C-18 cartridges, followed by continuous acid-urea polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography yielded one fraction with antibacterial activity associated with factors of < 6.5 kD. N-terminal amino acid sequencing identified these peptides as human neutrophil defensins (HD) 1 through 3. No hBD-1 was detected. Together with lysozyme, it appears that HD-1 through -3 are the most prominent antimicrobial factors in BALF. The contribution of epithelial defensins such as hBD-1 to antibacterial defense of human airway in vivo remains to be elucidated.     

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.     

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.     

Diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA): optimal conditions for quantitation of antibodies

In the diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) the quantitation of antibodies is based on their ability to form a diffusion gradient over an antigen-coated polystyrene surface. The antigen-antibody reaction is then visualized by an enzyme-conjugated anti-immunoglobulin. The enzyme-substrate reaction is finally performed by pouring a substrate-containing gel over the polystyrene surface. In this study with bovine serum albumin as antigen and a corresponding rabbit antiserum, the diffusion time of antiserum was shown to be the most critical variable of the method, while the antigen concentration used for coating, the conjugate binding time and the enzyme-substrate reaction time had a minor influence on the quantitation of antibodies. High antibody levels were measured with greater accuracy than low levels, but the standard deviation was below 10%. It was also shown that different sera containing antibodies to Salmonella typhi O LPS, Klebsiella pneumoniae K1 and O4 LPS, Escherichia coli O2 LPS, Yersinia enterocolitica Y3 LPS, cardiolipin and pneumococcus could be quantitated with the same accuracy.     

Comparison of two enzyme-linked immunosorbent assays and one rapid immunoblot assay for detection of herpes simplex virus type 2-specific antibodies in serum

The sensitivities and specificities of three immunoassays for the detection of herpes simplex virus type 2 (HSV-2)-specific immunoglobulin G antibodies in serum, including the one-strip rapid immunoblot assay (RIBA; Chiron Corporation) and two indirect enzyme immunosorbent assays (EIA; Gull Laboratories and Centocor), were compared by testing a panel of 1,250 serum samples from individuals attending an outpatient clinic for sexually transmitted diseases. A qualitative agreement among the three assays was observed with 1,080 serum samples (86.4%); 291 of the serum samples (23.3%) were positive, 789 samples (63.1%) were negative, and 170 serum samples (13.6%) gave a discordant result. Results were considered conclusive when a concordant result was obtained with two of three assays. The sensitivities and specificities of the RIBA, the Gull EIA, and the Centocor EIA proved to be 99.2, 99.7, and 89.9% and 97.1, 96.7, and 99.3%, respectively. These results indicate that the Chiron RIBA and the Gull EIA are especially useful and reliable for the detection of HSV-2-specific antibodies in serum.     

Application of double sandwich enzyme-linked immunosorbent assay for the diagnosis of foot-and-mouth disease in Saudi Arabia

A case of Hemangiopericytoma at the thigh is reported. The Hemangiopericytoma is a rare tumour made with pericyties. This neoplasia is usually benign and it is located in the soft tissues. Tumour is profusely irrigated. The most common locations of the Hemangiopericytoma are the lower limbs and the abdominal cavity. The Hemangiopericytoma is difficult to be recognized by histological criteria. The high number of relapses and metastasis involve to an extent surgical ablation of the tumour and its borders. Present literature is reviewed and the different diagnostic and therapeutic options are discussed.     

An enzyme-linked immunosorbent assay (ELISA) for monitoring guineapigs and rabbits for Bordetella bronchiseptica antibodies

An enzyme-linked immunosorbent assay (ELISA) for monitoring antibodies specific to Bordetella bronchiseptica in guineapigs and rabbits was developed. In conventional and SPF colonies of guineapigs and rabbits, the ELISA was equally successful in detecting infected animals when compared to selective cultivation from the respiratory tract. The ELISA showed a sensitivity of 100% and a specificity of 90% in guineapigs. In rabbits the sensitivity and specificity of the ELISA were 97% and 91% respectively. In rabbit sera from infected colonies, ELISA activity showed a statistically significant correlation with titres obtained in the micro-agglutination test. Since serologically unrelated strains of the bacterium exist, the monitoring of animals for B. bronchiseptica infection by ELISA should be performed with various antigens.     

Development of an enzyme-linked immunosorbent assay for measurement of serum-associated ALX40-4C

ALX40-4C is an antiretrovirus agent that has been found to have some inhibitory properties against human immunodeficiency virus (HIV) replication in vitro. The compound was designed as a competitor of the HIV Tat protein for TAR binding. In addition to its anti-HIV properties, it has demonstrated the ability to inhibit in vitro replication of herpes simplex virus types 1 and 2 as well as human cytomegalovirus. Subsequently, in vivo pharmacokinetic evaluation of ALX40-4C necessitated the establishment of a detection system for the measurement of ALX40-4C in subject serum. For this purpose, an indirect-competition enzyme-linked immunosorbent assay with generated rabbit anti-ALX40-4C antiserum was developed. The original assay took 12 h to complete and required many manipulations. Herein, we describe alterations to the system that resulted in the overall reduction in assay time and manipulation. We demonstrate that our alterations do not affect the specificity or sensitivity of the assay compared to that of the original system. ALX40-4C levels in spiked serum samples as well as drug levels from patient samples were used to validate the assay.     

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.     

Evaluation of enzyme-linked immunosorbent assay for the determination of polycyclic aromatic hydrocarbons in house dust and residential soil

Two commercially available enzyme-linked immunosorbent assays (ELISA) for total polycyclic aromatic hydrocarbon (PAH) and carcinogenic PAH (C-PAH) were evaluated. The testing procedures were refined for application to screening PAH and C-PAH in house dust and soil samples for human exposure studies. The overall method precision expressed as percent relative standard deviation (%RSD) of triplicate real world dust and soil samples was within +/- 29% (12-29%) for PAH ELISA and +/- 21% (5.9-21%) for C-PAH ELISA. Spike recoveries from real world dust/soil samples were 114 +/- 30% for phenanthrene from PAH ELISA and 120 +/- 8.2% for benzo[a]pyrene from C-PAH ELISA. The overall method accuracy for PAH and C-PAH assays cannot be assessed for multiple PAH components in dust/soil samples (which represent real-world samples), because of the assays' cross reactivities with other PAH components. Over 100 dust/soil samples from 13 North Carolina homes and 22 Arizona homes were analyzed by PAH and C-PAH assays, as well as by the conventional gas chromatography/mass spectrometry (GC/MS) method. Statistical analysis showed that dust/soil PAH data from ELISA and GC/MS methods are significantly different. In general PAH ELISA responses were higher than PAH GC/MS responses. The regression analysis showed that the linear relationship between ELISA and GC/MS measurements is not strong in the combined data. The relationship became stronger for the data from the same type of dust/soil samples. The screening performance of ELISA was evaluated based on the frequency distribution of ELISA and GC/MS data. The results indicated that the ELISA PAH and C-PAH assays cannot be used as a quantitative analytical tool for determining PAH in real-world dust/soil samples. However, the ELISA is an effective screening tool for ranking PAH concentrations in similar types of real world dust/soil samples.     

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.     

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.