Sm抗原纯化及ELISA检测Sm抗体的初步研究

从小牛胸腺中盐析出来的Sm/RNP提取物经DEAE Bio-Ge1 A层析即为Sm抗原。在PAGE检测时Sm抗原在BSA之前有几条染色带。 血清学研究显示,Sm抗原仅与Sm阳性和Sm/RNP阳性血清发生反应,不与RNP阳性血清发生反应。经Sm抗原吸收后,Sm阳性和Sm/RNP阳性血清与Sm抗原的反应降低至接近正常人水平,而上述血清与RNP抗原的反应设有明显降低。结果表明,Sm与RNP已完全分开。ELISA方法检测Sm抗体有10/18的SLE和2/4MCTD是阳性,在14例RA、硬皮病和其它疾病中为阴性。     

Lupus autoantibodies discriminate between the highly homologous Sm polypeptides B/B' and SmN by binding an epitope restricted to B/B'.

The ubiquitous Sm polypeptides B/B' (28 and 29 kD) and the highly homologous tissue-specific Sm N polypeptide (29 kD) share several autoepitopes recognized by systemic lupus erythematosus (SLE) sera. Previous studies on the antigenicity of nuclear antigens recognized by human autoantibodies have not discriminated between ubiquitous and tissue-specific forms. We set out to examine whether a tissue-specific nuclear antigen, Sm N, is autoantigenic in SLE by comparing the immunoreactivity of the most unique sequences in this polypeptide. Synthetic peptides from the two regions of least sequence homology that occur between Sm N and Sm B/B', a dodecamer (amino acid residues 179-190 containing five substitutions) and an undecamer (residues 203-213 containing four substitutions) were coupled to a carrier protein. These conjugates were used to quantify IgG anti-peptide antibodies in sera from patients with SLE. Of 43 sera with anti-Sm specificity, six bound to the B/B' 179-190 peptide but not to the N version. None of 17 anti-Sm-negative SLE sera bound these peptides. The second region of least sequence homology between N and B/B' (203-213) was not antigenic. Our data suggest that a subset of SLE patients with anti-Sm reactivity have IgG autoantibodies capable of discriminating between Sm N and SmB/B' polypeptides by binding a previously unreported SmB/B'-specific autoepitope. The data also indicate that brain and heart-specific anti-Sm antibodies do not exist in SLE sera, suggesting that these tissues do not participate in the induction or maintenance of the autoimmune anti-Sm response.     

Relationship of age and sex to autoantibody expression in MRL-+/+ and MRL-lpr/lpr mice: demonstration of an association between the expression of antibodies to histones, denatured DNA and Sm in MRL-+/+ mice.

Despite the protean nature of the clinical characteristics of systemic lupus erythematosus (SLE), autoantibodies represent an almost constant feature. Furthermore they are common to both human SLE and murine lupus. Nonetheless, the mechanism by which they arise has not been established. Amongst the several processes that have been proposed, evidence has emerged supporting specific antigen drive as a significant mechanism. We have documented the age- and sex-related differences in the prevalence of antibodies to both chromatin-related (histone and DNA) and non-chromatin-related (Sm) antigens in MRL mice. Our finding of an association between antihistone antibodies and anti-denatured DNA antibodies is consistent with chromatin being the putative antigen. Additionally, antibodies to the individual histones H1 and H2B, the most exposed histones in chromatin, were more prevalent than antibodies to the remaining histones (H2A, H3, H4). This, again, supports specific antigen drive as a mechanism for autoantibody production. However, associations were also found between antibodies to histone and DNA and antibodies to Sm. As Sm is a non-chromatin protein antigen, the associations between antibodies to Sm and those to histone and DNA suggest that mechanisms in addition to specific antigen drive are important in autoantibody production.     

Expression of the Epstein-Barr virus nuclear antigen-1 (EBNA-1) in the mouse can elicit the production of anti-dsDNA and anti-Sm antibodies

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder characterized by the production of anti-nuclear antibodies. The etiology of SLE is unknown, although several viruses including the Epstein-Barr virus (EBV) have been implicated. An increase in the frequency of EBV infection has been observed in SLE patients relative to normal individuals. Some patients with SLE develop antibodies that recognize a proline rich epitope in the ribonucleoprotein Sm B/B that is similar to an epitope in EBNA-1, a major nuclear antigen of EBV. In the present study we have cloned the EBNA-1 gene under the control of the CMV promoter in the vector pcDNA3. We now report for the first time that expression of the entire EBNA-1 protein in the mouse can elicit the production of IgG antibodies to Sm and to double-stranded DNA (dsDNA). Our data suggest that the anti-Sm response arises as a consequence of antigenic cross-reactivity by anti-EBNA-1 antibodies. These results support a possible association between EBV infection and SLE.     

Specific combinations of HLA-DR2 and DR3 class II haplotypes contribute graded risk for disease susceptibility and autoantibodies in human SLE

The human leukocyte antigen (HLA) Class II antigen presentation alleles DR and DQ are associated with susceptibility to systemic lupus erythematosus (SLE) and the production of lupus-related autoantibodies. Here, we explore the effect of different combinations of Class II risk haplotypes in a large, multi-center collection of 780 SLE families. Haplotypes bearing the DRB1*1501/DQB1*0602 (DR2) and DRB1*0301/DQB1*0201 (DR3) alleles were present in nearly two-thirds of SLE cases and were significantly associated with disease susceptibility in both family-based and case-control study designs. DR3-containing haplotypes conferred higher risk for disease than DR2, and individual homozygous for DR3 or compound heterozygous for DR3 and DR2 showed the highest risk profile. DR2 haplotypes were also found to be associated with antibodies to the nuclear antigen Sm, and, as previously observed, DR3 genotypes were associated with Ro and La autoantibodies. Interestingly, SLE cases and unaffected family members who were DR2/DR3 compound heterozygotes showed particularly strong risk of developing antibodies to Ro, and were enriched for La and Sm. These data provide convincing evidence that particular combinations of HLA Class II DR2 and DR3 haplotypes are key determinants of autoantibody production and disease susceptibility in human SLE.     

The measurement of antibodies to extractable nuclear antigen.

By the use of a combination of Ouchterlony immunodiffusion and hemagglutination, it was found that 30 of 81 sera (37%) from patients who had systemic lupus erythematosus (SLE) had antibodies to the ribonucleoprotein component of extractable nuclear antigen, and 37 (46%) had antibody to the Sm antigen. Immunodiffusion was more sensitive for detection of anti-ribonucleoprotein, while hemagglutination detected more anti-Sm. Both technics are necessary to define qualitatively the types of antibodies present in SLE sera.     

Lupus-like autoantibody development in rabbits and mice after immunization with EBNA-1 fragments

Epstein-Barr virus has been implicated in the etiology of systemic lupus erythematosus (SLE) through serologic and immunologic studies. A potential mechanism for this influence is through molecular mimicry. The EBV nuclear antigen EBNA-1 contains a region, PPPGRRP, with considerable homology to the initial sequence targeted by antibodies in Sm B' autoimmunity, PPPGMRPP. This study examined whether immunization of rabbits and mice with peptides containing the PPPGRRP sequence from EBNA-1 constructed on a poly-lysine backbone was able to drive the development of autoantibodies against the Smith antigen (Sm) and the related antigenic complex, the U1 nuclear ribonucleoproteins (nRNPs). PPPGRRP immunization, and immunization with an EBNA-1 fragment containing PPPGRRP, led to autoantibodies in both rabbits and mice at high frequency (83% of rabbits and 43% of mice). Five out of six immunized rabbits developed either leucopenia or lymphopenia or both. The fine specificity of antibody binding against the lupus-associated autoantigens Sm B', nRNP A, and nRNP C after immunization with the EBNA-1-derived peptides was very similar to the early antibody binding patterns against these proteins in human SLE. This similarity, as well as the prevalence of autoimmunity after immunization with these peptides, identifies PPPGRRP as a strong candidate for molecular mimicry in SLE etiology.     

Lupus-like autoantibody development in rabbits and mice after immunization with EBNA-1 fragments

Epstein–Barr virus has been implicated in the etiology of systemic lupus erythematosus (SLE) through serologic and immunologic studies. A potential mechanism for this influence is through molecular mimicry. The EBV nuclear antigen EBNA-1 contains a region, PPPGRRP, with considerable homology to the initial sequence targeted by antibodies in Sm B′ autoimmunity, PPPGMRPP. This study examined whether immunization of rabbits and mice with peptides containing the PPPGRRP sequence from EBNA-1 constructed on a poly-lysine backbone was able to drive the development of autoantibodies against the Smith antigen (Sm) and the related antigenic complex, the U1 nuclear ribonucleoproteins (nRNPs). PPPGRRP immunization, and immunization with an EBNA-1 fragment containing PPPGRRP, led to autoantibodies in both rabbits and mice at high frequency (83% of rabbits and 43% of mice). Five out of six immunized rabbits developed either leucopenia or lymphopenia or both. The fine specificity of antibody binding against the lupus-associated autoantigens Sm B′, nRNP A, and nRNP C after immunization with the EBNA-1-derived peptides was very similar to the early antibody binding patterns against these proteins in human SLE. This similarity, as well as the prevalence of autoimmunity after immunization with these peptides, identifies PPPGRRP as a strong candidate for molecular mimicry in SLE etiology.     

Immunization with the Sm nuclear antigen induces anti-Sm antibodies in normal and MRL mice.

The spontaneous occurrence of antibodies against the Sm nuclear antigen is a highly specific marker for the diagnosis of SLE. We have previously shown that anti-Sm can be elicited by immunization of SLE-prone mice with purified Sm antigen. In the present study, this autoantibody was induced in normal mice by a similar immunization protocol. Anti-Sm produced by normal strains was predominantly IgG1, which is similar to the isotype distribution in Sm-immunized MRL mice, but unlike the IgG2a-dominated response seen for spontaneous anti-Sm. Anti-Sm raised by immunization in most strains recognized epitopes not seen by spontaneous human and murine SLE anti-Sm; of the eleven normal strains tested, only C3H and AKR, strains from which MRL was partially derived, responded to these determinants. Further, immunoblot analysis of anti-Sm generated by immunization of MRL and normal mice revealed that the same proteins recognized by spontaneous human and murine anti-Sm were also seen by these sera. This study shows that an autoantibody highly characteristic of SLE can be produced in normal and MRL mice after appropriate immunization, and that the fine specificity of such experimentally induced antibody can be similar to that of spontaneous anti-Sm autoantibodies. The results imply a role for autoimmunization with Sm in the production of anti-Sm.     

Purification of the Sm nuclear autoantigen. Detection and clinical significance of IgM antibody

Sm antigen from rabbit thymus acetone powder was purified using a combination of ammonium sulphate precipitation, DEAE-Sephacel and hydroxyapatite chromatography. This preparation was devoid of previously identified nuclear antigens including ribonucleoprotein (U1-RNP), proliferating cell nuclear antigen (PCNA), Sjögren''s syndrome antigen A (SS-A/Ro), Sjögren''s syndrome antigen B (SS-B/La), Sjögren''s lupus antigen (SL), scleroderma antigen 70 (Scl-70), DNA and histones. The purified material was used in an enzyme linked immunosorbent assay (ELISA) to detect anti-Sm antibody. All sera with precipitating Sm antibody detected by immunodiffusion gave reactions in ELISA greater than 0.40 OD405 and contained predominantly IgG anti-Sm antibody. Of 112 sera which did not have anti-Sm by immunodiffusion there were five which gave reactions greater than 0.40 OD405. Four of these five sera contained only IgM antibody and the fifth contained both IgM and IgG. Of these five, one came from a ''normal'' control who had a positive anti-nuclear antibody (ANA), facial rash and diabetes, two were from patients with systemic lupus erythematosus (SLE) and two were from patients with mixed connective tissue disease (MCTD). These findings demonstrate that there are patients whose anti-Sm response may be restricted to IgM and in some of these patients the clinical presentation may be different from that of classical SLE.     

The anti-Sm immune response in autoimmunity and cell biology

Anti-Sm antibodies are found in greater than 30% of the patients with systemic lupus erythematosus (SLE) and are diagnostic of SLE. The Sm autoantigens are the small nuclear ribonucleoprotein (snRNP) common core proteins. The seven core proteins, B, D1, D2, D3, E, F and G, shared by a majority of the snRNP particles, form a heptamer ring approximately 20 nm in diameter, with the snRNA passing through the center. The Sm epitopes are distributed on the outside surface of the ring. A repeated proline rich motif with homology to an Epstein bar nuclear antigen in the B protein and a gly-arg-gly motif including a symmetrical dimethylarginine post translational modification in the B, D1 and D3 proteins are major Sm epitopes. The anti-Sm response has features typical of an antigen driven immune response. SnRNP proteins share several characteristics with other autoantigens including their assembly into ribonucleoprotein particles, homologies to known viral proteins, presence of post translational modifications, a high abundance and great stability and the presence of repeated motifs. Current work on the snRNP particles is attempting to identify the features that predispose the common core proteins to become autoantigens in vulnerable individuals.     

Longitudinal anti-nuclear antibody (ANA) seroconversion in systemic lupus erythematosus: a prospective study of Swedish cases with recent-onset disease

Serum immunoglobulin (Ig)G anti-nuclear antibodies (ANA) detected by indirect immunofluorescence (IF) microscopy remains a hallmark of systemic lupus erythematosus (SLE). Whether or not IF-ANA status varies over time is controversial. We therefore designed a prospective study with longitudinal follow-up of patients with recent-onset SLE. The study population consisted of 54 recently diagnosed SLE cases, all meeting the 1982 American College of Rheumatology (ACR) and/or the 2012 Systemic Lupus International Collaborating Clinics (SLICC) criteria. Clinical follow-up data, including disease activity, organ damage and sera, were collected from clinical onset of SLE and onwards, in most cases yearly (0‒96 months). IF-ANA was analysed on human epithelial cells-2 (HEp-2) cells and categorized regarding staining patterns. Using an addressable laser bead assay (FIDIS™ Connective profile), we measured IgG-ANA fine specificities against Ro52/SSA, Ro60/SSA, Sjögren’s syndrome type B antigen (La/SSB), Smith antigen (Sm), Smith antigen/ribonucleoprotein (Sm/RNP), U1 RNP (U1RNP), dsDNA, ribosomal-P protein and histone. At baseline, all patients were judged ANA-positive at an abnormal titre corresponding to the 95th percentile of healthy blood donors, but seven of 54 patients (13%) lost ANA-positivity over time. Homogeneous (AC-1; 46%) and speckled (AC-4 or 5; 31%) were the most frequently observed patterns at inclusion, whereas 7% switched pattern at least once during follow-up. Established associations between ANA fine specificities and clinical data were confirmed. Levels of anti-Sm/RNP, but not of anti-dsDNA, correlated with clinical disease activity [modified SLE disease activity 2000 (mSLEDAI-2K)]. Our data indicate that a considerable proportion of Swedish patients with SLE lose ANA-positivity over time, whereas consistent staining patterns were frequent. The clinical and mechanistic relevance of ANA seroconversion remains uncertain. Further prospective evaluations in larger SLE populations with more diverse ethnicities are warranted.     

Preparative isolation of p67, A, B, B' and D from nRNP/Sm and Sm antigens by reverse-phase chromatography. Use in a polypeptide-specific ELISA for independent quantitation of anti-nRNP and anti-Sm antibodies

The p67 (67 kDa) and A (33 kDa) polypeptides of nRNP/Sm antigen and the B, B' (28 and 29 kda) and D (16 kDa) polypeptides of 'free' Sm antigen were isolated and used in enzyme-linked immunoadsorbent assays (ELISA) for human autoantibodies. ELISA specificity was demonstrated using monoclonal antibodies. The ELISA using HPLC-purified polypeptides was found to be more sensitive than immunoblotting for detecting antibody. 86% of sera with precipitating anti-nRNP antibodies were positive in the ELISA, as were all sera with precipitating anti-Sm antibodies. Patients with rheumatoid arthritis (RA), Sj?grens syndrome (SS) and undifferentiated connective tissue disease (UCTD) had low levels of anti-p67 with a prevalence 11.6% and 18%, respectively, whilst patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) had high levels and prevalence rates of 55.2% and 80%, respectively. Anti-B or anti-D antibodies were detected at high levels in SLE (prevalence 30%) but were found rarely in UCTD and MCTD (prevalence 7% and 10%) and not at all in RA or SS sera.     

A common idiotype expressed on a murine anti-Sm monoclonal antibody and antibodies in SLE sera.

A rabbit anti-idiotypic antiserum made against a murine monoclonal anti-Sm autoantibody (Y2) was used in a solid-phase radioimmunoassay to investigate idiotypic cross-reactivity among anti-Sm antibodies present in sera from patients with systemic lupus erythematosus. Sera from 25 of 51 SLE patients (49%) containing anti-Sm antibodies were positive for this Y2 idiotype compared to only one of 22 normal human sera. Nine of 28 SLE patients (32%) whose sera were anti-Sm negative were also positive for the Y2 idiotype in low titre. Binding was not due to rheumatoid factor-like activity but was specific for the Y2 determinant and could be eliminated by absorption with Y2 monoclonal antibodies. The anti-idiotypic antibody blocked the ability of 12 of 25 anti-Sm positive lupus sera to bind Sm. Conversely, Sm antigen inhibited the binding of anti-idiotypic antibody in nine of 12 lupus sera.     

Characterization of the Autoimmune Antigenic Determinant for Ribonucleoprotein (RNP) Antibody

Small nuclear ribonucleoprotein complexes are antigens in various autoimmune diseases. The serological pattern of high titers of circulating antibody to nuclear ribonucleoprotein (RNP) antigen is a diagnostic marker for mixed connective tissue disease (MCTD); whereas antibody to Sm is prevalent in systemic lupus erythematosus (SLE). Both calf thymus and rabbit thymus are commonly used, excellent sources for preparation of the corresponding antigens RNP and Sm in clinical and research laboratories (A. M. Boak et al., accompanying paper). Thus, biochemical and structural characterization of the minimal antigenic determinant in these preparations is important for its use in the laboratory, as well as significant for understanding MCTD, SLE, and other examples of autoimmunity. Purification and biochemical analyses of immunologically active RNP from many different preparations of calf thymus extract has revealed that the majority of antibody in monospecific MCTD patient sera recognizes an antigen composed of the 165 nucleotide RNA, Ul RNA, and five peptides. Calf thymus Ul RNA was found to be identical in sequence to that of man. A sequence of 55 nucleotides within the 165 nucleotide RNA was the minimal RNA fragment found in RNP particles that were still immunologically active. Two of the RNP peptides react with patient sera monospecific for RNP and thus, are presumably the antigenic peptides complexed with the 55 nucleotide RNA sequence.     

Sequential autoantigenic determinants of the small nuclear ribonucleoprotein Sm D shared by human lupus autoantibodies and MRL lpr/lpr antibodies.

Autoantibodies directed against the Sm proteins of the spliceosome complex are found in approximately 25% of systemic lupus erythematosus (SLE) patients sera. To determine which regions of the Sm D polypeptide are involved in the lupus autoimmune response, binding to overlapping octapeptides of Sm D has been evaluated with sera from nine Sm D-positive patients, six patients with other autoimmune serology, and five normal human sera. Lupus patient sera which are Sm precipitin-positive bind various combinations of five regions of the peptide. The major antigenic region, Epitope 5 (REAVA(GR)10GGPRR), is bound by eight of nine Sm precipitin-positive sera tested. This region of Sm D shows significant sequence homology with Epstein-Barr nuclear antigen-1. To determine the fine specificity of the murine Sm response, four unique Sm D MoAbs derived from MRL lpr/lpr mice and three adult anti-Sm-positive MRL lpr/lpr mouse sera have been analysed. Two of these monoclonals, KSm 4 and Y12, as well as the MRL lpr/lpr sera tested, show binding with Epitope 5. Another of these monoclonals, KSm 2, binds octapeptides 84-91, DVEPKVKSKKREAVAG, which corresponds to Epitope 4 of this study. Antibodies from SLE patients with autoimmune serology other than anti-Sm bind the carboxyl glycine-arginine repeat (GR)10 peptides of Sm D. However, none of the antibodies tested from patients who do not have lupus and who have different autoimmune serology binds any of the Sm D octapeptides. Normal controls did not significantly bind any of the Sm D octapeptides. These results describe two major regions of shared antigenicity of Sm D between sera from SLE patients and MRL lpr/lpr mice, thereby establishing a basis for the cross-species similarity of autoimmunity to the Sm autoantigen in SLE.     

Expression of IgM and IgG autoantibodies in pediatric and adult systemic lupus erythematosus

To compare patterns of autoantibody responses in pediatric and adult patients with systemic lupus erythematosus (SLE). IgG and IgM antibodies to single-stranded DNA (ssDNA), Sm, and the 70-kDa protein component of the RNP antigen (70-kDa RNP) were measured in 29 pediatric and 36 adult patients by enzyme-linked immunosorbent assays. Antibodies of either isotype to ssDNA, Sm, and 70-kDa RNP were present in 64, 58, and 79% of pediatric patients, respectively, comparable to prevalences of these autoantibodies in the adult SLE patients. Pediatric SLE patients were more likely than adult patients to have IgM anti-Sm antibodies (41.4% vs 13.9%, P = 0.02) and tended to more commonly express IgM anti-70-kDa RNP and IgM anti-ssDNA antibodies. The prominence of IgM autoantibody responses among pediatric SLE patients was shown by multiple logistic regression analysis to be related to total IgM concentrations and not related to age or duration of disease. Sequential serum samples available from several pediatric patients revealed the maintenance of similar patterns of isotype responses over time in approximately one-half of patients. In those patients whose responses changed over time, the variations in isotype expression were consistent with maturation of antibody responses of each specificity. While these results demonstrate similarities in autoimmune reactivities between pediatric and adult SLE patients, the serologic study of pediatric patients may provide an opportunity to more readily investigate the evolution of autoantibody responses.     

Somatic mutations to arginine residues affect the binding of human monoclonal antibodies to DNA, histones, SmD and Ro antigen

Autoantibodies to a wide variety of antigens are associated with systemic lupus erythematosus (SLE). Antibodies to double-stranded DNA (anti-dsDNA) are thought to be particularly closely related to tissue damage and disease activity in SLE. Autoantibodies to histones, Sm and Ro are found in patients with SLE, but their role in pathogenesis is unclear. Using a transient expression system, we previously showed that particular sequence motifs in CDRs of light chains derived from the human Vlambda gene 2a2 are very important in determining their ability to form a DNA-binding site, when paired with the heavy chain of the human monoclonal anti-dsDNA antibody B3. These motifs are often sites of somatic mutation and/or contain arginine residues. In the experiments reported in this paper, the same expression system was used to show that these CDR motifs also affect binding to histones, Ro antigen and Sm antigen, but that binding to different antigens is affected in diverse ways by particular changes in the sequence of the CDRs. The heavy chain also plays a role in binding to these antigens. Pairing of the same range of 11 2a2 derived light chains with the heavy chain of a different anti-DNA antibody, 33.H11, gave reduced ability to bind DNA in comparison with the results obtained using the B3 heavy chain. Computer-generated models of the three-dimensional structures of these heavy/light chain combinations were used to define the positions occupied by the important sequence motifs at the binding sites of these antibodies, and to explain the different effects exerted by arginine residues at different positions in the light chains.     

Analysis of variable region genes encoding anti-Sm and anti-cardiolipin antibodies from a systemic lupus erythematosus patient.

We have analysed the heavy and light chain variable region genes of two monoclonal antibodies, specific for the Sm antigen (RSP1; IgG kappa) and for cardiolipin (RSP4; IgM lambda), derived from a patient with active systemic lupus erythematosus (SLE). We have established that the variable region genes of the RSP1 autoantibody are somatic mutants of two germ line genes from the VH4 and V kappa 1 gene families. RSP4 antibody uses gene segments closely related to a VH3 gene member and to a V lambda 1 gene. The presence and distribution of the somatic mutations on both monoclonal autoantibodies are compatible with an antigen-driven immune process. These data suggest that in SLE a common antigenic stimulus may govern the autoantibody response against a wide spectrum of unrelated antigens, including native DNA, cardiolipin or Sm antigens, and provide further evidence that disease-associated autoantibodies are generated through antigen-selected somatic mutations.     

Application of immunoperoxidase staining in the cytodiagnosis of herpes simplex keratitis

In corneal scraping smears from 13 patients with clinically suspected herpes simplex keratitis (HSK), HSK is demonstrated by means of peroxidase-antiperoxidase (PAP) technique with antisera to herpes simplex virus (HSV) in Papanicolaou-destained cellular samples. The staining for HSV antigen was present in seven cases of corneal scraping smears with superficial keratitis (dendritic and geographic ulcers) while six cases of stromal keratitis (deep keratitis) failed to show HSV antigen except in one case. Specific antigen for HSV was predominantly present in the cytoplasm rather than in the nucleus. Immunoreactions were negative with HSV antisera in patients with other infections and in those in a normal control group. Using the PAP technique, detection of HSV antigen in corneal scraping smears was of great value in the diagnosis of HSK, especially in cases of superficial keratitis.