Serological evidence for variation in the incidence of herpesvirus infections in different species of apes.

Sera from captive lowland gorillas, chimpanzees, orangutans, and gibbons were screened by enzyme-linked immunosorbent assay (ELISA) for antibody to herpesviruses serologically related to human herpes simplex virus types 1 and 2 (HSV-1, HSV-2), a baboon virus (SA8), and a macaque herpesvirus (B virus). The incidence of herpesvirus antibodies varied considerably among the different species, gorillas having the highest incidence of seropositivity (65.4%) and orangutans the lowest. The virus specificity of positive sera was further analyzed by examining the kinetics of virus neutralization, competition of reactivity in ELISAs, and immunoblotting against HSV-1, HSV-2, SA8, and B virus antigens. Using these assays, the majority of positive gorilla sera (49 of 53, 92%) were determined to react in a manner identical to human HSV-1 immune sera. The remaining four positive gorilla sera reacted as HSV-2-positive sera. In contrast, the majority of positive chimpanzee sera (5 of 7, 71%) reacted as HSV-2 immune rather than HSV-1 immune. All positive sera from gibbon apes reacted as HSV-1 positive. No orangutan sera were identified which gave positive reactions by ELISAs to any of the four primate herpesviruses tested. Although four orangutan sera gave equivocal results against HSV-1 antigen, further analysis by immunoblotting could not confirm any specific reactivity with any of the primate herpesvirus antigens. Varied reactivity among individual animals with both SA8 and B virus proteins was observed, but none of the seropositive primates detected appeared to be infected with either of these simian viruses. Three gorilla sera had antigen recognition patterns slightly different from those of HSV-2-positive human and chimpanzee sera and another HSV-2-positive gorilla serum, raising the possibility that these animals harbor an indigenous virus related to HSV-2.     

Isolation of the major herpes simplex virus type 1 (HSV-1)-specific glycoprotein by hydroxylapatite chromatography and its use in enzyme-linked immunosorbent assay for titration of human HSV-1-specific antibodies.

A 131,000 molecular weight herpes simplex virus type 1 (HSV-1) glycoprotein designated antigen number 6 (Ag-6) was previously shown to possess almost exclusively HSV-1-specific antigenic sites. Fused rocket and crossed immunoelectrophoresis of fractions obtained from hydroxylapatite chromatography of crude HSV-1 antigen (Triton X-100-solubilized, infected tissue culture cells) showed that a subfraction of Ag-6 could be separated from the other HSV antigens. Enzyme-linked immunosorbent assay with the isolated Ag-6 showed that sera from rabbits infected with HSV-1 and HSV-1 human antisera contained antibodies to Ag-6, whereas sera from HSV-2-infected rabbits and sera from patients with primary HSV-2 infections did not react with Ag-6. Enzyme-linked immunosorbent assay of 852 human sera for antibodies to HSV type-common glycoproteins, Ag-6, and HSV 2-specific antigens showed that 139 sera which reacted negatively with HSV type-common glycoproteins also did not react with Ag-6 with HSV-2 specific antigens. The 713 sera reacting positively to HSV type-common antigens either reacted with Ag-6 (328 sera) or with HSV-2-specific antigens (31 sera) or both (354 sera). This means that Ag-6 might be useful in large-scale human serology for the detection of past infection with HSV-1, irrespective of whether or not past infection with HSV-2 has occurred.     

Antibody responses in humans to individual proteins of herpes simplex viruses.

Sera from 231 women were used to examine their frequency of precipitation of various herpes simplex virus type 1 and 2 (HSV-1 and HSV-2) proteins and to determine if there was a rank order of immune responsiveness of humans to these HSV antigens. Radiolabeled viral proteins were reacted with serum and immune complexes isolated with staphylococcal protein A. Individual antigens were resolved by polyacrylamide gel electrophoresis and visualized by fluorography. As a group, these sera precipitated 31 HSV-1 and 27 HSV-2 proteins. HSV-1 polypeptides with molecular weights of 133,000, 99,000, and 82,000, as well as HSV-2 polypeptides with molecular weights of 131,000 and 101,000, were precipitated by essentially all sera that contained antibodies to HSV-1 and HSV-2. When attempts were made to order the viral proteins by constructing precipitation profiles ranking the antigens in patterns according to their frequency of precipitation, it was observed that the antigens were generally not ordered. Demographic analysis of the sera suggested that the differences in the number of proteins precipitated were associated with differences in age, education, age at first marriage, and income, which collectively may reflect the frequency of exposure to the virus.     

The immune response to herpes simplex virus: comparison of the specificity and relative titers of serum antibodies directed against viral polypeptides following primary herpes simplex virus type 1 infections

Employing an immunoblotting technique, the polypeptide specificity and relative titers of anti-HSV IgG reactive with denaturation-resistant epitopes on HSV proteins were determined in patients experiencing primary HSV-1 infections at various anatomical sites. Early sera from previously seronegative patients with primary HSV-1 infections were found to have comparatively low levels of antibody directed against the major viral glycoprotein antigens (gB, gC, and gD) relative to titers present in sera of individuals with long-standing, latent orofacial HSV-1 infections. Patients with primary infections did however have high titers of antibody directed against a series of low molecular weight HSV polypeptide antigens. These antigens were found to be antigenically related to a structural component of virion nucleocapsids. At later times postinfection, titers of antibodies directed against other viral polypeptides including the major glycoproteins increased to levels more closely approximating those observed in latently infected individuals. These results indicate that the anti-HSV IgG detected by immunoblot analysis which appears earliest following primary infection is not directed against the known major infected cell or virion glycoprotein surface antigens but rather against an internal capsid protein of HSV.     

Analysis of the HSV-2 early AG-4 antigen

Summary Genital herpes simplex virus type 2 (HSV-2) infections can be distinguished from present or past HSV-1 infections by an AG-4 antigen complement fixation assay. The assay which utilizes a 4 hour HSV-2 infected cell extract prepared at a multiplicity of infection (MOI) of 1.0 PFU/cell, appears to consist of several viral proteins. Studies using monoclonal antibodies, polyclonal rabbit hyperimmune serum, HSV-1×HSV-2 intertypic recombinant viruses and polyacrylamide gel electrophoresis suggest that ICP8 may be one of the major antigens involved in the complement fixing reaction. It is probable that the success of the assay is not due to a true type specificity but rather a threshold phenomenon in which HSV-2 extracts contain more early viral antigens (including ICP8) and sera from HSV-2 patients contain more complement fixing antibody to these antigens.With 4 Figures     

Herpes simplex virus type-selective enzyme-linked immunosorbent assay with Helix pomatia lectin-purified antigens

Helix pomatia lectin-purified antigens with specific reactivity to herpes simplex virus type 1 (HSV-1) and HSV-2 antibodies in human sera were used in an enzyme-linked immunosorbent assay. The type specificity of the antigens was assessed by double immunodiffusion precipitation in gel against rabbit HSV-1 and HSV-2 hyperimmune sera, and by enzyme-linked immunosorbent assay with human reference sera containing antibodies to either type of HSV. Fifty-two sera from patients with documented infection with either HSV-1 or HSV-2 were assayed for HSV type-specific immunoglobulin G antibodies. The reactivity of the sera against lectin-purified antigens correlated completely with the results of virus typing. We conclude that HSV type-specific immunoglobulin G antibodies can easily be measured by enzyme-linked immunosorbent assay with the use of Helix pomatia lectin-purified HSV-1 and HSV-2 antigens.     

Immunochemical studies of polioviruses: identification of immunoreactive virus capsid polypeptides

Investigation of the immunological reactions with individual poliovirus capsid polypeptides of antisera and monoclonal antibodies raised against poliovirus type 3 antigens are described. Virus polypeptides were separated by electrophoresis, transferred electrophoretically to nitrocellulose sheets and treated with antibody preparations. Antibody binding specifically to the virus polypeptides was then detected by application of 125I-labelled anti-immunoglobulin followed by autoradiography. The technique readily enabled the identification of the polypeptides recognized by the antibody. Antibodies present in polyclonal, type-specific neutralizing sera to poliovirus type 3 bound to the two largest capsid polypeptides (VP1 and VP2) of the homotypic poliovirus, and also to the VP1 of poliovirus type 1 and type 2. There was no obvious difference between the antibody binding patterns obtained with neutralizing and non-neutralizing antisera or between C-specific and D-specific antisera. VP1 appeared to be the immunodominant virus polypeptide. Among monoclonal antibodies specific for the C antigen of poliovirus type 3, a proportion reacted homotypically with the VP1 of poliovirus type 3. Other monoclonal antibodies of C antigen or D antigen specificity, or which reacted both with D and C antigens, some of which had potent virus-neutralizing activity, failed to give demonstrable binding reactions. The non-correlation of neutralization and immunoblot reactivity suggests that sequence determinants alone do not mediate virus neutralization which may depend on antigenic determinants specified by complex conformational arrangements of the virus capsid proteins.     

Identification of antigenic determinants by polyclonal and hybridoma antibodies induced during the course of infection by vaccinia virus

In order to extend the understanding of determinants involved in the humoral response in the infected host, mice were subjected to an immunization regimen using both active and uv-killed vaccinia virus. The spectrum of antibody specificity in hyperimmune sera was followed by Western blotting. Comparable studies involving Western blotting and immunofluorescence were conducted with a panel of monoclonal antibodies derived from hybridomas selected from similarly immunized animals. Hyperimmune sera contained circulating antibodies primarily against three polypeptides of 28K, 35K, and 62K. These antigens were shown to be located both at the surface and within the virion. The repertoire of monoclonal antibodies included some that reacted with the 28K and 35K antigens and others that recognized a 32K core complex component or a nonvirion cell surface component, corresponding to the viral hemagglutinin. Within the panel of monoclonal antibodies was a large group which reacted with a 32K antigen found in the IHD-J virion but absent from the IHD-W strain. This finding correlates with the absence of a 32K polypeptide from the IHD-W particle. Overall, the current findings reveal the absence of any particular correlation between the incidence of polyclonal antibodies in the circulation of the immune host and the frequency of selected hybridomas against vaccinia antigens. Application of this type of immunological analysis should provide useful data concerning the detection and mapping of the antigens and their epitopes which are significant for humoral immunity.     

Immunochemical studies of microsomal membranes of rat preneoplastic and neoplastic hepatocytes

Heterogenous rabbit antisera were prepared against microsomal proteins of hyperplastic hepatic nodules (HPN) induced by chemicals, and were utilized to assess the antigenic differences of microsomal polypeptides within a normal liver, HPN, and hepatocellular carcinomas (HCC), utilizing immunodetection of antigens separated electrophoretically and transferred to nitrocellulose. Although most antigens were common to all microsomes, differences (increase or decrease) were noted in some polypeptides not only between the normal liver and HPN, but also between HPN and HCC. On the other hand, monoclonal antibodies against epoxide hydrolase (EH), which was initially found as the PN antigen, reacted to a single polypeptide with a molecular weight of 49,000 in all the microsomes. These results suggested that there is little molecular modification of EH during hepatic carcinogenesis.     

Preparation and evaluation of anti-species and antiviral peroxidase conjugates

Several antispecies peroxidase conjugates from human and rabbit immunoglobulins G (IgG) and a conjugate from IgG fraction of hyperimmune rabbit serum against the membranes of herpes simplex virus type 1 (HSV-1) infected cells have been prepared and characterized. The conjugates when tested in enzyme immunoassay for detection of antiherpetic antibodies in human and hyperimmune rabbit sera, as well as for detection of HSV-1 antigen in infected Vero cells appeared active and highly specific. Comparison with the peroxidase conjugates from antiherpetic IgGs prepared by means of ion- exchange and affinity chromatography has shown similar activities.     

Characterization of the Epstein-Barr virus-induced early polypeptide complex p50/58 EA-D using rabbit antisera, a monoclonal antibody, and human antibodies

A polypeptide complex (p52) belonging to the D-subspecificity of the EBV-induced early antigens (EA-D) was purified from chemically induced P3HR-1 cells. Rabbit antisera raised against the isolated polypeptides reacted with components of EA-D as could be shown by indirect immunofluorescence and immunoperoxidase staining of IdU-induced EA positive Raji cells, ELISA, and immunoblotting. In one-dimensional immunoblots the rabbit antisera detected a predominant polypeptide complex of 52 kDa. Two-dimensional immunoblots prepared with proteins from IdU-induced Raji cells showed that the rabbit sera detect three series of polypeptides of 52 kDa (pl 8.5-6.2), 55-58 kDa (pl 6.2-4.5), and 48-50 kDa (pl 6.0-4.5). These three groups of polypeptides could also be identified by 50 high titered anti-EA-D positive human sera and a specific monoclonal antibody (R3) as being the main components of EA-D in Raji and B95-8 cells. All polypeptides of the p50/58 complex showed DNA binding properties either by themselves or by an interaction with other proteins. When TPA or IdU-induced Raji cells were labeled with 2Pi, two phosphorylated polypeptides pp50 and pp58 could be immunoprecipitated with the rabbit sera and a high anti-EA titered human serum. The time course of the synthesis of polypeptides associated with the EA-D complex was studied by 2-D immunoblots: EA polypeptides of 52 kDa appeared as early as 6 hr after the addition of IdU to Raji cells in culture, polypeptides of 55-58 and 48-50 kDa after 18 and 25 hr, respectively. The coordinated appearance of these groups of polypeptides and their similar size and reactivity with human sera and rabbit antisera produced against the isolated p52 as well as with a monoclonal antibody (R3) suggested that most of these polypeptides are derived from post-translational modifications of one or a few initially synthesized polypeptides, possibly p52. Phosphorylation seems to be at least one possibility of post-translational modification.     

Antigenic specificity of recombinant polypeptides, containing fragments of hepatitis E virus proteins]

Antigenic specificity of recombinant polypeptides HE40 and HE60 containing fragments of gene ORF2 and ORF3 protein products of hepatitis E, strain Burma, produced in E. coli cells, is analyzed. Blood sera from patients with acute hepatitis from an endemic region in Uzbekistan were tested for IgG to recombinant antigens by solid-phase enzyme immunoassay with a protein fragment coded by PRF3 gene, a synthetic peptide previously characterized in a commercial test system, as the positive control. 93% sera reacting with recombinant polypeptide HE60 and 32% reacting with HE40 reacted with the synthetic peptide. No antibodies to the studied polypeptides were detected in the sera of Moscow patients with hepatitis A, B, or C confirmed by laboratory findings. Antigenic specificity of recombinant polypeptide HE60 was confirmed by competitive enzyme immunoassay with the same peptide as the competitive antigen. Test system based on recombinant polypeptides HE40 and HE60 was used for deciphering the etiological structure of acute viral hepatitis which occurred in a hepatitis E endemic region of Uzbekistan in 1993.     

Circulating immune complexes in experimental syphilis: identification of treponemal antigens and specific antibodies to treponemal antigens in isolated complexes.

As a prelude to characterization of the host and treponemal antigens present in purified immune complexes from the sera of rabbits with disseminated syphilis, autoradiographic and immunoenzymatic analyses of solubilized extracts of Treponema pallidum, Treponema phagedenis biotype Reiter, and Treponema refringens were performed on electroblots of polypeptides first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electroblots of purified immune complexes were developed with the same panel of antisera so that protein profiles could be compared. Eight treponemal antigens were consistently present in isolated complexes; four of these cross-reacted with antisera prepared against avirulent treponemes. The average molecular weights of these antigens were 87,000, 76,000, 66,000, and 45,000. Antibodies dissociated from isolated immune complexes, when used for the development of T. pallidum electroblots, reacted with four antigens of comparable molecular weight. Antibodies to those polypeptides were also present in the sera of animals immunized with immune complexes. The demonstration of treponemal antigens in purified immune complexes convincingly argues that their occurrence in experimental syphilis is not merely due to tissue destruction and responses to endogenous host antigens.     

Characterization of BK virus-specific antibodies in human sera by Western immunoblotting: use of a zwitterionic detergent for restoring the antibody-binding capacity of electroblotted proteins

After initial problems related to denaturation of antigenic epitopes, we developed a Western immunoblotting method for the characterization of antibodies reacting with BK virus (BKV) structural polypeptides. When a zwitterionic detergent, Empigen BB, was added to the running buffer during electroblotting, the antibody-binding capacity of electrophoretically separated BKV polypeptides was partially restored. Antibodies reacting with different BKV antigens were detected and visualized by biotinylated anti-species-specific antibodies, peroxidase-conjugated streptavidine, and diaminobenzidine staining. Human sera containing anti-BKV antibodies reacted with VP1, but a serum containing antinuclear antibodies also reacted with VP4, -5 and -6 (histones). Serum from a rabbit inoculated with purified BKV reacted with VP1, and also with VP4, indicating that BKV inoculation may imply production of antibodies against histones.     

Heterogeneity of thyroid autoantigens identified by immunoblotting

Autoimmune thyroid disease in man is commonly associated with autoantibodies against thyroglobulin, microsomes, and the TSH receptor, and the character and specificity of these antithyroid antibodies have been extensively utilized in investigating these conditions. In the present study we have asked whether other thyroid-related antigens exist, against which autoantibodies may be directed. A crude thyroid extract was separated by polyacrylamide gel electrophoresis followed by immunoblotting with serum obtained from patients with Graves' disease or Hashimoto's thyroiditis. Antibodies in sera from patients with Graves' disease and Hashimoto's thyroiditis reacted with many antigenic determinants in immunoblots of the thyroid membrane preparation (2000g supernatant). These determinants were disease specific in that sera from normals and patients with Addison's disease and rheumatoid arthritis did not react, but there was no difference between the patterns of reactivity with Graves' disease or Hashimoto's thyroiditis sera. Thyroglobulin produced two predominant bands of reactivity at 320 and 200 kDa, whereas purified microsomal antigen produced a triplet of bands around 105 kDa, when these preparations were reacted with appropriate autoimmune sera. Nonetheless, some sera produced additional bands with the microsomal antigen blots, indicating that some of the antigens which were detected using crude thyroid membrane remained in the microsome preparation to produce multiple antibody binding reactivities. We were unable to inhibit any of the antibody binding with TSH. Purification of individual thyroid antigens on the basis of their molecular weights should standardize current antibody assays and permit more detailed evaluation of the cellular immune responses in Graves' disease and Hashimoto's thyroiditis.     

Identification of a Plasmodium chabaudi antigen present in the membrane of ring stage infected erythrocytes

A novel antigen of asexual blood stages of the rodent malaria parasite Plasmodium chabaudi, was detected by means of a modified indirect immunofluorescence assay (IFA), using glutaraldehyde fixed and air dried monolayers of P. chabaudi infected erythrocytes. P. chabaudi hyperimmune sera gave a distinct surface immunofluorescence of erythrocytes infected with early stages of the parasite. Fixation and drying of the erythrocytes was necessary for the antigenic activity to be exposed. The antigens were species specific as P. chabaudi hyperimmune serum only stained P. chabaudi but not P. yoelii or P. falciparum infected erythrocytes. The antigenic activity involved in the IFA was resistant to trypsin, phospholipases and neuraminidase but not to pronase, suggesting that the antigens were polypeptides. The surface immunofluorescence was inhibited by an extract of parasitized erythrocytes, but not by similar extracts of normal erythrocytes. The inhibitory antigens were soluble and heat stable (100 degrees C, 5 min). For identification and characterization of the antigens, antibodies were isolated by acid elution from monolayers of infected erythrocytes and monoclonal antibodies were produced. Probing in immunoblotting of extracts of parasitized erythrocytes separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis with the eluted antibodies, showed that they reacted consistently with a polypeptide of Mr 105 000 (Pch105). The Pch105 antigen shares many characteristics with Pf155, a P. falciparum antigen considered as a candidate for a vaccine against that parasite.     

Analysis of polypeptide composition and antigenic components of Rickettsia tsutsugamushi by polyacrylamide gel electrophoresis and immunoblotting.

Polyacrylamide gel electrophoresis of lysates of purified Rickettsia tsutsugamushi revealed as many as 30 polypeptide bands, including major bands corresponding to molecular sizes of 70, 60, 54 to 56, and 46 to 47 kilodaltons. Compared with the polypeptide composition of the rickettsiae of Gilliam, Karp, and Kato strains and a newly isolated Shimokoshi strain, the major polypeptide in the Kato strain (54-56K) and in the Karp strain (46-47K) migrated a little faster and slower, respectively, than the corresponding polypeptides in the other strains. The largest major polypeptide (54-56K) was digestible by the treatment of intact rickettsiae with trypsin and variable in content in separate preparations, suggesting that the polypeptide exists on the rickettsial surface and is easily degraded during the handling of these microorganisms. Several surface polypeptides of rickettsiae, including the 54-56K and 46-47K polypeptides, were detected by radioiodination of intact rickettsiae followed by polyacrylamide gel electrophoresis of the lysate; however, the 70K and 60K polypeptides were not labeled. Immunoblotting experiments with hyperimmune sera prepared in guinea pigs against each strain demonstrated that the 70K, 54-56K, and 46-47K polypeptides showed antigenic activities. The 54-56K polypeptide appeared to be strain specific, whereas the 70K and 46-47K polypeptides cross-reacted with the heterologous antisera.     

Biological activities of monoclonal antibodies to Mycoplasma pneumoniae membrane glycolipids.

A purified preparation of membranes was obtained by using a unique method of treating Mycoplasma pneumoniae with the ATPase inhibitor, diethylstilbestrol. This method was shown to yield highly purified membranes with little or no cytoplasmic contamination. These membranes were used to immunize mice for subsequent productions of monoclonal antibodies (MAbs). Hybridoma culture supernatants were screened by enzyme-linked immunosorbent assay with whole-cell M. pneumoniae and lipid extract antigens. Four stable MAbs were obtained and characterized. MAb CP3-46F5 reacted with a protein of a molecular weight of approximately 52,000 as determined by Western blot (immunoblot). MAbs CP3-50C2, CP3-53C5, and CP3-53C8 did not react with any antigens on Western blots but did bind to at least 10 distinct glycolipid bands as determined by orcinol staining on thin-layer chromatograms of M. pneumoniae lipid extracts. The MAbs did not react with similarly prepared lipid extracts from Mycoplasma genitalium, Mycoplasma neurolyticum, and Mycoplasma gallisepticum. These MAbs did not inhibit M. pneumoniae metabolism or attachment to WiDr cell cultures. The anti-glycolipid MAbs recognize determinants specific to M. pneumoniae, unlike polyclonal hyperimmune sera against M. pneumoniae, which cross-react with lipid extracts of M. genitalium.     

Characterization of three different human T cell membrane antigens,two being present on T lymphocyte subpopulations

Heteroantisera were raised in rabbits to thymocytes, HSB2 cells, and Sezary cells. Following absorption with Ia-positive leukemia cells, these sera appeared to be specific for different T cell antigens. Both the anti-HSB2 and the anti-Sezary sera reacted with approximately 50% and the antihymocyte serum with 100% of normal peripheral blood T lymphocytes. None of the sera reacted with B cells. The apparent molecular weights of the antigens being derected were determined by immunoprecipitation followed by SDS polyacrylamide gel eletrophoresis. A dimer of 170,000 daltons consisting of two similar 85,000-dalton polypeptide chains was immunoprecipitated by the anti-HSB2 serum whereas single polypeptides of 53,000 and 64,000 daltons were immunoprecipitated by the anti-Sezary and antithymocyte sera, respectively.     

Quantitation of antibodies to IgM, IgD and beta2-microglobulin in antisera to chronic lymphatic leukemia lymphocytes.

Rabbit antisera were prepared to chronic lymphatic leukemia (CLL) lymphocytes and were tested for their reaction with radioiodinated, solubilized, cell surface proteins of normal and CLL lymphocytes. A pooled, hyperimmune antiserum precipitated at least 16 polypeptides from both normal and CLL lymphocytes as shown by sodium-dodecyl sulfate polyacrylamide gel electrophoresis. These polypeptides varied in molecular weight from 11,000 to 180,000. None was prominent or unique to the CLL lymphocytes, although four peptide bands in the CLL cells usually showed more radioactivity than their counterparts in normal cells. Precipitation with antisera of known specificity showed that the cell surface proteins from CLL and normal lymphocytes included HLA antigens and beta2-microglobulin. IgM and IgD were found in preparations of normal cells and in cells of 3 of 5 CLL patients. Of cells from the other 2 patients, one showed only IgD and the other no Ig. An antigen-binding capacity test was employed to quantitate the antibody content of a pooled anti-CLL lymphocyte serum. The antiserum reacted with all Ig classes; however, after absorption with light chains and F(ab')2 fragments, the serum retained activity only for IgM and IgD. The absorbed antiserum bound 45 microgram IgM, 1.5 microgram IgD and 4.5 microgram beta2-microglobulin per milliliter. These data indicate that rabbit anti-CLL lymphocyte sera fail to detect a qualitatively unique tumor-specific polypeptide on the surfaces of CLL cells. However, such antisera contain antibodies to many cell surface proteins including IgM, IgD, HLA antigens and beta2-microglobulin.