Heterophile infectious mononucleosis antigen used in the latex diagnostic test. I. A method of antigen isolation and its serologic activity

The aim of this study was to elaborate a method of heterophile mononucleosis antigen preparation useful for latex coating. This antigen was isolated from bovine red blood cells stroma by the technique of Schwarzweiss and Tomcsik with author's own modification, in which introductory extraction of erythrocytes stroma ++ was performed by means of trichloracetic acid, aqueous extraction and elution of active substance with 80% ethanol. Besides of heterophile antigen preparation obtained by the method of Schwerzweiss and Tomcsik (preparation S-T) two serologically++ active preparations were obtained (fraction I and IV), which ability to inhibit PBD agglutinating reaction and bovine red blood cells haemolysis was 16 and 8 times lower, respectively, than S-T preparation. The preparation of heterophile mononucleosis antigen obtained differed in latex coating efficacy. In order to prepare latex reagent MZ-I (from fraction I) a solution of preparation of 125 micrograms/ml concentration was used, for MZ-II (from fraction IV)--50 micrograms and for MZ-III (from preparation S-T)--15 micrograms/ml. The reagent MZ-I showed, the highest activity in agglutinating test with human serum containing heterophile mononucleosis antibodies while two others reacted with 2-4 times lover serum dilutions. Similar differentiated reactivity with these reagents was found in latex test with 15 sera from patients suspected of having infectious mononucleosis.     

Hanganutziu-Deicher antibodies in infectious mononucleosis and other diseases

An enzyme immunoassay (EIA) was developed for the detection of heterophile, Hanganutziu-Deicher (H-D) antibodies in sera of patients with infectious mononucleosis (IM) and various other diseases. The EIA with a high m.w. glycoprotein (HMWGP) isolated from bovine erythrocyte stromata was shown to detect H-D antibodies directly, in spite of higher titers of Paul-Bunnell (P-B) antibodies in the IM sera. Absorption and inhibition studies of IM sera demonstrated H-D specificity of the antibodies combining with HMWGP in the EIA. The H-D antibodies were found in sera of 56% Caucasians and 27% of Japanese suffering from IM. The vast majority of the H-D antibodies in IM sera was of IgM class. Sera of patients with various other diseases also gave positive results: rheumatoid arthritis, 22%; syphilis, 19%; cancer of the gastrointestinal tract, 13%; and lepromatous leprosy 9.7%. The incidence of positive results in control sera from apparently healthy subjects was less than 4%. Results of this study confirmed our previous observation that whereas P-B antigens appear in immunogenic form in only IM, the H-D antigen is expressed as an immunogen in various diseases including IM.     

Specificities of human heterophile Hanganutziu and Deicher (HD) antibodies to glycosphingolipids and a glycoprotein

The specificities of five heterophile Hanganutziu and Deicher (HD) antibody-containing sera from four different cancer patients and one other diseased patients were compared. Three glycosphingolipids and one glycoprotein antigens and their chemically modified derivatives were used. The antibodies of all whole sera showed similar specificities. IgG and IgM antibody fractions of each serum were separated. Although antibodies of the same class showed similar specificities, differences were detected between the specificities of IgG and IgM. IgG antibody specificities were dependent on the hydrophobic (ceramide) group while IgM antibodies were directed more to the terminal sialic acid moiety of the glycosphingolipid antigens. The results suggested that a similar population of IgG-producing lymphocytes is stimulated in patients. Due to the similarities in specificities of HD antibodies, the results of this study will facilitate the future isolation of either IgG or IgM antibody-producing lymphocyte(s) from a patient with HD antibodies and the establishment of a monoclonal antibody through hybridization with a human myeloma cell line.     

Heterophile antibodies to rabbit erythrocytes in human sera and identification of the antigen as a glycolipid

Normal human sera contain heterophile hemagglutinins to rabbit erythrocytes which are different from anti-B isoantibody and other heterophile antibodies such as Hanganutziu-Deicher antibody or Paul-Bunnell antibody. The antigen to this antibody was purified from rabbit erythrocyte stroma, and identified as pentaglycosyl ceramide, Gal(alpha 1-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc-Cer.     

Conglutinin, immunoconglutinins and heterophile antibodies in the sera of apparently healthy moose (Alces alces) and caribou (Rangifer tarandus) in Quebec

Serum samples from apparently healthy wild populations of moose and caribou in the province of Quebec, Canada were screened for the presence of conglutinin (K), immunoconglutinins (IKS) and heterophile antibodies. The conglutinating factor in moose and caribou sera was characterized utilizing the necessity of calcium ions for its reaction with sensitized sheep erythrocytes which had been alexinated with horse complement. The conglutinating substance in these animals did not require calcium ions for its activity. The conglutinating activity in both moose and caribou sera was characterized due to IKS as those present in sheep, dog and rabbit sera. Both moose and caribou had non-agglutinating type of heterophile antibodies. Their titres varied from 0 to 80. None of the animals tested had K in their blood. The titre of IKS varied from 0 to 640 with a mean value of 41 in moose, whereas it varied from 0 to 80 with a mean value of 18 in caribou. About 75% of all the animals in both the groups were positive for IKS. The specificity of IKS was demonstrated by the total removal of its activity on absorption with alexinated cells. Presence of IKS in these animals is suggestive of latent infection(s) possibly of bacterial, viral or parasitic origin.     

Antigen of "serum sickness" type of heterophile antibodies in human sera: indentification as gangliosides with N-glycolylneuraminic acid.

Antigen of “serum-sickness” type of heterophile antibodies in pathologic human sera was purified from equine and bovine erythrocyte stroma. The chemical nature of this antigen was glycosphingolipids with N-glycolylneuraminic acid. The antigen of equine erythrocytes was identified as hematoside with N-glycolylneuraminic acid, GlNeu(α, 2–3)Gal(β, 1–4)Glc(β,1-1) ceramide and the antigen of bovine erythrocytes was N-glycolylneuraminyl-paragloboside, GlNeu (α,2–3)Gal(β,1–4)GlcNAc(β,1–3)Gal(β,1–4)Glc(β,1-1) ceramide. The results indicate that “serum-sickness” antibodies react with a common disaccharide moiety of non-reducing end of the both glycosphingolipids.     

Human heterophile antibodies recognizing epitopes present on insect glycolipids.

As a consequence of detecting an IgM M-protein (naturally occurring diseased-state monoclonal antibody) immunoreactive to insect acidic glycolipids in a patient with demyelinating peripheral neuropathy, normal human sera were examined for the occurrence of heterophile antibodies directed against carbohydrate epitopes present on glycosphingolipids of Calliphora vicina (Insecta: Diptera). The insect glycolipids can be separated into neutral, zwitterionic, and acidic types, according to whether the oligosaccharide chains consist of neutral monosaccharides only, or carry an additional phospho-ethanolamine side chain and/or a beta-glucuronic acid residue, respectively. Natural antibody activity to these three classes of insect glycosphingolipids was detected in all normal human sera examined. The antibody activities were separated by sequential chromatography on affinity columns of octyl-Sepharose 4B-bound neutral and zwitterionic glycolipids into three populations with differing epitope-type specificities. As expected for heterophile antibodies, they are mainly of the IgM class. Population I recognized epitopes present on the three types of insect glycolipids, i.e., the neutral oligosaccharide chain backbone, the main determinant of which contains a terminal N-acetylhexosamine. Immunoreactivity is separable into at least four subpopulations of differing carbohydrate epitope specificity. Population II recognized epitopes containing phosphoethanolamine in zwitterionic and some acidic insect glycolipids. There are two subpopulations, the majority of which require the free amino group of phosphoethanolamine for immunoreactivity. Population III antibodies showed immunoreactivity to terminal beta-glucuronic acid-containing epitopes present only on acidic insect glycolipids.     

Heterophile antibodies to the antigens of myocardial interstitial connective tissue and erythrocytes in animals of different species

Reactions of heterophilic antibodies with antigens of the interstitial connective tissue (ICT) of the myocardium of different animals were studied and compared. The reactions were identical on bovine, pig, sheep and rabbit myocardium. The heterophilic antigen of the ICT of bovine myocardium was also found on bovine, rabbit and rat red cells and was absent on sheep red blood cells. It is suggested that heterophilic antigen of the ICT of the bovine myocardium is a novel heterophilic antigen, the specificity of which is linked with D-galactose and which differs from antigens of other heterophilic systems.     

The presence of anti-sheep red blood cell heterophile antibodies and their characteristics in murine schistosomiasis japonica

Sensitive methods of an enzyme-linked immunosorbent assay (ELISA) using red blood cells (RBC) have been developed and were applied to the detection of anti-sheep red blood cell (SRBC) heterophile antibodies (Ab) present in sera of Schistosoma japonicum (SJ)-infected mice. The indirect hemagglutination test (IHA) was used for the purpose as well. By these methods a significant increase in the heterophile Ab levels was demonstrated in the mice particularly after 6-10 weeks of infection. The heterophile Ab in SJ-infected mice were predominantly immunoglobulins resistant to 2-mercaptoethanol treatment and had temperature-independent reactivity. In an attempt to investigate the immunological specificity of the heterophile Ab, various absorption tests were performed; Davidsohn's differential absorption test revealed that the heterophile Ab were distinct from Forssman antibody, Paul-Bunnell antibody and heterophile agglutinins known to appear in serum sickness. The heterophile Ab were absorbed only with SRBC and goat red blood cells, not with other species of RBC such as human O Rh-, O Rh+, A Rh+, B Rh+, mouse, ox, chicken, horse, rabbit, guinea pig, and rat RBC, or syngeneic tissue homogenates including brain, lung, liver, spleen, kidney, muscle, and thymus. This heterophile antibody response is not a consequence of a specific immune response directed to the antigens of SJ parasites, since absorption of the heterophile Ab with SJ adult worms or an egg preparation did not reduce the heterophile Ab level.     

Demonstration of heterophile antibody in chicken antiserum against Marek's disease tumor-derived cell line, MSB-1

Sheep red blood cells (SRBC) were agglutinated by all six chicken anti-MSB-1 sera examined, but not by sera of thirty specific pathogen-free chickens. The SRBC-agglutination titer was greatly reduced by absorption with SRBC, bovine red blood cells (BRBC) or guinea-pig kidney cells (GPKC). The dissociation of heterophile antibody and antibody to so-called Marek's disease tumor-associated surface antigen (MATSA) is discussed.     

Antibodies to different isoforms of the heavy neurofilament protein (NF-H) in normal aging and Alzheimer's disease

Sera of normal controls and of patients with neurological diseases contain antineurofilament antibodies. Recent studies suggest that biochemically and immunologically distinct subclasses of neurofilaments occur in different types of neurons. Alzheimer's disease (AD), the major cause of dementia, is associated with a marked degeneration of brain cholinergic neurons. In the present work we characterized the repertoire and age dependence of antineurofilament antibodies in normal sera and examined whether the degeneration of cholinergic neurons in AD is associated with serum antibodies directed specifically against the neurofilaments of mammalian cholinergic neurons. This was performed by immunoblot assays utilizing neurofilaments from the purely cholinergic bovine ventral root neurons and from the chemically heterogeneous bovine dorsal root neurons. Antibodies to the heavy neurofilament protein NF-H were detected in normal control sera. Their levels were significantly higher in older (aged 70–79) than in younger (aged 40–59) subjects. These antibodies bound similarly to bovine ventral root and dorsal root NF-H and their NF-H specificity was unchanged during aging. In contrast, the levels of IgG in AD sera that are directed against ventral root cholinergic NF-H were higher than those directed against the chemically heterogeneous dorsal root NF-H. Immunoblot experiments utilizing dephosphorylated ventral root and dorsal root NF-H and chymotryptic fragments of these molecules revealed that AD sera contain a repertoire of antimamalian NF-H IgG. A subpopulation of these antibodies binds to phosphorylated epitopes that are specifically enriched in ventral root cholinergic NF-H and that are located on the carboxy terminal domain of this molecule. The level of these anticholinergic NF-H IgG are significantly higher in AD sera than in those of both normal controls and patients with multi-infarct dementia.     

Paul-Bunnell antigen in lymphoma and leukemia spleens.

Lymphoid cells obtained from spleens of patients with lymphomas or leukemias were studied for the presence of heterophile (Paul-Bunnell (P-B)) antigen. A mixed agglutination (MA) test was established utilizing monolayers of cells attached to poly-L-lysine-coated wells of plastic U plates. After incubation of the monolayers with infectious mononeucleosis (IM) sera, indicator cells, sheep, or trypsinized bovine erythrocytes were added. The results were assessed according to sedimentation patterns of the indicator cells on the monolayers. Positive MA reactions were shown to be due to specific binding of P-B antibodies to the corresponding antigens on the spleen cells. Positive results were obtained with 15 of 37 spleens from patients with Hodgkin's disease, 5 of 8 lymphoma spleens, 4 of 15 chronic myelocytic leukemia spleens and 2 of 4 chronic lymphocytic leukemia spleens. Only 2 of 25 spleens from patients with various other diseases and 1 of 26 apparently normal thymus specimens gave positive results. This study confirmed demonstration of P-B antigen in lymphoma and leukemia by means of absorption experiments, which was reported previously.     

Heterophilic infectious mononucleosis antigen used in the latex diagnostic test. II. Preliminary evaluation of the usefulness of latex reagents for detection of serum heterophile antibodies in patients with infectious mononucleosis

The aim of this study was to evaluate the usefulness of laboratory made reagent of polystyrene latex coated with three preparations of mononucleosis antigen (reagent MZ-I, MZ-II, and MZ-III) for detection of heterophile antibodies in patients sera with clinical symptoms of infectious mononucleosis. These studies were carried out on 153 serum samples taken from persons suspected of being infected with EB virus and on 100 healthy controls--blood donors. The results of latex tests were compared to the results of Paul-Bunnell-Davidsohn (PBD) and hemolytic tests. Out of three latex reagents evaluated only one (reagent MZ-I) showed sensitivity equal to PBD and haemolytic tests. The sensitivity reached 91.1%. Agglutination reaction when appeared in latex test at serum dilution 1:5, is considered positive and diagnostically significant result.     

Formation of Paul-Bunnell antibodies by cultures of lymphocytes from infectious mononucleosis.

Short-term cultures of peripheral blood lymphocytes obtained from 20 infectious mononucleosis patients 2–4 weeks after the onset of the disease were studied for formation of heterophile antibodies. In studying pooled supernatant fluids of lymphocytes from three patients cultured for 3–20 days, lytic antibodies for red blood cells of bovine (BRBC) and sheep (SRBC) origin were demonstrated. These hemolysins were shown to be of IgM nature and Paul-Bunnell specificity. Subsequently, plaque-forming cell (PFC) assays were performed with lymphocyte cultures of 15 patients. Significant numbers (60–750/2 × 10⁷ cells) of PFC secreting antibodies against BRBC were demonstrated in lymphocyte cultures of 12 patients. The number of PFC apparently reached its peak after 5 to 10 days of culturing. No or a very few PFC were observed in the lymphocytes that were not cultured or in lymphocytes cultured for 3 weeks or longer. Lymphocyte cultures prepared in a similar fashion from normal individuals or patients suffering from sore throat and submandibular lymphadenopathy of other than infectious mononucleosis origin did not produce PFC. Production of lytic zones by antibodies to BRBC secreted by PFC was inhibited by preincubation of lymphocytes of infectious mononucleosis patients with solubilized Paul-Bunnell antigen but not with other heterophile antigens, indicating that antibodies involved in the PFC formation are of Paul-Bunnell specificity. An increased number of PFC against BRBC were obtained in two of three lymphocyte cultures after cultivation with BRBC or solubilized Paul-Bunnell antigen.     

Rabbit antibodies against the human milk sialyloligosaccharide alditol of LS-tetrasaccharide a (NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4GlcOH)

The sialyloligosaccharide, NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc (LS-tetrasaccharide a), a minor component of human milk, is obtained in relatively large quantities from autohydrolysates of the major milk disialyloligosaccharide, NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc (disialyllacto-N-tetraose). Rabbits immunized with an oligosaccharide-protein conjugate prepared from keyhole limpet hemocyanin and LS-tetrasaccharide a produce antibodies directed against the corresponding oligosaccharide alditol. The anti-LS-tetrasaccharide a sera bind 3H-labeled LS-tetrasaccharide a in a direct-binding radioimmunoassay on nitrocellulose filters. The specificities of these antibodies are determined by comparing inhibitory activities of structurally related oligosaccharides. Strong hapten-antibody binding (Ka greater than 10(6) M-1) requires sialic acid linked alpha 2-3 to the nonreducing terminal galactose residue of reduced lacto-N-tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4GlcOH). Specificities of antibodies prepared against keyhole limpet hemocyanin conjugates of LS-tetrasaccharide b (Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc) and LS-tetrasaccharide c (NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc) differ only slightly from rabbit antibodies prepared against the corresponding bovine serum albumin conjugates described previously [D. F. Smith and V. Ginsburg (1980) J. Biol. Chem. 255, 55-59].     

The reactivities of human erythrocyte autoantibodies anti-Pr2, anti-Gd, Fl and Sa with gangliosides in a chromatogram binding assay.

The thin layer chromatogram binding assay was used to study the reaction of several natural-monoclonal autoantibodies which recognize sialic acid-dependent antigens of human erythrocytes. Immunostaining of gangliosides derived from human and bovine erythrocytes was achieved with four autoantibodies designated anti-Pr2, anti-Gd, Sa and Fl, each of which has a different haemagglutination pattern with untreated and proteinase-treated erythrocytes and with cells of I and i antigen types. From the chromatogram binding patterns of anti-Pr2 with gangliosides of the neolacto and the ganglio series, it is deduced that this antibody reacts best with N-acetylneuraminic acid when it is alpha 2-3- or alpha 2-6-linked to a terminal Gal(beta 1-4)Glc/GlcNAc GlcNAc sequence and to a lesser extent when it is alpha 2-3-linked to a terminal Gal(beta 1-3)GalNAc sequence or to an internal galactose and when it is alpha 2-8-linked to another, internal N-acetylneuraminic acid residue. The other three antibodies differ from anti-Pr2 in their lack of reaction with glycolipids of the ganglio series. They react with the NeuAc(alpha 2-3)Gal(beta 1-4)Glc/GlcNAc sequence as found in GM3 and in glycolipids of the neolacto series, but show a preference for the latter, longer sequences. Thus all four antibodies react with sialylated oligosaccharides containing i type (linear) and I type (branched) neolacto backbones. Fl antibody differs from the other three in its stronger reaction with branched neolacto sequences in accordance with its stronger agglutination of erythrocytes of I rather than i type. The four antibodies show a specificity for N-acetyl- rather than N-glycolyl-neuraminic acid.     

Antibodies against synthetic fragments of the prion protein for the diagnosis of bovine spongiform encephalopathy

Seven peptides matching fragments of the prion protein and containing from 17 to 31 amino acid residues were synthesized to obtain antibodies for diagnostics of bovine spongiform encephalopathy. Rabbits were immunized with either free peptides or peptide-protein conjugates to result in sera with a high level of antipeptide antibodies. Immunohistochemical assay revealed sera against four free peptides and a protein-peptide conjugate, which effectively bind to the pathogenic isoform of the prion protein in brain tissue preparations from cattle afflicted with bovine spongiform encephalopathy and do not interact with normal brain preparations. The resulting antipeptide sera can be used in developing a diagnostic kit for bovine spongiform encephalopathy.     

An enzyme-linked immunosorbent assay (ELISA) for measurement of heterophile antibody

An enzyme-linked immunosorbent assay (ELISA) test has been developed for measurement of heterophile antibody. The microtiter test utilizes a bovine erythrocyte monolayer as antigen and anti-human IgM antiserum conjugated with horseradish peroxidase to measure the degree of binding of the heterophile antibody in the test serum with the erythrocytes. A single serum dilution yields quantitative results when read in a spectrophotometer. The ELISA test showed a sensitivity comparable with the immune adherence hemagglutination assay (IAHA) and other heterophile tests, good reproducibility, and high specificity.     

Two different adenylyl cyclases in brain distinguished by monoclonal antibodies

Four monoclonal antibodies, raised against the 115-kDa adenylyl cyclase from bovine brain [Pfeuffer, E. et al. (1985) EMBO J. 4, 3675-3679] have been selected and designated BBC-1 to BBC-4. BBC-1 and BBC-3 are highly specific for the 115-kDa enzyme from bovine brain. The two other antibodies, BBC-2 and BBC-4, recognize an additional 150-kDa adenylyl cyclase in bovine brain, but also in brain tissue from other species. In membranes from lung and myocardium (bovine and rabbit) only the 150-kDa species is detected by the crossreacting antibodies BBC-2 and BBC-4. The two adenylyl cyclases from brain can be separated by calmodulin-Sepharose: only the enzyme of 115 kDa but not that of 150 kDa was retained by the affinity resin and could be stimulated by Ca2+/calmodulin. The data obtained with these antibodies of defined specificity provide for the first time direct evidence for the presence of two distinct adenylyl cyclase species in brain tissue.     

Differentiation of lymphoid cells. A selective anti-mitogenic component of normal serum which inhibits the induction of immunoglobulin production.

Rabbit lymph node cell populations cultured in vitro in the presence of fetal calf serum are induced to produce immunoglobulin M-secreting cells. The induction of such immunoglobulin production, measured by the capacity of the cell population to secrete immunoglobulins, was inhibited when cells were cultured with sera from a variety of species despite the presence of fetal calf serum. The addition of such inhibitory serum 36 hours after initiation of the cell culture or thereafter was without effect on the extent of induction of immunoglobulin production. On the other hand, the presence of inhibitory serum in culture during only the first 24 hours yielded the same inhibition as when serum was present throughout the 72-hour culture period. Inhibitory sera also suppressed the incorporation of thymidine into DNA. The induction of immunoglobulin production and the incorporation of thymidine into DNA were essentially equally inhibited by the same range of serum concentrations. Unlike conventional inhibitors of DNA synthesis, the inhibitory sera exhibited selective specificity with regard to the kind of cells that could be affected. Thus, such sera inhibited the DNA synthesis of lymph node cells cultured in the presence of fetal calf serum but did not inhibit concanavalin A-stimulated DNA synthesis of such cultured cells and, similarly, serum did not inhibit DNA synthesis of thymus cells cultured in the presence of fetal calf serum. The sera of all species examined were inhibitory except for fetal sera. As judged from a quantitative assay, bovine and porcine serum contained the highest titer of inhibitor, whereas sera from human, rat, mouse, and rabbit were clustered in a group exhibiting less inhibitor. Ascites fluid and lymph node extracellular fluids contained less inhibitor than found in the serum of the same animal and lysates of washed lymph node cells were devoid of inhibitor. Although fetal bovine serum and newborn bovine serum did not contain the inhibitor, it was detectable within 24 hours of parturition. The inhibitor is of relatively large apparent molecular weight (about 300,000) and has been purified about 70-fold.