Effective production of mouse monoclonal antibodies against influenza virus by repeated intrasplenic immunization

Two immunization techniques that enable production of mouse monoclonal antibodies were evaluated in terms of small quantities of antigen. Various amounts of purified influenza A virus particles were applied either for in vitro sensitization in cultured splenocytes or for intrasplenic immunization, followed by hybridization of the immunized cells with mouse myeloma cells. Hybridomas producing specific antibodies for influenza viral proteins were detected by enzyme-linked immunosorbent assay when more than 50 micrograms of antigens was used for the in vitro immunization method, and at least 5 micrograms was necessary for a single intrasplenic immunization. On the other hand, as little as 60 ng of antigen administered in two intrasplenic injections was sufficient to produce specific hybridomas. Two out of six randomly selected monoclonal antibodies obtained using the repeated intrasplenic immunization method were IgG and the other four were IgM. Immunoprecipitation analyses revealed that the recognized antigens involved a viral inner protein (nucleocapsid protein), as well as an envelope glycoprotein (hemagglutinin). We conclude that immunization by two direct injections of antigen into the spleen is the most effective method for sensitization with nanogram quantities of insoluble antigen such as influenza viruses.     

Generation of hybridomas producing human monoclonal antibodies against human cytomegalovirus

In vitro stimulation of human lymphocytes were studied in connection with cell fusion. When splenic lymphocytes were stimulated with human cytomegalovirus (CMV), they produced IgG but not IgM antibody against CMV. The stimulation with 50 ng/ml of CMV antigen induced the maximum antibody response, and higher concentrations of CMV antigen decreased antibody response and increased nonspecific IgG production. Human splenic lymphocytes were stimulated for 6 days with CMV antigen (50 ng/ml) and/or B-cell growth factor (BCGF), and then fused with mouse myeloma cells. Stimulation with a combination of antigen and BCGF were able to generate CMV-specific hybridomas synergistically. Two of these hybridomas were cloned by limiting dilution. The human monoclonal antibodies produced by them, C1 and C23, bound to CMV but not to other herpesviruses. C23 neutralized virus infectivity C1 did not at all. This method for generation of hybridomas producing human monoclonal antibodies against a predefined antigen may be applicable to a variety of viral antigens.     

Primary anti-trinitrophenyl memory cells investigated by plaque-forming cells and ELISA.

Primary anti-trinitrophenyl antibody production was investigated from spleen cells of mice immunized with trinitrophenylated-keyhole limpet hemocyanin, using the plaque-forming cell method and ELISA. Cells taken 5 days after antigen injection do not produce IgE, but do produce IgM and IgG1 anti-trinitrophenyl antibodies as demonstrated by plaque-forming cells. Substantial increase of IgM, IgG1, and IgE antibody production was seen from cells taken 7 days after immunization, followed by a rapid decline. By ELISA it was seen that cells taken 3 days after immunization already produce small amounts of anti-trinitrophenyl antibodies. Presence of antigen from the start of the cultures did not increase antibody production from cells taken 3 days after immunization, but potentiated antibody secretions from cells taken 5 days or later after immunization. This potentiation was interpreted as recruitment of antibody-forming cells from early memory B cells. The presence of IL-4 from the start of the cultures had no appreciable effect. Cell sorting with specific antibody-coated magnetic beads showed that plaque-forming cells from nonsorted cells, membrane IgE+ or membrane IgE- cells secreted similar amounts of anti-trinitrophenyl IgG1 and IgE antibodies. No difference in anti-trinitrophenyl IgM, IgG1, or IgE production was found in controls; cells sorted negatively or positively for CD23. The data show that memory B cells can be demonstrated already on Day 5 after immunization, and their antigen-induced antibody secretion is IL-4 dependent.     

Characterization of Mono- and Polyclonal Antibodies Against Highly Purified Choline Acetyltransferase: A Monoclonal Antibody Shows Reactivity in Human Brain

Choline acetyltransferase (ChAT) from porcine brain was purified by immunoaffinity chromatography, and the highly purified enzyme was subsequently used for immunization of mice and rabbits. After fusion of mouse spleen cells, 32 cultures producing monoclonal antibodies directed against ChAT were detected by an enzyme-linked immunosorbent assay (ELISA) with immunoaffinity-purified ChAT. Of these original 32, the most active 11 cultures were cloned and used for ascites production. The 11 clones generated monoclonal antibodies of the immunoglobulin (Ig) M class (three), the IgG1 subclass (seven), and the IgG2b subclass (one). The isoelectric points of the antibodies of the IgG class were different in each case. The monoclonal antibodies exhibited different binding characteristics in the above ELISA and on western blots. Two monoclonal antibodies demonstrated excellent immunohistological results with neurons of rat brain and spinal cord. One of them reacted well immunohistochemically with neurons of human brain and also recognized partially purified human placenta ChAT in the ELISA.     

Production of antigen-specific human monoclonal antibodies from in vitro-primed human splenocytes

We have developed culture conditions for human lymphocytes that support primary in vitro immune responses to protein Ag of either human or nonhuman origin. We now show that these primed B cells can be efficiently immortalized by fusion with a heterohybrid fusion partner to generate human, Ag-specific IgM or IgG antibody-producing heterohybridomas at a rate of 17 to 50 hybrids/10(6) lymphocytes fused. Approximately 50% of the Ig-secreting clones were stable with respect to Ig secretion. Levels of secretion attained with terminal cultures ranged from less than 1 to 100 micrograms/ml. Fusions of cells between 2 and 5 days after initiation of in vitro exposure to Ag produced more Ag-reactive and Ag-specific antibodies than fusions at 1 day or fusions performed after 5 days. Ag-reactive hybrids could be isolated at frequencies of 3 to 10%, depending on antigenicity of the immunogen. Foreign proteins, horse spleen ferritin, and a murine monoclonal Ig, induced higher percentages of Ag-reactive mAb than immunization with the human-derived ferritin. Ag-reactive IgG mAb were produced at relatively high frequency, depending on immunization conditions and the nature of the Ag. The strategy for identification of the best hybrids included early elimination of unstable hybridomas and of hybridomas producing broadly cross-reactive antibody, followed by evaluation of units of Ag reactivity/micrograms Ig. Ferritin-specific mAb selected according to these criteria showed immunocytochemical reactivity with ferritin-containing tissues and apparent affinities in the range of 10(7) to 10(8)/mol.     

小鼠抗人胰岛素受体单克隆抗体的制备、纯化及鉴定

 M11D杂交瘤细胞株是由人胎盘细胞膜纯化所得胰岛素受体免疫BALB/C小鼠后,取其脾细胞与同系小鼠骨髓瘤细胞株NS-1细胞融合所得。该杂交瘤细胞分泌的抗体经ELISA及放射免疫沉淀法证实为胰岛素受体特异的单克隆抗体。该抗体经Protein A-Sepharose亲和层析分离、纯化,SDS-聚丙烯酰胺梯度凝胶电泳鉴定得分子量分别为53000及23000的两条区带,免疫双扩证明为IgGl。该抗体特异地沉淀125Ⅰ-人胎盘细胞膜胰岛素受体,沉淀经SDS-聚丙烯酰胺凝胶电泳后放射自显影得分子量为135000的特异显影带,与胰岛素受体α亚基分子量相同,说明M11D为抗胰岛素受体α亚基的单克隆抗体。     

The in vitro antibody response to cell surface antigens. I. The xenogeneic response to human leukemia cells.

An in vitro spleen fragment culture system has been developed for the production and analysis of xenogeneic antibody responses to cell surface antigens. Depending on the methods of immunization and in vitro stimulation employed, mouse spleen fragments can produce antibody of both IgG and IgM classes directed against human cell surface antigens for more than 30 days in culture. A saturation binding analysis of the antibody products indicates that their range of specificities was more restricted than that of serum antibody. Approximately 5% of the in vitro antibody products raised against a homogeneous population of human leukemia cells could distinguish between the antigens present on the leukemia cells and those present on normal human lymphocytes. Methods previously employed to influence the range of serum antibodies expressed against complex immunogens, such as suppression of certain responses by passive administration of antibody at the time of immunization, were tested in the in vitro spleen culture system and resulted in successful modulation of the antibody response patterns observed.     

Inhibition of experimental autoimmune thyroiditis in mice by anti-I-A antibodies

Experimental autoimmune thyroiditis is induced in mice by immunization with thyroglobulin emulsified in Freund's complete adjuvant. The disease is characterized both by thyroid infiltration with mononuclear cells and by circulating thyroglobulin antibodies. The magnitude of the thyroid infiltration and the titer of thyroglobulin antibodies are controlled by genes in the I-A subregion of the major histocompatibility complex (H-2). We investigated the in vivo effect of monoclonal anti-Ia antibodies on experimental autoimmune thyroiditis in susceptible mice. Antibodies were given around the time of immunization, later after immunization, and to mice with established disease. Monoclonal antibody produced by the hybridoma line 10-3.6 (anti-I-Ak, s, u, v, z, f) completely prevented both production of thyroglobulin antibodies and thyroid infiltrates, when given shortly before or at the time of antigen administration. This effect was dose-dependent and this monoclonal antibody decreased the severity of the disease when given after the antigen challenge but did not fully suppress established thyroiditis. The same antibody markedly decreased the number of B lymphocytes in the spleen and decreased the thyroglobulin-induced spleen cell proliferation when either given in vivo or added in vitro to cell cultures. Antibodies produced by the hybridoma line 11.2.12 (anti-I-Ak) did not show an inhibitory effect on the disease. These experiments suggest that in this model of murine thyroiditis anti-Ia antibodies act on antigen-presenting cells. Furthermore, only one monoclonal antibody, anti-Ia, suppressed the immune response to thyroglobulin, suggesting a possible role for the isotype and specificity of anti-Ia antibody.     

A modified hybridoma technique for production of monoclonal antibodies having desired isotypes

In the present study, we describe a modified hybridoma technique for production of monoclonal antibodies (mAbs) having a desired isotype. Mice were immunized with the antigen of interest. After having reached a high antibody titer, cells expressing IgM or IgG molecules were isolated from spleen cells of the immunized mice using a Magnetic Cell Sorting System. The isolated cells were fused with myeloma cells using the conventional fusion protocol. With the isolated IgM⁺ spleen cells, more than 75% (85 ± 7%; means ± SD) were IgM producing cells and a large number of IgM mAbs specific to the protein of interest were obtained. With the isolated IgG⁺ spleen cells, 41 ± 40% of the generated hybridomas produced IgG antibody and no IgM producing hybridoma was generated. A large number of IgG mAbs specific to the protein of interest could be produced. The results indicate that the generated hybridomas produce corresponding antibody isotypes as expressed on the surface of their starting cells. The technique that we have developed will be very useful for production of desired mAbs having a specific isotype.     

Xenoantisera against a murine T cell tumor product cross-react with immunoglobulin Fab determinants

Some murine monoclonal T lymphoma cells express a surface component that reacts with chicken antisera produced against the Fab fragment of normal mouse IgG. In the present study, we use a solid phase immunoadsorbent consisting of affinity-purified chicken anti-Fab coupled to Sepharose to isolate a product produced by the in vitro T cell line, WEHI-7.1. The affinity-purified T cell surface molecule (IgT) migrated on SDS-PAGE as a single band of approximately 65,000 daltons. The object of these studies was to produce xenoantisera against the purified T cell product cross-reactive with Ig determinants and to characterize the antisera. Rabbits immunized with this purified molecule produced antibodies that reacted with Fab fragments of polyclonal mouse IgG and with the myeloma proteins MOPC-104E and MOPC-41, as detected by enzyme-linked immunosorbent assay (ELISA). This binding was eliminated by adsorption of the antisera with normal polyclonal IgG; however, adsorption with fetuin did not significantly affect the reactivity of the antisera. Radioimmune precipitation assays revealed that the rabbit anti-IgT bound to normal murine spleen and thymus cells; this reactivity was abrogated by adsorption with insolubilized polyclonal IgG. Competition radioimmunoassays demonstrated that detergent extracts of the thymus and the spleen contained material that inhibited the precipitation of MOPC-41; nonlymphoid cells lacked such material. The rabbit anti-IgT serum blocked the binding of antigen by normal T cells; adsorption of the antiserum with polyclonal IgG-Sepharose abrogated this blocking capacity. A solid phase immunoadsorbent prepared from the IgG fraction of the rabbit anti-IgT isolated a single component from formic acid-solubilized mouse thymus. This molecule had an approximate mass of 65,000 to 70,000 daltons. The anti-IgT serum isolated surface IgM and IgD from lactoperoxidase-catalyzed radioiodinated B cells. The anti-IgT serum detected IgM and IgG in mouse serum with the use of immunoelectrophoresis. The anti-IgT immunoadsorbent isolated several components from normal mouse serum, that, when analyzed by SDS-PAGE under reducing conditions, revealed bands corresponding to mu-, gamma-, and light chains as well as components that migrated between mu- and gamma-chains, and another component with an approximate mass of 45,000 daltons. Our results with antibodies to a purified T cell product indicate that a surface component of normal T cells and certain monoclonal T cell tumor lines is serologically related to the Fab fragment of serum Ig and is implicated in the binding of antigen.     

Protection against Campylobacter jejuni infection in suckling mice by anti-flagellar antibody

We obtained two monoclonal antibodies of IgM class and IgA class of immunoglobulin prepared from mouse spleen cells immunized with crude flagellar preparation, and a polyclonal antibody raised against purified flagellin monomer of Campylobacter jejuni in a rabbit. The specificity of the reaction of these antibodies for flagellar filament was confirmed by Western blotting and by immunoelectron microscopy. These antibodies caused agglutination of the bacteria and inhibited the motility of the bacteria. When a strain of C. jejuni was treated with IgM class monoclonal antibody before being inoculated into suckling mice, it reduced colonization of the intestinal tract by this bacteria. Inhibition of the colonization by IgA class monoclonal antibody was less effective than that of IgM class, and the polyclonal antibody consisting mostly of IgG class immunoglobulin was without effect.     

Antibody-mediated modulation of the immune response

We have studied the ability of monoclonal IgM and IgG antibodies to enhance or suppress immune responses and attempted to dissect the underlying mechanisms. Both IgM and IgG1 antibodies increased the rate of clearance of antigen from the circulation. Monoclonal IgM antibody to SRBC was found to specifically increase antibody responses, enhancement being insensitive to low doses of irradiation (150 R). IgM antibody specifically depressed the delayed hypersensitivity response to SRBC in vivo. Following administration of IgM in vivo, in vitro responses to SRBC were also enhanced. This in vitro enhancement appeared to depend on both T cells and B cells. In contrast, monoclonal IgG1 antibody to SRBC specifically depressed antibody responses in vivo. Such depressed antibody responses were also seen in vitro following IgG1 in vivo and did not appear to be due to the induction of suppressor T cells.     

Monoclonal antibodies to human prolyl 4-hydroxylase

Monoclonal antibodies against human prolyl 4-hydroxylase (EC 1.14.11.2), an intracellular enzyme of collagen biosynthesis, were produced by fusing spleen cells from BALB/c mice hyperimmunized with human prolyl 4-hydroxylase and mouse myeloma cells (P3/NS 1/1-AG 4-1). Hybridomas from 14 different primary microtiter-plate well cultures produced antibodies to human prolyl 4-hydroxylase; six of them with the highest antibody titer were cloned and antibodies produced by one clone from each of the six lines were further characterized. All of the six cloned hybrids produced antibodies of the IgG class as detected by immunodiffusion. The enzyme antigen used in the present study was a tetramer composed of two pairs of different subunit proteins, alpha and beta. Only one clone which produced antibodies to the alpha subunit was obtained, the other five antibodies being directed against the beta subunit. All the antibodies reacted with the tetramer form of the enzyme. Species cross-reactivity of the antibodies was tested using cultured human, mouse and chick fibroblasts and purified prolyl 4-hydroxylase from chick and mouse sources. None of the antibodies cross-reacted with chick or mouse fibroblasts, as determined by immunofluorescence, whereas one antibody reacted with purified chick and mouse prolyl 4-hydroxylase when examined by the western blotting technique. This antibody caused a strong inhibition of human prolyl 4-hydroxylase activity, but the other five antibodies had negligible inhibitory effect on the activity of the enzyme.     

Chicken developmental antigens: analysis of erythroid populations with monoclonal antibodies

Fusions were performed between the mouse PAI myeloma cell line and spleen cells from Balb/c mice immunized with intact erythrocytes from 1-day Cornell K-strain White Leghorn chickens. Following single cell cloning, four hybridoma clones were found to secrete erythroid specific monoclonal antibodies. Based on its pattern of reactivity, the antibody (IgG2a, kappa) secreted by clone 10C6 detects a specific avian oncodevelopmental antigen associated with the hematopoietic system: chicken fetal antigen (CFA). Two other clones, designated as 3F12 and 4C2, produced antibodies (IgM, kappa) that recognize another avian developmental antigen: chicken adult antigen (CAA). A fourth clone, 9F9, produced an antibody (IgM, kappa) that reacts with all peripheral erythrocytes from both Japanese quail and chicken regardless of age. Clone 10C6 antibody apparently detects an erythrocyte specific (ES) determinant of CFA associated with determinant #8 while antibodies of clones 3F12 and 4C2 recognize a chicken specific determinant of CAA. Analysis by complement mediated microcytotoxicity indicated that the epitopes detected by 10C6 vs 3F12 and 4C2 antibodies were expressed on erythrocytes in a reciprocal fashion during development. Furthermore, strain variations in the incidence of erythrocytes carrying these epitopes were observed. The usefulness of these monoclonal antibodies for the study of erythroid populations is discussed.     

IgG antibodies to asialoGM1 are more sensitive than IgM antibodies to kill in vivo natural killer cells and prematured cytotoxic T lymphocytes of mouse spleen

IgG and IgM antibodies, which were isolated from the anti-asialoGM1 (GA1) serum, had different effects against natural killer (NK) and prematured cytotoxic T cells (CTLs) in in vivo administration and in in vitro treatment. In in vitro treatments, the IgM antibody killed NK cells of nude mouse spleen in the presence of complement (C') 12 times more potently than the IgG antibody did, and either antibody with C' killed pre-CTL. In in vivo administrations, only the IgG antibody was effective in diminishing NK activity of the nude mouse spleen cells and in suppressing antigen-specific CTL induction from primed spleen cells by in vitro stimulation with X-irradiated tumor cells. The IgM antibody was not effective at all in either system. The in vivo effect of the IgG on NK activity was blocked by preadministration with silica or carrageenan but not by that with cobra venom factor (CVF). These results indicate that in vivo administration of anti-GA1 antiserum leads to macrophage-mediated depletion of CTL precursors as well as NK cells.     

Anti-schistosome monoclonal antibodies of different isotypes--correlation with cytotoxicity

Five monoclonal antibodies specific towards Schistosoma mansoni antigens were prepared by fusion of spleen cells of infected and immunized mouse with the murine myeloma NS-1 cells. Three of the five antibodies belonged to the IgG1 class, one was an IgM and the fifth one was an IgE. The IgE monoclonal antibody designated 54.10, induced antigen-specific degranulation of rat basophilic cell line, a property which served as the basis for the screening assay. Its biological function was demonstrated by a specific macrophage activation that led to killing of schistosomula; no such killing was obtained with anti-schistosome antibodies of other classes or with IgE of different antigenic specificity. The second monoclonal antibody of biological significance was an IgG1, designated 27.21 which is reactive in the immunofluorescence staining of surface antigens on intact schistosomula. All three monoclonal antibodies that belonged to the IgG1 class were effective in mediating killing of schistosomula by complement, with the highest effect exerted by 27.21. It is thus apparent that the 27.21 monoclonal antibody is directed against a densely distributed surface antigen on the schistosomula membrane which is possibly involved in the protective immunity. Preliminary data showed that immunoprecipitation with the 27.21 antibodies results in the isolation of three major protein bands, of 60 kd, 50 kd, 19 kd, respectively.     

Analyses of Idiotypes on Anti-α-1, 6-Dextran Antibodies in Mice

When mice of strains C57BL/6, C3H/He, and BALB/c were immunized with native dextran B512, only a small amount of IgM antibody was produced, but a substantial amount of anti-dextran antibody of IgG class was produced after immunization with a conjugate of dextran T10 and keyhole limpet hemocyanin regardless of the mouse strain used. Isoelectric focusing (IEF) spectra revealed limited heterogeneity of anti-dextran antibody of IgG class with strict consistency in all individual sera from C57BL/6 mice, even after secondary immunization, whereas antibodies from C3H/He and BALB/c mice showed more heterogeneous IEF spectra with some individual variations. Rabbit anti-idiotypic (Id) antibodies were raised by immunization with a subfraction of anti-dextran antibody of IgG class from C57BL/6 mice, which showed major bands focused at around pH 7.7 upon IEF. It was found by using the anti-Id antibodies that virtually all anti-dextran antibody molecules of both IgG and IgM classes from C57BL/6 mice possessed common Id determinants which can be classified into two specificities, one specific for antibody from C57BL/6 mice and the other cross-reactive with antibodies from BALB/c and C3H/He mice. About 80% of the antibody molecules from BALB/c and less than 20% of those from C3H/He mice were positive for the interstrain cross-reactive Id. Both Id determinants seemed to be closely related to the antigen binding sites, or at least to reside in the vicinity of the antigen binding sites of anti-dextran antibody.     

Serological analysis of early mouse embryo with rat monoclonal antibodies produced against mouse teratocarcinoma cells

Rat-mouse hybridoma antibodies were produced against mouse teratocarcinoma F9 or PCC4 aza1 cells, and four clones were established. Both the F11 (IgM) and F20 (IgG2c) antibodies showed a similar specificity, reacting only with nullipotential teratocarcinoma cells. They were also found to agglutinate sheep red blood cells. Solid-phase enzyme-linked immunofluorescence assay showed that, among the neutral glycolipids studied, they only reacted with the Forssman antigen. P2 antibody (IgG2b) reacted with the undifferentiated-type and embryonal endodermtype teratocarcinoma cells. During the preimplantation stage, this antibody did not stain mouse embryos, but it reacted very weakly with the inner cell mass of blastocysts cultured in vitro. In the 5th-day embryo, the embryonic ectoderm as well as the visceral and parietal endoderm were positive, but the extraembryonic ectoderm was not. Mesoderm of the 7.5th-day embryo also reacted with this antibody. However, P2 antigen was not observed in the 16th-day embryo or in adult tissues. F2 antibody (IgG2a), which was reactive with all of the cultured cell lines tested, showed an immunoreaction with mouse embryos throughout the preimplantation stage. However, in the 7.5th-day embryo, the presence of F2 was limited to the cells forming the parietal endoderm. This antigen was present in some epithelial tissues of the 16th-day embryo and adult mouse. Of these antigens, P2 and F2 are probably novel differentiation antigens of the early mouse embryo. Together with the Forssman antigen, these will be important markers for analyzing cell-surface antigens of mouse teratocarcinoma cells as well as embryos.     

Anti-major histocompatibility complex immunity detected prior to intentional alloimmunization. II. Monoclonal H-2-specific antibodies obtained from an unimmunized C57BL/KaLwRij (H-2b) mouse

A monoclonal 'natural' anti-H-2 IgM antibody produced by a hybridoma cell line OL-3.17 (H-2 m. 209) is described. The OL-3.17 monoclonal antibody was obtained by hybridization of spleen B cells from an unimmunized C57BL/Ka (H-2b) mouse in the serum of which simultaneously an IgM kappa paraprotein of high concentration and a natural H-2-specific antibody of high titer was detected. The monoclonal antibody OL-3.17 reacted strongly with H-2d and H-2s and weakly with H-2k,q,r lymphocytes, thereby detecting a hitherto unknown H-2 public determinant. The target molecules for OL-3.17 cocapped with class-I H-2 antigens, but immunoprecipitation of H-2 antigens was not achieved. This is the first monoclonal H-2-specific antibody obtained from a mouse without intentional immunization and, with high probability, was derived from a B-cell clone which produced natural H-2-specific antibodies detectable in the serum of the original mouse.     

Monoclonal IgM antibody protection in mice against infection with an encapsulated strain of Staphylococcus epidermidis.

Passive protective activities of three different classes of monoclonal antibodies in mice against challenge with strain ATCC 31432 (capsular type I) of Staphylococcus epidermidis were examined. Monoclonal IgM antibody passively protected mice against challenge with the homologous strain, whereas monoclonal IgG1 and IgG2b antibodies did not. The protective activity of IgM was absorbed by the cell surface antigen extracted from the homologous strain but not by the antigen from heterologous strains. Rapid reduction of viable cells took place in the peritoneal cavity of mice immunized with monoclonal IgM as early as 6 h after the challenge with the homologous strain. An enzyme-linked immunosorbent inhibition assay showed there was remarkable inhibition with the homologous cell surface antigen but not with heterologous preparations from other strains. Results suggest that in the mouse the major passive protection against the S. epidermidis strain is provided by the IgM antibody to the cell surface antigen.