Determination of DDT in model samples by using solid-phase extraction and indirect competitive enzyme immunoassay

Two-step indirect competitive enzyme immunoassay of DDT has been developed. The limit of detection and limit of quantification were 0.3 nmol l^-1 and 4.2 nmol l^-1, respectively. To shorten the analysis time, the one-step enzyme immunoassay has been developed by replacing anti-DDT antibody with the complex anti-DDT antibody-HRP. The anti-DDT antibody-HRP conjugate was prepared by periodate method. A concentration of the preparation was 1.9 mg·ml^-1 and molar ratio Px/IgG was 1.87. The detection limit of the one-step ELISA reached 0.3 nmol l^-1, but the sensitivity of assay was very poor. The two-step enzyme immunoassay of DDT has been adapted for the chemiluminescent detection of the complex antigen-antibody. The detectability of chemiluminescent ELISA was comparable with that of chromogenic ELISA. A limited number of the artificially contamined samples have been tested. All samples were prepared by solid-phase extraction by using the commercial Agilent Zorbax SPE C₁₈ columns. A background was observed in most tested samples. The recoveries obtained from the analysis of DDT spikes reached values between 89.0-161.0%, and 81.5-111.6% at standard addition 50 μg·kg^-1, and 200 μg·kg^-1, respectively. The overestimated amounts of DDT were determined in spinach and nectarines at standard addition 50 μg·kg^-1.     

Determination of DDT in model samples by using solid-phase extraction and indirect competitive enzyme immunoassay

Two-step indirect competitive enzyme immunoassay of DDT has been developed. The limit of detection and limit of quantification were 0.3 nmol l^?1 and 4.2 nmol l^?1, respectively. To shorten the analysis time, the one-step enzyme immunoassay has been developed by replacing anti-DDT antibody with the complex anti-DDT antibody-HRP. The anti-DDT antibody-HRP conjugate was prepared by periodate method. A concentration of the preparation was 1.9?mg·ml^?1 and molar ratio Px/IgG was 1.87. The detection limit of the one-step ELISA reached 0.3?nmol l^?1, but the sensitivity of assay was very poor. The two-step enzyme immunoassay of DDT has been adapted for the chemiluminescent detection of the complex antigen-antibody. The detectability of chemiluminescent ELISA was comparable with that of chromogenic ELISA. A limited number of the artificially contamined samples have been tested. All samples were prepared by solid-phase extraction by using the commercial Agilent Zorbax SPE C₁₈ columns. A background was observed in most tested samples. The recoveries obtained from the analysis of DDT spikes reached values between 89.0–161.0%, and 81.5–111.6% at standard addition 50 μg·kg^?1, and 200 μg·kg^?1, respectively. The overestimated amounts of DDT were determined in spinach and nectarines at standard addition 50 μg·kg^?1.     

Evaluation of a competitive enzyme immunoassay for detection of Coxiella burnetii antibody in animal sera.

A competitive enzyme immunoassay (CEIA) was established and compared with other serological techniques for detecting Coxiella burnetii antibody in camels, goats, and sheep. This technique was evaluated because a conjugated anti-camel immunoglobulin was not available to serve as a direct signal for the demonstration of antigen-antibody reaction. A C. burnetii antibody-positive human serum and a peroxidase-conjugated anti-human immunoglobulin G were used as an indicator system competing against antibody in animal serum or as an indicator of the absence of antibody. Sera were considered antibody positive when the A414 of the test sera plus the competing positive antibody was less than or equal to 50% of the A414 of the negative-control serum plus the competing antibody. Antibody to C. burnetii was repeatedly demonstrated in 66% of camel serum samples (n = 200) by the CEIA. Among 48 camel serum samples, 71% were positive for antibody by CEIA versus 65% by EIA using peroxidase-labeled protein A. The CEIA detected C. burnetii antibody in 63% of sheep serum samples (n = 40) and in 50% of goat serum samples (n = 96), while the indirect fluorescent-antibody technique detected antibody in 38% of sheep and 34% of goat serum samples and the EIA detected antibody in 50% of sheep and 35% of goat serum samples. These data indicate that the CEIA is a reliable and sensitive technique for demonstrating C. burnetii antibody in camels, sheep, and goats.     

Determination of human rotavirus VP4 using serotype-specific cDNA probes

Summary The VP4 genetic groups of 151 field strains of human rotaviruses obtained from infants and young children with diarrhea from four locations in Malaysia were analyzed. The strains were adapted to growth in tissue culture and studied further by molecular hybridization of northern blotted RNA to PCR-generated cDNA probes representing amino acids 84–180 of the KU strain VP4, 83–181 of the DS-1 strain VP4, and 83–180 of either the 1076 or K8 strain VP4, representing VP4 genetic groups 1–4 (P1A, P1B, P2, and P3), respectively. The majority (79% of the field strains hybridized with the KU VP4 genetic group 1 probe and were associated with G1, G3, G4, untypable, or mixed G serotypes. VP4 genetic group 1 (P1A) strains were the most common in all locations in Malaysia between 1978–1988. Three strains which exhibited G3 and subgroup I specificity hybridized with the K8 VP4 genetic group 4 probe. These three VP4 genetic group 4 (P3) strains were detected in two different years and locations, extending the initial detection of this VP4 genetic group (the K8 strain) in Japan to a larger geographical area of Asia.     

Use of serotype-specific monoclonal antibodies to study the epidemiology of rotavirus infection

The development of an enzyme immunoassay (EIA) capable of serotyping human rotavirus (HRV) in faecal extracts has enabled us to retrospectively study the epidemiology of rotavirus infection in Melbourne. Of 552 stored specimens obtained from individuals with rotavirus-associated gastroenteritis between 1975 and 1986, the serotype could be determined in 62%. Infection was most prevalent in two groups, neonates and children aged between 12 and 24 months. In these groups infection was due to different serotypes, type 1 in older children and an untypable virus in infants. Serotype 1 strains were detected in greater numbers than the other serotypes and circulated in each year of the study. Serotype 2 rotaviruses were associated with a large epidemic in 1978, but have been detected only rarely since.     

Epitope specificity of monoclonal antibodies against Newcastle disease virus: competitive fluorogenic enzyme immunoassay.

A test to determine the epitope specificity of monoclonal antibodies (MCA) was developed for hybridoma clones producing antibodies against Newcastle disease virus (NDV). The virus was first immobilized on nitrocellulose membranes of Millititer HA plates. Dilutions of MCA were then added, singly, or simultaneously in pairs, and bound antibody was quantitated with alkaline phosphatase-labelled detector antibody and a fluorogenic substrate, 4-methylumbelliferyl phosphate (4-MUP). Fluorescence count was measured fluorometrically. Additivity indices were calculated and plotted against dilutions of paired MCA. Antibodies that recognized identical epitopes displayed non-additivity at saturating antibody dilutions, followed by partial additivity and by total additivity at low, non-saturating dilutions. In contrast, MCA that recognized distinct epitopes exhibited total additivity throughout the curve. MCA that exhibited partial additivity were interpreted as competing for overlapping shared epitopes, or, distinct epitopes in close proximity, resulting in steric hinderance.     

Detection of antibodies to HLA1 antigens by enzyme immunoassay

A modification of the MAILA (monoclonal antibody specific immobilization of lymphocyte antigens) method has been developed for the detection of antibodies to class 1 histocompatibility antigens. Russian biotin-treated monoclonal antibodies IKO-53 (Medbiospektr, Moscow) were used. In a complex with monoclonal antibodies, lymphocyte HLA antigen was found to retain its antigenic properties when stored for a long time. High specificity and sensitivity of the method were demonstrated. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 9, pp. 315–317, September, 1995 Presented by V. I. Shumakov, Member of the Russian Academy of Medical Sciences     

Evaluation of new commercial enzyme immunoassay for rotavirus detection.

We evaluated a new commercial enzyme immunoassay (EIA) for rotavirus (Rotavirus EIA; International Diagnostic Laboratories, Chesterfield, Mo.). A total of 161 consecutive stool samples (including 18 from infants less than 30 days old) submitted to the diagnostic laboratory at Children's Hospital, Washington University Medical Center, St. Louis, Mo., for rotavirus detection were tested by Rotavirus EIA and by Rotazyme II (Abbott Laboratories, North Chicago, III.) according to the instructions of the manufacturer. In addition, 16 samples from infants less than 30 days old without diarrhea were tested by both assays. Samples showing discrepant results after repeat testing were examined by electron microscopy. Nine samples yielding discrepant results were also tested by using a reference EIA directly on the specimen and on culture supernatants from two passages in MA 104 cells. Rotavirus EIA and Rotazyme II yielded concordant results for 85% of the samples. All of the 26 discrepant samples tested negative by Rotavirus EIA and positive (15 samples) or equivocal (11 samples) by Rotazyme II. These samples included 11 from symptomatic infants more than 30 days old, 2 from symptomatic infants less than 30 days old (neonates), and 2 from neonates without diarrhea. Rotavirus was not detected in any of the 24 that were examined by electron microscopy or in any of the 9 that were tested by the reference EIA. The sensitivity, specificity, positive predictive value, and negative predictive value were 100% for Rotazyme EIA and 100, 90, 70, and 100%, respectively, for Rotazyme II. Rotavirus EIA was comparable to Rotazyme II in ease of performance. We conclude that Rotavirus EIA is equally sensitive and more specific than Rotazyme II for detecting rotavirus. Rotavirus EIA is a practical and accurate rotavirus assay for use in clinical laboratories.     

Determination of mycobacterial antigens in sputum by enzyme immunoassay.

An enzyme-linked immunosorbent assay (ELISA) was examined for its usefulness in detecting mycobacterial antigens in sputum. A double-antibody sandwich procedure was set up by using a commercially available hyperimmune serum directed against Mycobacterium bovis, BCG. The ELISA was able to detect 10 ng of protein per ml of BCG sonic extract. The system also clearly distinguished Mycobacterium tuberculosis organisms from Mycobacterium avium and Mycobacterium kansasii organisms. A total of 68 unknown sputum specimens submitted to the clinical laboratories for examination for tuberculosis were tested by ELISA. Of the 20 specimens that were smear positive and culture positive, 12 (60%) were positive by ELISA; 6 of the 11 (55%) smear-positive culture-negative samples were positive by ELISA; 1 of 2 (50%) of the smear-negative culture-positive samples was positive by ELISA; and only 3 of 35 (9%) of the smear-negative culture-negative samples were positive by ELISA. This approach offers promise as an aid in the presumptive differentiation of nontuberculous mycobacteria from the M. tuberculosis complex.     

Peroxidase-labelled monoclonal antibodies for use in enzyme immunoassay

Desirable characteristics of enzyme-antibody conjugates for use in enzyme immunoassay are labelling uniformity, permanent availability and stability. The use of monoclonal antibodies (McABS) for preparation of enzyme conjugates, in place of polyclonal antibodies, ensures labelling uniformity and permanent availability. The problem of stability still exists. Monoclonal antibody-horseradish peroxidase (McAB-HRPO) conjugates produced in our laboratory showed variable stability. After extensive testing of McAB-HRPO conjugates it became obvious that sodium borohydride, used as a reducing agent, did not result in the production of stable conjugates without enzyme pretreatment with fluorodinitrobenzene (FDNB). Ascorbic acid or ethanolamine used as the reducing agent, resulted in McAB-HRPO conjugates which were stable for periods of ten months or more when stored filter sterilized at 4 degrees C.     

IgG and IgM antibodies to rubella quantitated by enzyme immunoassay

A solid-phase enzyme immunoassay for rubella antibodies (of IgG, H & L type, IgM-type) is described that requires assay at usually one dilution of serum. Results are reportable in milligrams IgG (or IgM)--equivalents per liter serum and in approximate hemagglutination inhibition (HAI) titers. The method uses purified rubella virus immobilized by a special process in excess, a 16 to 18 h first incubation at 20 degrees C, and a glucose-oxidase labeled second antibody to obtain 97% agreement with HAI. IgM-class antibodies are assayed after a preliminary separation from IgG by ion exchange to obtain results equivalent to those obtained after sucrose gradient sedimentation.     

Discrepant rotavirus results in two laboratories using the same enzyme immunoassay

Rotavirus testing was performed on 66 stool samples in two separate laboratories using the same enzyme immunoassay. Sixteen of 46 of the positive results reported by the reference laboratory were negative in the hospital laboratory. In addition, RNA gel electrophoresis had negative results in the 16 discrepant samples. This indicates the need to repeat or confirm positive results.     

Detection of specific antibodies toToxoplasma gondii by a competitive enzyme immunoassay using a monoclonal antibody against the P30 antigen

An inhibition EIA using a monoclonal antibody against the major P30Toxoplasma gondii surface protein was designed for detection of specific antibodies in human sera. The assay was based on the inhibition of binding of peroxidase labelled monoclonal antibody toToxoplasma gondii crude antigen coated plates by the corresponding antibodies present in human sera. This rapid and simple assay was compared to indirect immunofluorescence, direct agglutination and an immunosorbent agglutination assay using 435 human sera. The specificity and sensitivity were 100 % and 97 % respectively. This test was found to be as sensitive as the dye test.     

Solid-phase enzyme immunoassay for Chlamydial antibodies.

An enzyme immunoassay (EIA) for chlamydial immunoglobulin G antibodies was developed by using microtiter wells coated with partially purified reticulate bodies of Chlamydia trachomatis serotype L2, grown in McCoy cells, and uninfected McCoy cells as a control. Duplicate testing of a single serum dilution, 1:500, was found to be sufficient. A good correlation between positive reactions was observed in a comparative study of 421 patient sera with the EIA and an inclusion immunofluorescence test. A good correlation between positive reactions was also observed in a comparative study of 140 patient sera with EIA and microimmunofluorescence tests in which chlamydial elementary or reticulate bodies were used as antigens. Sera of 77 healthy control individuals with low titers in inclusion immunofluorescence or complement fixation tests gave negative results in the EIA. Immunoblotting experiments showed that the major antigenic component in the EIA antigen was a protein with an Mr of 39,000.     

Investigation of sera reactive to hepatitis C virus by second-generation enzyme immunoassay.

Hepatitis C virus antibodies were detected by a second-generation enzyme immunoassay and investigated with a second-generation recombinant immunoblot assay. Most sera with optical density values of > or = 2.0, particularly those with optical densities of > or = 2.0 after a further 10-fold dilution, were positive in the second-generation recombinant immunoblot assay. Repeat testing of hemodialysis patients revealed excellent reproducibility and increased sensitivity of the second-generation enzyme immunoassay.     

Magnetic enzyme immunoassay of anti-grass pollen specific-IgE in human sera.

This paper reports a magnetic solid-phase sandwich enzyme immunoassay for specific IgE antibodies in human sera. Crude extracts of grass pollen bound to magnetic polyacrylamide agarose beads were mixed with human serum to be tested. After washing in a magnetic rack, the beads were incubated with the glucose-oxidase-labelled sheep anti-IgE. The enzyme activity associated with the beads was measured by colorimetric assay. Results obtained from sixty-one human sera, as measured by the magnetic enzyme immunoassay, gave a linear correlation coefficient of 0.98 with the values as determined by radio-immunoassay. This procedure, which allows the grass pollen specific IgE in human sera, to be measured, is easy to perform, reproducible and may avoid the use of radioactive compounds.     

Simultaneous detection of platelet and lymphocyte antibodies by slide enzyme immunoassay

The feasibility of using an enzyme immunoassay (EIA) to detect simultaneously the antibodies bound to platelets and lymphocytes on glass slides was investigated. A peroxidase-antiperoxidase (PAP) immune complex method was used in which lymphocytes and platelets were attached to glass slides by poly-L-lysine and the preparations were stored at -20 degrees C for subsequent assays. Test sera were incubated with the cells. Reagents linking the platelet and/or leukocyte antibody to the PAP enzyme marker were added, followed by a staining step to localize the antigen-antibody complex. Any brown staining of the perimeter of the cells and in the cytoplasm was considered indicative of a positive reaction. A total of 146 sera were assayed; 36 were from Roswell Park Memorial Institute (RPMI) plasmapheresis donors, 75 were from RPMI oncology patients, and 35 were from other laboratories. The PAP method agreed with complement lysis inhibition assay (CLIA) by 97 percent in detecting antibodies capable of reacting with platelets while concordance with complement dependent lymphocytotoxicity (CDL) was 81 percent. With further investigation, this method could be adapted as a screening procedure in the clinical laboratory. It is easy, inexpensive, and could be performed in a few hours.     

Detection of antimitochondrial antibodies: characterization by enzyme immunoassay and immunoblotting

Mitochondrial antigens were purified from rat liver and characterized by immunoblotting. Sera from 19 well defined patients with primary biliary cirrhosis (PBC) reacted with two mitochondrial polypeptides of 68 Kd and 45 Kd. Antibodies to these antigens were not detected in any of the sera of patients with cirrhosis of the liver, chronic active hepatitis or other autoimmune diseases. The two polypeptides were derived from the soluble fraction of the mitochondrial matrix. An enzyme-linked immunosorbent assay (ELISA) employing these rat liver mitochondrial antigens is described. Positive results were obtained with all except one PBC sera (95%), five out of 47 patients with cirrhosis (11%), one out of 20 patients with chronic active hepatitis (5%), and two out of 19 patients with various autoimmune disorders (11%). The titers detected in PBC were markedly higher than those recorded in patients with other liver and autoimmune diseases. Strong correlation was found between immunoblotting and the ELISA in determining antimitochondrial antibodies. The ELISA presented is easily performed and seems to be a useful diagnostic tool for antimitochondrial antibodies in patients with PBC.     

Ability of TESTPACK ROTAVIRUS enzyme immunoassay to diagnose rotavirus gastroenteritis.

TESTPACK ROTAVIRUS, a simple 10-min enzyme immunoassay, was compared with electron microscopy and Pathfinder enzyme immunoassay on feces from 172 patients of various ages with gastroenteritis. The percent sensitivities and specificities before blocking with antiserum were as follows: TESTPACK, 100% sensitivity and 99% specificity; Pathfinder, 95% sensitivity and 98% specificity. After blocking, the sensitivity and specificity, respectively, were 100% and 100% for TESTPACK and 95% and 99% for Pathfinder. TESTPACK ROTAVIRUS was more sensitive, but not significantly, than Pathfinder (P greater than 0.1) and the direct electron microscopy technique (P greater than 0.1).     

化学发光法检测受血者梅毒螺旋体特异性抗体的方法学评价

目的 通过与梅毒螺旋体微量血凝试验(TPHA)和蛋白印迹试验法(WB)比较评估梅毒血清学筛查法化学发光法(CLIA)的性能.方法 回顾性研究18 494例受血者血清标本CLIA和TPHA检测梅毒螺旋体特异性抗体结果,对CLIA和(或)TPHA结果阳性177例标本用WB检测确认.同时用CLIA、TPHA和WB检测了81例各期梅毒患者血清、55例有潜在干扰的患者血清和250例阴性对照血清梅毒抗体.结果 以WB检测结果为金标准,CLIA方法的灵敏度为98.4％,显著高于TPHA(94.4％)(x2=5.76,P＜0.05);CLIA方法特异性为100％,高于TPHA方法特异性(99.7％),但差异无统计学意义(x2=1.0,P＞0.05).结论 CLIA法具有高度敏感性和特异性,适合于临床实验室进行梅毒筛查.