Development and evaluation of a capture ELISA for IgM antibody to the human cytomegalovirus major DNA binding protein.

A new capture ELISA (ELAb) for determination of the IgM antibody response to the human cytomegalovirus major DNA binding protein (p52) was developed by using a p52-specific monoclonal antibody. As a reference test, a capture ELISA using in parallel viral- and cell-control labeled antigens (ELA) was employed. General specificity, which was determined on 180 unselected IgM-negative sera from an adult population was 100%; stringent specificity, which was evaluated on 108 potentially interfering sera from patients with Epstein-Barr virus infectious mononucleosis, autoimmune diseases, rheumatoid factor or treated with radioimmunotherapy, was 96.3%; finally, clinical specificity, determined on 79 IgM-negative sera drawn prior to onset of primary HCMV infection, was 100%. Thus, the overall specificity was 98.9% (363/367 IgM negative tested sera). Sensitivity assayed on 277 IgM-positive sera was 100%. The study of the kinetics of the IgM antibody response in sequential blood samples from 9 immunocompetent and 9 heart transplanted patients showed that, while in the immunocompetent p52-specific IgM titer fell sharply 2-3 months after onset and was virtually undetectable 12 months after onset, in the immunocompromised the IgM response persisted for longer than a year. Recurrent HCMV infections were associated with a high titer IgM response in 6 (30%), and with a low IgM response in another 6 (30%) heart transplanted patients within a group of 20 patients sequentially examined. Finally, IgM antibodies were detected in all 4 infants with congenital infection and in 5 of 6 infants with neonatal infection. The results show that the HCMV p52-specific IgM antibody response parallels that obtained by ELA, thus representing a major component of it. ELAb is highly sensitive, specific and reproducible. It represents a major advance among capture ELISA techniques, allowing detection of IgM antibody reactive to a specific viral protein.

The "soluble sandwich" approach for immunoassays: methodological and instrumental implications.

The simultaneous presence of homogeneous and heterogeneous reactions at different binding sites of a multiepitope antigen makes the description of the kinetic parameters of the so called "one step" solid phase immunometric assays complex. The authors extended the "one step" approach to the concept of the "soluble sandwich" methodology which differs from the former by the delayed solid phase capture of the biotinylated immunocomplex to a streptavidin coated solid support. Using prolactin monoclonal IEMAs as a model, the equilibria involved in the reactions have been studied on a thermodynamic basis through a description of the kinetics of the interactions between biotinylated Mabs and solid phase streptavidin both in presence and/or in absence of the antigen and HRP-conjugated antibody. A comparative evaluation of models in which the biotinylated antibody was previously insolubilized on the streptavidin solid phase has been performed as well. The experimental work was carried out by using 125I labelled McBiot and Prolactin to trace individual interactions and peroxidase/H2O2/TMB systems to develop the enzymatic analytical signals. A new instrument/data reducing system was also optimized to expand the OD reading range provided by conventional, single wavelength colorimeters. The greater flexibility theoretically expected for the "soluble sandwich" approach and the possibility to extend the analyte working range without detrimental effects on the readability of low doses responses have been experimentally confirmed.

Development and evaluation of a capture enzyme-linked immunosorbent assay for determination of rubella immunoglobulin M using monoclonal antibodies.

A capture enzyme-linked immunosorbent assay (ELISA) for detection of virus-specific immunoglobulin M (IgM) antibody was developed which used a panel of labeled monoclonal antibodies to rubella virus hemagglutinin. The rapidity of the test system was increased by using, after 1-h incubation of the test serum, a second 1-h incubation of the serum with a mixture of viral antigen and labeled monoclonal antibody. The new assay was tested for specificity on 371 human sera from people without any recent contact with rubella virus; of these, 66 were sera selected from people with rheumatoid factor or IgM antibody to human cytomegalovirus, Epstein-Barr virus, or other viruses. In parallel, the new assay was performed on 191 sera from patients having recent contact with rubella virus. Results were compared with those obtained by an indirect ELISA method on IgM serum fractions, using purified rubella virus as a solid phase. Of the 371 sera tested for specificity, 5 (1.3%) gave false-positive results with indirect ELISA (1 rheumatoid factor, 2 heterophil antibody, and 2 human cytomegalovirus sera positive for IgM), and none were false-positive with the capture assay. Two sera from a patient with primary cytomegalovirus infection, which were positive for rubella IgM antibody with both methods and were initially interpreted as false-positive, were finally considered to be true-positive, since they were reactive only in the presence of IgM antibody and viral antigen. Of the 191 sera from 92 patients (84 patients with acute rubella, four newborns from mothers with rubella during pregnancy, and four vaccinees), 136 (71.2%) were found to be positive for IgM by direct ELISA, and 128 (67.0%) were positive by capture ELISA; 12 sera drawn during the first 2 days of disease, or at least 40 days after onset (or after vaccination), were detected only by indirect ELISA, and 4 sera were detected only by capture ELISA. Thus, specificity and sensitivity, respectively, were 100 and 91.4% for capture ELISA and 98.6 and 97.1% for indirect ELISA. However, when the number of patients was considered, 86 were detected as IgM positive by indirect ELISA, and 87 were detected positive by capture ELISA. The overall agreement between the two assays was 96.2%. Capture ELISA using monoclonal antibody appears preferable over indirect ELISA on IgM serum fractions because of its higher specificity and shorter time for test performance; furthermore, there is no need for serum fractionation or virus purification for the capture ELISA.

ELISA for specific anti-toxoplasma IgM antibodies: aspects related to serum interference.

Two different methods were used to prepare solid-phase antigen (Ag) from soluble extracts of tachyzoites of Toxoplasma gondii: (A) physical adsorption on polystyrene beads; and (B) formaldehyde fixation of Ag previously dried in microtitration wells. In both cases a horseradish peroxidase conjugate with anti-IgM IgG was used as tracer. The assay scheme consisted of sequential incubations of diluted serum samples and tracer solution (1 or 2 h, 37 degrees C), colour development in the presence of substrate (10 min at room temperature), addition of H2SO4, and absorbance reading at 492 nm. In procedure A no cut-off value for positives could be determined owing to a large overlap between positive and negative sera. The extent of overlap directly correlated with the total IgM content of samples. With negative sera similar values were obtained with sensitized and untreated beads: thus a correction could be made by directly subtracting absorbance values determined in parallel runs with uncoated beads. Results with negative sera correlated with total IgM concentration in procedure B also, but much less variability of blank values allowed negative and positive sera to be effectively discriminated. A series of reference positive and negative sera was correctly classified by both procedures A and B. However, the latter appeared preferable, as not requiring blank correction.

ELISA for antibody measurement: aspects related to data expression.

In an ELISA for antitoxoplasma IgG (antigen-coated polystyrene beads, horseradish peroxidase-coupled IgG or staphylococcal protein A), 3 modes of expressing the analytical results were considered, i.e. end-point antibody titre, untransformed absorbance reading at a single sample dilution, and antibody-activity unit from a calibration response curve (reference sera as calibrators). Criteria of merit for evaluation were defined as (a) stability of data under various conditions relating to both changes in assay design and minor variability of experimental conditions, and (b) linearity of response with dilutions of positive sera in negative serum, i.e., with positive sera sequentially defined as to antibody activity. Conclusions emerging were: single absorbance readings have some validity as indicators of trends but are very prone to systematic and random variability and inconsistent in response-antibody activity parallelism; parallelism of response proved to be an advantage of titration, but disadvantages are lack of practicability (manipulation and reagent costs involved) and lack of reliability (high levels of systematic and statistical error) The introduction of a reference scale allowing data to be expressed in activity units eliminates systematic components of error and gives analytical consistency. Use of the latter appears mandatory for between-run, between-laboratory and between-method normalization.

Rapid detection of antibodies to infectious bovine rhinotracheitis by 'macro' and 'micro' ELISA.

Two kinds of enzyme-linked immunosorbent assay were evaluated in their ability to detect specific antibodies against Bovine Rhinotracheitis Virus (IBR-IPV). The tests were called MACROELISA and MICROELISA, according to the kind of the solid support used for antigen insolubilization, polystyrene beads and microtitration plates respectively. Partially purified virus was used to coat both beads and plates; a single dilution of examples was tested and protein A linked to peroxidase was employed as enzyme tracer. Quantitative instrumental results from MACROELISA and qualitative visual results from MICROELISA were compared with serum neutralization titers. The results clearly show that ELISA tests are suitable for IBR serologic detection, being sensitive, specific and accurate over serum neutralization method.

Radioimmunoassay of trypsin-like substance in human serum.

A radioimmunological method for circulating trypsin-like substances was set up, using cathodic trypsin (isolated from human pancreas) as immunogen, reference standard and material to label, and bovine serum to stabilize the system. The assay scheme included a bound-free separation by polyethylene-glycol precipitation after a 1-h incubation at room temperature. Some relevant points emerged from the validation experiments: a) the assay proved simple and reliable, meeting both requirements of a prompt clinical response and of a high sensitivity level (1.5 ng/ml or less); b) evidence was added to the view that cathodic trypsinogen is the immunoassayable species present in the blood stream; c) the parallelism of the radioimmunological responses of cathodic trypsin and trypsinogen demonstrated that a self-consistent analytical information may be derived using either references; d) the absence of detectable amounts of trypsin-like substances in sera from totally-pancreatectomized patients (3 cases) indirectly confirmed the organ-specificity of the measurement; e) a "normal" level of 27 +/- 8 ng trypsin equivalent/ml (X +/- SD, n = 82) resulted: some much higher literature data, all related to a single radioimmunoassay system (RIA-Gnost trypsin kit, Hoechst, FRG), are seemingly explained by the different purity observed for the reference materials.

Radioimmunoassay of lactate dehydrogenase, H forms.

Antisera to H4-lactate dehydrogenase (LDH) were elicited in rabbits, against both human (h) and porcine (p) isoenzymes. 125I-labelled H4-LDH was prepared by electrolytic iodination. A simple and fast procedure (1-h incubation for clinical assays) was set up by using polyethylene glycol for the bound-free separation. The results obtained in the antiserum characterization indicated that the heterologous homotetramer, M4 was completely discriminated in the porcine system, while a weak cross-reaction with human antisera resulted. In both cases, for the hybrid forms, a cross-reactivity level related to the stoichiometric contents of the H-subunit in the tetramers was observed. The H4-LDH from other species was found to be much more effectively disinguished in the porcine than in the human system. The assay for human LDH was further validated in terms of analytical suitability and clinical response. For healthy subjects the mean concentration was 0.46+/-19 micrograms/ml (mean+/-SD). Patients with acute myocardial infarction had levels ranging from 1.2 to 5.9 micrograms/ml.

Direct radioimmunoassay of plasma cortisol.

The simplification of the measurement of circulating cortisol by direct radioimmunoassay of plasma samples sets the problem of inhibiting the carrier proteins competing with antibodies. This was accomplished by exploiting the much higher effectiveness of pH and temperature variations on steroid binding to carrier proteins than to antibody sites. A solid-phase system was set up, using antisera to cortisol-21-BSA conjucates coupled to CNBr-activated cellulose. The standardized procedure consisted of an incubation at pH 3.5 and room temperature, directly assaying 10 mul of plasma. A methodological and clinical validation of the measurement was carried out through a series of tests aimed at assessing the reliability of results (assay of steroid-deprived plasma, recovery test and serial dilution of samples, comparison between different antisera and with different methods including extraction, responsiveness to well-established physiological situations). The results obtained are reported and the validity of the method discussed in terms of more general applicability to steroid assay.