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.     

Isolation and partial characterization of the heterophile antigen of infectious mononucleosis from bovine erythrocytes

The heterophile antigen (Paul-Bunnell antigen, PBA) of infectious mononucleosis was isolated by extraction of an aqueous suspension of bovine erythrocyte stromata with chloroform-methanol (2:1). The upper aqueous layer contained gangliosides, PBA, and a high-molecular-weight glycoprotein. PBA and gangliosides were separated from the high-molecular-weight glycoprotein by extraction of lyophilized upper layer with chloroform-methanol solvents. Separation of PBA from gangliosides was carried out by chromatography on DEAE-cellulose with chloroform-methanol solvents. PBA appeared to be a minor glycoprotein component of the erythrocyte membrane and had both hydrophobic and hydrophilic properties. It was soluble in either organic or aqueous solvents. On SDS-polyacrylamide gel electrophoresis, it migrated as a single component that stained for protein with Coomassie blue, for carbohydrate with periodic acid-Schiff reagent, and for lipid with oil red 0; it had an apparent molecular weight of 26,000. It was composed of 62% protein with major amino acids: glutamic acid, proline, glycine, isoleucine, leucine, and threonine (158, 116, 98, 90, 85, and 82 residues per 1,000 residues, respectively). Carbohydrate content was 9.2% with major sugar constituents: sialic acid, galactosamine, and galactose. Serologic activity of PBA was destroyed by pronase but not by trypsin.     

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.     

Use of heterophilic mononucleosis antigen in the latex diagnostic test. III. Diagnostic field studies

The aim of this study was to evaluate usefulness of latex coated with a preparation of heterophilic mononucleosis antigen for the detection of antibodies present during a course of infectious mononucleosis. Studies were performed in district serological laboratories. Sanitary-Epidemiological Stations in conditions of routine diagnostics. Studies were performed on 656 blood serum samples collected from individuals with a clinical suspicion of infectious mononucleosis. Latex and Paul-Bunnell-Davidsohn (PBD) tests were run in parallel. Out of 154 blood serum samples which contained antibodies detected by PBD test in diagnostically significant titer of 1:56 or higher, 151 were reactive in latex test in the dilution 1:5 or higher, while in the remaining three cases agglutination appeared with undiluted serum only. Out of 268 serum samples tested in latex test 167 reacted in 1:5 titer - diagnostically accepted as a significant or in higher titers. The sensitivity of latex test amounted to 98.1% and specificity was 96.9% as compared to reference Paul-Bunnell-Davidsohn test. The results of our study suggest the possibility of replacing Paul-Bunnell-Davidsohn test by latex test performed as a method providing the possibility of determination of the presence of heterohilic antibodies in blood serum samples and the follow up of their dynamics.     

Paul-Bunnell antigen and a possible mechanism of formation of heterophile antibodies in patients with infectious mononucleosis

Sera of patients with infectious mononucleosis contain heterophile anti-Paul- Bunnell (PB) antibodies to erythrocytes of numerous mammalian species. Evidence is presented that the corresponding antigen of bovine erythrocytes is not, as previously described, a single molecule, but a series of glycoproteins with glycans terminated with N-glycolylneuraminic acid (Neu5Gc). The latter compound should be an important part of the PB epitope because, in agreement with the results of others, we found that desialylation of the PB antigen abolishes almost completely its activity. We examined three different preparations of GM3 ganglioside for their capacity to bind anti-PB and found that only GM3 from horse erythrocytes containing Neu5Gc exhibited a low although ELISA measurable PB activity. The other two GM3 preparations, from bovine milk and dog erythrocytes, containing N-acetylneuraminic acid (Neu5Ac) bound little if any anti-PB antibodies. This finding confirms a previous report that human erythrocyte Neu5Ac containing sialoglycoprotein with similar O-linked glycans as the PB-antigen of bovine erythrocytes exhibits only very low PB activity (Patarca & Fletcher, 1995, Crit Rev Oncogen., 6: 305). In conclusion, we present a hypothesis that anti-PB antibodies in patients with infectious mononucleosis are formed against infection-induced cell membrane glycoconjugates containing highly immunogenic Neu5Gc.     

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.     

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.     

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.     

Reactivity of Paul-Bunnell type heterophile antibody in sera from infectious mononucleosis patients with the surface of lymphoid cells carrying Epstein-Barr virus genomes.

The possible presence of Paul-Bunnell (PB) antigen on Epstein-Barr virus (EBV)-transformed lymphocytes were investigated. Of 23 EBV genome-positive lymphoblastoid cell lines tested all but one absorbed PB type antibody from the serum of an infectious mononucleosis patient. The one EBV-negative B cell line tested did not absorb the heterophile antibody. PB antibody, purified by an immunoadsorbent procedure using beef cell antigen, reacted with the EBV producer P3HR-1 cell line in an indirect membrane immunofluorescence test and was shown to be IgM antibody. Titers of heterophile agglutinin and reactivity with the cell surface were reduced to the same degree by absorption with beef cell antigen but not with guinea pig kidney antigen. PB antibody was distinct from IgM antibody against the EBV-determined membrane antigen, since the latter was not absorbed by beef cell antigen. PB antibody was also reactive with other EBV-positive B cell lines (QIMR-WIL, NC-37, and Raji) which were free of surface IgM. No reaction occurred with the nonproducer P3HR-1 line, a null cell line, and two T cell lines. The results suggest the presence of PB antigen on most EBV-transformed B lymphocytes, and its appearance in each of the transformed lymphocytes of patients with acute infectious mononucleosis.     

Inhibition by alpha-D-galactose of heterophilic antibodies against the antigens of the interstitial connective tissue of the myocardium

The reaction of heterophile antibodies with interstitial connective tissue cells of bovine myocardium was inhibited with alpha-D-galactose. D-glucose, D-mannose, L-fucose, N-acetylgalactosamine and other monosaccharides demonstrated the lack of inhibitory activity. Disaccharide melibiose and trisaccharide raffinose containing terminal alpha-galactose also had an inhibitory effect on the reaction of sera with bovine myocardium. The results show that heterophile antibodies to heterophile bovine antigen revealed in rheumatic patients' sera differ from antibodies to I and i antigens and T-antibodies, whose specificity is associated with terminal beta-galactose. They also differ from other heterophile antibodies described earlier.     

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.     

Hanganutziu-Deicher antigen and antibody in pathologic sera and tissues.

Heterophile, Hanganutziu-Deicher (H-D) antigen was studied in pathologic sera by means of inhibition of agglutination of bovine erythrocytes by H-D antibodies. H-D antigen was demonstrated in 38% of random cancer sera, 25% of lymphoma or leukemia sera, 25% of leprosy sera, 8% of infectious mononucleosis sera, 6% of rheumatoid arthritis sera, and 27% of synovial fluids of rheumatoid arthritis patients. None of 134 normal human sera gave positive results. Some of the inhibition-positive cancer sera formed precipitation lines with H-D antibody-containing sera. Over 50% of various extracts of cancer tissues as well as spleens of lymphoma or leukemia patients were shown to contain H-D antigen by means of the inhibition test.     

The reactivity of canine red cells with heterophile agglutinins having anti-A activity*

Heterophile A-reactive agglutinins, obtained from the seeds of Dolichos biflorus and from the snails Helix aspersa, Helix pomatia and Cepaea nemoralis, were tested for reactivity with the saliva and red cells of a random series of dogs. Saliva from dogs with the A-like Tr red cell antigen inhibited the agglutination of human A red cells by each of the reagents. However, none of the heterophile agglutinins distinguished Tr+ from Tr— red cells: the Dolichos agglutinin was non-reactive, while all of the snail reagents agglutinated both Tr+ and Tr— cells. It is concluded that the canine red cell bears a series of heterophile receptors which mask reactivity with the canine homologue of the red cell A-antigen, and that the restricted size of the binding sites of the agglutinins limits the informational significance of cross-reactivity obtained with these reagents.     

Blood group H like activity in crude preparations of bovine lung galactan.

In crude preparations of bovine lung galactan blood group H-like activity, which does not occur on bovine red cells, has been detected with heterophile anti-H-like reagents from different origin. These H-like receptors, associated with pneumococcus type XIV cross-reactivity, belong to a glycoprotein fraction and not to the galactan itself.     

Physiological studies on the pneumococcal Forssman antigen: a choline-containing lipoteichoic acid.

The cell concentration and possible biological activities of the pneumococcal Forssman (F) antigen (membrane lipoteichoic acid) were examined in a number of physiological situations. In test tube cultures of pneumococci the concentration of the Forssman antigen per bacterium showed no significant fluctuations within a typical culture cycle. Purified F antigen had no effect on the activation of pneumococci to competence for genetic transformation, DNA mediated genetic transformation or adsorption of the pneumococcal phage Dp-1 to bacteria. Pneumococci grown in the presence of different amino alcohols (ethanolamine, N-monomethylethanolamine, or choline) exhibit differences with regard to both their ability to stimulate heterophile (haemolytic) antibody production in rabbits and in their ability to bind such antibodies. Choline-grown bacteria seem to cross-react with sheep red blood cells better than do the analogue-grown bacteria.     

G1 antigen: a cell-surface immunoprotective 96 kDa glycoprotein from the virulent fish pathogen Enterococcus seriolicida, its purification and characterization

Strains of the fish pathogen Enterococcus seriolicida were identified as agglutinating and non-agglutinating, according to their reaction with anti-serum raised against type strain YT-3 (ATCC49156). The non-agglutinating strains are highly pathogenic in contrast to agglutinating strains. A 96 kDa immunoprotective glycoprotein G1 antigen from non-agglutinating Ent. seriolicida strain SS91-014 (N) was purified and characterized. The purification procedure entailed extraction of antigen by glass bead agitation, 80% (NH4)(2)SO4 precipitation, gel filtration and electroelution. An immunofluorescence microscopy study using monoclonal antibody M3A5 raised against G1 antigen revealed that G1 antigen is present only on the cell surface of non-agglutinating strains. Therefore, the G1 antigen of virulent Ent. seriolicida could be a potential candidate for protective vaccine against enterococcosis in fish.     

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.     

Solubilization and comparative analysis of mammalian erythrocyte membrane glycoproteins

SDS-acrylamide gel electrophoresis reveals the presence of a major glycoprotein in human, ox, horse, swine, and sheep erythrocyte membranes. Their apparent molecular weights differ among the various species. The major glycoproteins and additional minor glycoproteins can be recovered in the aqueous phase after extraction of the membranes with a mixture of CHCl₃-CH₃OH at room temperature. The extracted glycoproteins remain in the supernatant after centrifugation at 100,000 g for 60 min. in Tris-EDTA buffer. These glycoprotein preparations possess high activities for the phaseolus vulgaris phytohemagglutinin receptor, the infectious mononucleosis heterophile antigen, and the myxovirus receptor. Their specific activities and yields differ markedly from species to species.     

Enterobacterial common antigen: isolation from Shigella sonnei, purification and immunochemical characterization.

In the studies presented the effective procedure of isolation and purification of enterobacterial common antigen from Shigella sonnei has been elaborated. The method is based on sonification of bacterial suspension in the presence of lysozyme and EDTA and subsequent extraction of the pellet with boiling water. The crude extract of common antigen was purified by fractionation with ethanol and chromatography on silica gel and Sephadex LH-20. The comparison of several extraction procedures of enterobacterial common antigen from Shigella sonnei proved that the method described above is most effective. The purified enterobacterial common antigen preparation obtained preserved full biological activity: antigenicity (precipitation and activity in enzyme-linked immunosorbent assay), immunogenicity in rabbits, ability to coat erythrocytes (passive hemagglutination) and inhibitory activity in passive hemagglutination. The pure enterobacterial common antigen was identified to 90% as a polymer of N-acetyl-D-mannosaminuronic acid and N-acetyl-D-glucosamine (2:1, molar ratio), O-acetylated and containing 3.2% fatty acids (C16:0 and C18:1, not oleic). It contains 5.3% nitrogen, less than 4% protein, less than 0.5% phosphorus and less than 1.6% neutral sugar; glycerol and RNA were not found in the preparation.     

Usefulness of the latex test for rapid detection of Salmonella O antigen in bacterial culture fluids and clinical specimens. III. Diagnostic field studies]

The aim of this study was to evaluate a latex reagent prepared in our laboratory for a routine diagnosis of Salmonellosis in humans. Liquid cultures in selenite broth (SF) (18-24 hr), previously inoculated with faeces samples of individuals suspected of being infected with Salmonella were subjected to the study. In these cultures, after 15 min. of heating at boiling temperatures, group antigens of Salmonella with an aid of polyvalent latex reagent A-E and monovalent reagents B, C1, C2, D, and E were searched. The results of latex test were compared to the results obtained by routine bacteriological examination. Studies performed in 13 laboratories of Sanitary Epidemiological Stations included 5246 faeces samples. Out of these samples 1835 (35%) reacted with monovalent latex reagent and 1897 (36.2%) samples were positive, for Salmonella by culture technique and belonged to 14 genera of group B, C1, D, and E. S. enteritidis was the most frequently isolated and encountered for 98.6% of all isolated strains. Latex test with A-E reagent was positive in 2246 (42.8%) of culture samples in SF medium, of which 1736 were positive by culture and 510 samples were negative for Salmonella in routine bacteriological examination. The samples positive in culture and with A-E latex reagent reacted in 97.2% with one monovalent reagent. Out of bacteriologically negative samples and reacting with A-E latex reagent 28.8% were positive with monovalent latex reagents. In summary, we can conclude that latex test used in a survey studies can be an usefull test in addition to routine bacteriological examination, since after 18-24 hr it allows with high credibility of 95% to confirm or exclude Salmonella in a tested sample. Such a procedure due to a shortening of routine diagnostic course brings significant savings. Moreover, latex test makes possible rapid detection of mixed infections with Salmonella of different serological groups. The use of extremely carefully, properly prepared selenite broth constitutes a basic condition for agreement between results of latex test and routine bacteriological investigation.