Use of bifunctional hybrid beta-lactamases for epitope mapping and immunoassay development

Mapping of epitopes is a crucial step for the study of immune pathways, the engineering of vaccines and the development of immunoassays. In this work, the Bacillus licheniformis beta-lactamase BlaP has been engineered to display heterologous polypeptides in a permissive and solvent-exposed loop. When combined with phage display, this modified enzyme can be used for epitope mapping by cloning random gene fragments. The procedure presented in this paper allows the selection of large infectious phage libraries with high diversity and efficient beta-lactamase activities. A useful aspect of the proposed technique results from the possibility of using the beta-lactamase activity carried by phages to evaluate the proportion of immobilised phages during the successive enrichment steps of the library or competition experiments with the selected phages. Another advantage of the technique derives from the fact that the epitope is selected as a bifunctional hybrid protein, which can be overproduced and purified. The resulting recombinant protein associates an epitope with a specific and efficient enzymatic activity. This constitutes an original tool for immunoassay development. A virus influenza hemagglutinin (HA1)-gene fragment library has been generated with this system and used to identify a linear epitope.     

Production of monoclonal antibodies to calcitonin and development of a two-site enzyme immunoassay

A procedure is described for the production of calcitonin-specific hybridomas which involves primary and secondary immunization of Balb/c mice in the hind footpads with free, synthetic human calcitonin and cell fusion of lymphocytes from popliteal lymphonodes with a P3 x 63 myeloma line. This protocol offers the following advantages: (a) it is short and easy to perform, (b) it requires small amounts of unconjugated antigen, and (c) it gives a high yield of antigen-specific IgG-secreting hybridomas. Routine screening was carried out by ELISA on solid phase calcitonin and binding of the monoclonals to free antigen was studied with calcitonin linked to biotin through a 13 carbon atom spacer. Monoclonal couples capable of simultaneously binding calcitonin in solution were found by pairing in all possible combinations 25 purified antibodies, in their unlabelled and biotinylated form, in a checkerboard matrix experimental system. Of the over 30 positive pairs identified, four were used in a one-step enzyme immunoassay for calcitonin determination on microtiter plates in a concn range between 0.1 and 5 ng/ml. With the detecting monoclonal directly conjugated to peroxidase with a heterobifunctional crosslinker, the range of the assay with one monoclonal pair was between 25 and 1000 pg/ml with an 18 hr incubation at 4 degrees C.     

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.     

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.     

An enzyme immunoassay for rapid isotyping of monoclonal antibodies

An enzyme-immunoassay has been developed for rapid isotyping of murine monoclonal antibodies. This assay has several advantages over the commonly used typing methods. It does not require the presence of specific antigen. Typing plates can be prepared in advance and stored frozen. Unequivocal results can be obtained in 2 h, and the urease indicator system produces a vivid colour change that can be read visually.     

Use of biotin-labeled monoclonal antibodies and avidin-peroxidase conjugates for the determination of epitope specificities in a solid-phase competitive enzyme immunoassay

The epitope specificities of monoclonal antibodies against carcinoembryonic antigen (CEA) were determined in a solid-phase, biotin-avidin enzyme immunoassay. Constant amounts of biotin-labeled antibodies were incubated with the immobilized antigen in the presence of decreasing amounts of unlabeled antibodies. The biotinylated antibodies bound to the antigen were then reacted with avidin-peroxidase conjugates. The activity of the bound peroxidase was determined by the use of o-phenylenediamine and hydrogen peroxide.     

An enzyme immunoassay for the screening of monoclonal antibodies to cyclosporin

An enzyme-linked immunosorbent assay (ELISA) has been developed which allows hybridoma cell cultures to be tested for the presence of monoclonal antibodies specific for cyclosporin A. Immunization of mice with free cyclosporin was found to be preferable to immunization with cyclosporin-carrier conjugates. It is hoped that the availability of monoclonal antibodies to cyclosporin will clarify the contribution of cyclosporin metabolites to immunosuppression and nephrotoxicity.     

Polyethylene glycol enzyme immunoassay for screening anti-haptoglobin monoclonal antibodies

An enzyme immunoassay for the screening of anti-haptoglobin activities in supernatants of hybridoma cell cultures is described. The sandwich technique was greatly improved by the addition of 2% polyethylene glycol 6000 to all the reagents. It allowed a six-fold shortening in the incubation time compared with the standard method without sacrificing either sensitivity or accuracy.     

Human-isotype-specific enzyme immunoassay for antibodies to pneumococcal polysaccharides.

A simple enzyme immunoassay has been developed to allow the quantitation of the human response to immunization with pneumococcal polysaccharide. The assay uses the 14-valent vaccine (Pneumovax) as a convenient antigen to adsorb to the solid-phase microdilution plate wells and commercially available isotype-specific antibody conjugates. The results have been expressed as arbitrary pneumococcal polysaccharide antibody units by reading off a standard curve constructed by using heterogeneous pooled serum. All nonimmunized subjects tested had immunoglobulin G (IgG) antibodies present in serum. All six control subjects who were immunized with Pneumovax demonstrated an IgG response, and the majority responded with a rise in IgA- and IgM-specific antibody concentrations at a mean of 6 weeks postimmunization. Five out of six cord sera tested contained IgG antibodies only, which were present in concentrations similar to those seen in adults, whereas in 6- to 12-month-old infants only low levels of IgG and IgM and no IgA antibodies were detected. Serum taken from 10 hypogammaglobulinemic patients immediately prior to infusion of immunoglobulin showed low to negative IgG antibody concentrations, and no IgA or IgM antibody was present.     

Reverse enzyme immunoassay for the determination of Dermatophagoides pteronyssinus IgE antibodies

Sera from patients with suspected mite sensitivity were assayed using both the radioallergosorbent test (RAST) and a reverse enzyme immunoassay for IgE antibodies (REIA). Of the 133 sera studied, the RAST gave positive results in 48 cases and the REIA in 59. Negative results for both assays were obtained in 72 of the sera. The immunoenzymatic assay described here, using micro-plates as solid phase, has proven to be IgE- and antigen-specific. The allergen conjugate has a prolonged shelf life and the method is easy to perform. The sensitivity of the REIA for Dermatophagoides pteronyssinus IgE antibodies is limited by the small amount of IgE bound by the well surface (less than 100 ng). However, we think that the method offers a reliable alternative for the diagnosis of D. pteronyssinus-allergic patients.     

Solid-phase enzyme immunoassay for determination of antibodies to cytomegalovirus.

A solid-phase enzyme immunoassay for the determination of immunoglobulin H (IgG) and IgM antibodies to cytomegalovirus is described. The enzyme immunoassay gave reliable and consistent results which were in concordance with those obtained by the complement fixation test and the indirect immunofluorescence test. Antibodies to herpes simplex and varicella-zoster viruses did not interfere in the enzyme immunoassay for cytomegalovirus IgM antibodies. In a few patients with IgM antibodies to Epstein-Barr virus, cytomegalovirus IgM antibodies were also detected. False-positive cytomegalovirus IgM antibody results were observed in sera containing both the rheumatoid factor and cytomegalovirus IgG antibodies. This rheumatoid factor interference was overcome by the absorption of sera with polymerized human gamma globulin. The absorption did not affect true cytomegalovirus IgM antibody titers. Also described is a simple enzyme immunoassay that makes possible a more sensitive detection of the rheumatoid factor than the latex agglutination test.     

Comparison of an enzyme immunoassay to an indirect fluorescent immunoassay for the detection of antinuclear antibodies

The standard method for detecting antinuclear antibodies (ANAs) is by immunofluorescence assay (IFA), a method that is labor intensive and subjective. In an attempt to overcome these limitations, several commercial enzyme immunoassays (EIAs) have been developed. We report the results of our evaluation of the ANA Microplate EIA (Sanofi Diagnostics Pasteur, Chaska, MN). For the evaluation, 808 serum samples were tested by EIA and IFA; 52 specimens were positive by both assays, 561 were negative by both assays, 91 were positive by EIA only, and 3 were positive by IFA only. Borderline results (not positive or negative) were obtained for 101 specimens, which were excluded when calculating the sensitivity, specificity, and positive and negative predictive values of this assay, which were 94.6%, 86.0%, 36.4%, and 99.5%, respectively. Because of its high negative predictive value, this assay can be used reliably to detect ANA-negative samples; however, the low positive predictive value indicates that EIA-positive specimens should be retested by an IFA to determine the final result.     

Rapid dot enzyme immunoassay for the detection of antibodies to cytomegalovirus.

Cytomegalovirus (CMV) antigen was coated onto a white opaque plastic card as small dots inside circles marked in the microtiter plate well pattern. The card with antigen dots could be cut according to the number of test samples to be assayed. Small drops of undiluted serum samples, goat antibodies to human immunoglobulin G labeled with alkaline phosphatase, and finally substrate (5-bromo-4-chloro-3-indolyl phosphate) were sequentially added to the antigen spots and incubated in the open air at room temperature for 5 min each. The antigen dots showed blue color for sera with immunoglobulin G antibodies to cytomegalovirus but no color for those without. The developed antigen dots could be rinsed with water and kept as permanent records. For the assay of a large number of serum samples, a modified procedure with serum diluted 1:10 and longer first two incubations (20 min each) was found to be more comfortable to perform. The results of this assay for 123 undiluted and 256 diluted serum samples revealed very good correlations with those obtained by a commercially available test kit for immunoglobulin G antibodies to cytomegalovirus with 97 and 99% agreement, respectively. This dot test was very reproducible and required no instrumentation. The reagents, including coated antigen dots, are stable at room temperature for at least 2 months and are ready for use.     

Solid-phase enzyme immunoassay for immunoglobulin M antibodies to cytomegalovirus.

A sensitive, solid-phase enzyme immunoassay for the detection of immunoglobulin M antibodies to cytomegalovirus is described. The results of the enzyme immunoassay correlated well with those obtained by an indirect immunofluorescence method. Horseradish peroxidase proved to be a more sensitive label than alkaline phosphatase. Nonspecific reactions, occurring with commercially available cytomegalovirus antigens, could be avoided by using a nuclear antigen prepared from sonically disrupted nuclei of cytomegalovirus-infected cells.     

Latex enzyme immunoassay for measuring IgG antibodies to rubella virus

A latex enzyme immunoassay (LEIA) for detecting IgG class antibody to rubella virus was compared with the latex agglutination test (LA) and a haemagglutination inhibition assay (HI). Of 243 sera tested, four discrepant results were observed among all three techniques, corresponding to borderline values. Except for one sample containing specific IgM class antibody, the difference between quantitative results from each pair of tests was always within a value corresponding to two doubling dilutions and was considered to have acceptable variation. The LEIA technique allowed 10 seroconversions to be detected, as determined by the other techniques. The LEIA required a single 1 in 8 sample dilution, took 30 minutes, and provided a useful alternative assay for the quantitation of rubella antibodies.     

Immunotherapeutic perspective for bispecific antibodies

Bispecific antibodies (BsAb) can, by virtue of combining two binding specificities, improve the selectivity and efficacy of antibody-based treatment of human disease. Recent studies underline the importance of both the 'anti-trigger' and 'anti-target' modalities of BsAb for therapeutic efficacy. Several BsAb induce effective cytotoxicity as well as 'vaccine effects' in vivo. Here, Annemiek van Spriel and colleagues discuss how these results have catalysed renewed efforts to translate BsAb concepts into effective therapies.     

Use of epidemiologically well-defined subjects and existing immunofluorescence assays to calibrate a new enzyme immunoassay for human herpesvirus 8 antibodies

Agreement between assays for the detection of human herpesvirus 8 (HHV-8) antibodies has been limited. In part, this disagreement has been because assay calibration (i.e., differentiating positive from negative results) has not been done in a standardized fashion with reference to a wide spectrum of HHV-8-infected (true-positive) and HHV-8-uninfected (true-negative) persons. To describe the performance of an assay for HHV-8 antibodies more accurately, we used epidemiologically well-characterized subjects in conjunction with testing on two existing immunofluorescence assays for HHV-8 antibodies to define two groups: a group of 135 HHV-8-infected individuals (true positives), including Kaposi's sarcoma patients and those asymptomatically infected, and a group of 234 individuals with a high likelihood of being HHV-8 uninfected (true negatives). A new enzyme immunoassay (EIA), using lysed HHV-8 virion as the antigen target, was then developed. With the above true positives and true negatives as references, the sensitivity and specificity of the EIA associated with different cutoff values were determined. At the cutoff that maximized both sensitivity and specificity, sensitivity was 94% and specificity was 93%. When the EIA was used to test a separate validation group, a distribution of seropositivity that matched that predicted for the agent of Kaposi's sarcoma was observed: 55% of homosexual men were seropositive, versus 6% seropositivity in a group of children, women, and heterosexual men. It is proposed that the EIA has utility for large-scale use in a number of settings and that the calibration method described can be used for other assays, both to more accurately describe the performance of these assays and to permit more-valid interassay comparison.     

Two distinct monoclonal antibodies raised against mouse beta nerve growth factor. Generation of bi-specific anti-nerve growth factor anti-horseradish peroxidase antibodies for use in a homogeneous enzyme immunoassay

Two hybridomas producing monoclonal antibodies against mouse beta nerve growth factor (NGF) were obtained from the fusion of hyperimmune splenocytes from rats immunized with polymerized beta-NGF and Sp2/0.Ag mouse myeloma cells. The monoclonal antibodies coded IgG 24 and 30 produced and secreted by the hybrid cells are both of the IgG2a subclass. Both monoclonal antibodies are capable of recognizing native NGF coated on microassay plates as well as the denatured factor on Western blots. However, only IgG 30 has been found to block NGF-induced process outgrowth from the rat pheochromocytoma cell line (PC12) as well as NGF-induced increase in choline acetyltransferase activity in rat primary septal cell cultures. In addition, only IgG 30 was able to detect immunocytochemically NGF-immunoreactive sites in fixed tissue. And, finally, IgG 24 could not compete for IgG 30 binding to immobilized native NGF. Consequently, it appears that these antibodies are recognizing different epitopes on the NGF molecule. Neither monoclonal antibody displayed any crossreactivity with serum albumin, aprotinin, epidermal growth factor or insulin. A hybrid-hybridoma producing bi-specific anti-NGF anti-horseradish peroxidase (HRP) monoclonal antibodies was generated from the fusion of an azaguanine resistant anti-HRP hybridoma, coded RAP2.Ag and the anti-NGF IgG 30 hybridoma treated with emetine. The potential merits of using these bi-specific antibodies in combination with their mono-specific anti-NGF parent in a homogeneous sandwich immunoassay for the quantitation of NGF are discussed.     

Evaluation of a commercial enzyme immunoassay for detection ofMycoplasma pneumoniae specific immunoglobulin G antibodies

A commercial enzyme immunoassay (Platelia Mycoplasma) infections was evaluated and found not to be suitable for the purpose. More than 80 % of healthy persons and patients with non-Mycoplasma pneumoniae respiratory infection, all with a negativeMycoplasma pneumoniae complement fixation test, had a positive EIA. Paired sera did not show the positive correlation between a rise in complement fixation titre and the EIA ratio reported by the manufacturer.