Monitoring cancer antigen 125 in serum of ovarian cancer patients after administration of 131I-labeled F(ab')2 fragments of OC125 antibody.
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We evaluated the effect of repeated administration of OC125 F(ab')2 fragments on cancer antigen (CA) 125 determination in 210 serum samples from 30 patients. We found falsely high CA 125 concentrations in 142 (68%) samples, using a homologous CA 125 enzyme immunoassay (EIA) with OC125 antibodies. The Truquant OV2 method, which involves two other murine antibodies, and the IMx CA 125 method, which uses sheep antibodies as capture antibodies, resulted in only slightly increased (false-positive) values in some samples with exceptionally high CA 125 EIA values. We measured falsely low CA 125 values in 37 (18%) samples with the Truquant OV2 method. Interferences could be eliminated by removal of serum IgG. Our results suggest that interferences are to some extent caused by anti-idiotypic IgG induced by OC125 administration. Assays involving nonmurine anti-CA 125 antibodies as capture antibodies seem to be most suited for CA 125 determination after OC125 treatment, but in every case an apparent increase of CA 125 after OC125 infusion should be validated.Keywords:
Cancer antigen
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Four EIA (enzyme immunoassay) test systems for the detection of salmonella O-antigens in various biological tissues were studied. To find the optimum test system two types of affinity purified antibodies were used to coat the microtitre plates: (i) monospecific antibodies isolated on immunoadsorbent bearing synthetic O-antigen factor 4 (O:4; salmonella serogroup B) as ligand, (ii) antibodies specific for lipopolysaccharide (LPS) B isolated from the IgG fraction of hyperimmune sera. The use of affinity purified antibodies led to an increase in the sensitivity of 'sandwich' EIA by an order of magnitude and to improved specificity. Competitive EIA was ten to twenty times less sensitive. It is demonstrated for the first time that salmonella O-antigens can be detected in the sera of animals within a day after challenge. For patients with salmonellosis, O-antigen could be detected after the fifth day of illness, but in the urine and faeces only (not in the blood serum), in 30%-70% of cases. This substantially improves the identification of salmonellosis from among other enteric infections. In competitive EIA, inhibition of standard antibodies by the sera under study was caused not by the presence of O-antigen but by antibodies homologous to the coating antigen. This resulted in "blocking' of the latter and led to false-positive results. The results obtained enable the optimum EIA technique to be selected to improve the serological diagnosis of salmonellosis with both synthetic salmonella O-antigens and LPS.
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A sensitive four-layer modification of an enzyme immunoassay for the detection of soluble antigens of Toxoplasma gondii is described. Microtiter plates were sensitized with rabbit anti-toxoplasma immunoglobulins (6 micrograms/ml) used as the primary antibodies; guinea pig anti-toxoplasma immunoglobulins (6 micrograms/ml) were used as the secondary trapping antibodies. Horseradish peroxidase-conjugated anti-guinea pig immunoglobulins were used as the indicator antibodies. The specificity of the antigen assay was confirmed by using guinea pig immunoglobulins from preimmunization sera. The sensitivity of the antigen assay was found to be at least 10 ng of antigen protein per ml. The suitability of the method for detecting antigens of T. gondii in different specimens was studied by experimental toxoplasma infection in mice. Antigenic components of T. gondii could be detected in different tissue specimens from infected animals from the first day after infection onwards. Toxoplasma antigen in serum and urine samples from infected mice reached detectable levels on day 2 after infection followed by a linear increase in antigen concentration in succeeding samples. This method might offer a valuable aid for a rapid etiological diagnosis also in human cases of acute toxoplasmosis.
Toxoplasmosis
Horseradish peroxidase
Microtiter plate
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Previously, immunological detection of a small hapten was only possible in competitive format, which needed a competitor antigen either labeled by a reporter or attached to a carrier protein. Recently, we proposed the open sandwich (OS) immunoassay, a simple immunoassay that can noncompetitively determine monovalent antigen concentration by measuring the antigen-dependent change in a heavy-chain variable region (VH)/light-chain variable region (VL) interaction of an antibody. However, there was a limitation in the assay that the antibody used should have a suitable property such that the VH/VL interaction would become fairly strong along with the addition of antigen. Here, we devised a phage-based "split-Fv system" to rapidly evaluate and select antibody variable region (Fv) fragments that are suitable to OS immunoassay. When three antibodies raised against endocrine disruptor bisphenol A were tested with this system, all were more or less suitable to OS-ELISA. Among them, the best Fv selected was used to construct fusion proteins of VH tethered to an alkaline phosphatase and a tagged VL that can be site-specifically biotinylated to perform direct OS-ELISA. The results showed that the OS-ELISA detects bisphenol A with higher sensitivity than the corresponding competitive assay, also implying that many antibodies to small haptens have suitable properties for OS-ELISA.
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Abstract The objective of this study was to analyze apparently discrepant results that arose during the use of an indirect immunofluorescence (IIF) assay using transfected HEp‐2 cells to detect anti‐SS‐A/Ro autoantibodies in human sera. Fourteen sera that had SS‐A/Ro antibodies as detected on this commercial substrate, but did not have antibodies to SS‐A/Ro as determined by double immunodiffusion (ID) or enzyme‐linked immunosorbent assay (ELISA), were studied by immunoprecipitation (IP) of radiolabeled cell extracts and full‐length recombinant SS‐A/Ro. A multi‐antigen strip immunoblotting (IB) assay containing both the 52‐ and 60‐kDa antigens was included in the analysis. We confirmed that 12 of 14 of the sera under study had antibodies to SS‐A/Ro protein antigens as determined by at least one other immunoassay. One serum had antibodies to hyRNA but no detectable reactivity with the 52‐ or 60‐kDa antigens. One serum remained negative in all assays for SS‐A/Ro autoantibodies. J. Clin. Lab. Anal. 16:103–108, 2002. © 2002 Wiley‐Liss, Inc.
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Immunofluorescence
Immunoprecipitation
Immunodiffusion
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Tritrichomonas foetus
Polyclonal antibodies
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A new solid-phase antigen immunoassay was developed for the detection of anti-zona pellucida-antibodies. The assay was validated in a double blind study with clinically defined sera. The new assay is easy to perform and large numbers of sera can be processed in one day. The antigen can be prepared in advance and stored until use. This represents an advantage over other methods, such as immunofluorescence and RIA, which require fresh antigen preparation for each set of assays. With this new test ca. 7% of females and ca. 20% of males with unexplained infertility have shown elevated antibody concentrations compared with fertile controls. The estimated intra-assay and inter-assay CV were 8.5% and less than 10% respectively.
Unexplained infertility
Immunofluorescence
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Microtiter plate
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Antibodies to different cytomegalovirus (CMV) polypeptide antigens, captured by monoclonal antibodies coated on the solid phase of an enzyme immunoassay test, were analyzed in 42 serum pairs submitted for serodiagnosis of CMV infection. Three CMV antigens, captured on the solid phase by three monoclonal antibodies of different specificities, designated CH92-1, CH65-1, and CH16-1, were glycoproteins A (gA), gC, and gD, respectively; and one antigen, captured by CH23, was a polypeptide with an apparent molecular weight of 150,000, possibly associated with the nucleocapsid. Of these four CMV antigens, gA captured by CH92-1 was most effective in eliciting an antibody response. Antibody to this antigen was present in serum samples at a higher concentration in primary and reactivated infection and persisted longer than did antibody to the other tested antigens. In contrast, antibody to antigen captured by CH23 was at a lower concentration, rose more slowly in infection, and persisted for a shorter time than did antibody to the other antigens. Antibody response to gC and gD was intermediate in concentration and temporal appearance compared with the antibody response to gA and to the polypeptide bound by CH23. An enzyme immunoassay on paired serum samples with the captured glycoproteins as antigen was equal for the detection of current infection to an enzyme immunoassay with the whole CMV antigen from infected cell lysates. Enzyme immunoassays with either the CMV glycoproteins or the whole CMV antigen from infected cell lysates were superior to a complement fixation test with a glycine extract antigen for serodiagnosis of current infection.
Complement fixation test
Cytomegalovirus
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Whether purified antigens of Lamblia intestinalis trophozoites can be used to detect these antibodies by immunoassay. The drugs of immunodominant Lamblia antigens were prepared by anion-exchange chromatography of solubilized trophozoite components and they are mainly presented by proteins having molecular weights of 70, 56, and 49 kD. Immunoassay using these antigens revealed antibodies to Lamblia trophozoite antigens in sera of 87.6% of patients with lambliasis (its diagnosis was established on the basis of microscopic data on the duodenal content) and only in 16.2% of clinically healthy blood donors. Twenty six sera from patients with trichomoniasis having high levels of antibodies to trichomonad antigens were studied to evaluate the specificity of this method for detection of antibodies. It has been found that the proportion of subjects in this group who have also antibodies to Lamblia antigens does not greatly differ from that of healthy blood donors (19.2 and 16.2, respectively).
Giardia lamblia
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