The premenstrual symptoms screening tool (PSST) for clinicians

A variety of instruments have been used in an attempt to operationalize DSM-IV criteria for premenstrual dysphoric disorder (PMDD) and to understand clinically significant premenstrual syndrome (PMS). The objectives of this research were to devise a simple user friendly screening tool to identify women who suffer from severe PMS/PMDD and who are likely to benefit from treatment. Five hundred and nineteen women, between the ages of 18 and 55 yrs, who were seen at a primary care facility completed "The Premenstrual Symptoms Screening tool" (PSST). The PSST reflects and 'translates' categorical DSM-IV criteria into a rating scale with degrees of severity. The results are in line with reported prevalence rates from several recent large prospective studies. We believe that the PSST applies a necessary degree of measure of severity and impact of premenstrual symptoms, establishes quickly if women qualify for PMDD, and is less time consuming and more practical than two cycles of prospective charting. This fast simple tool is an effective screening tool and an important starting point for further assessment.     

A biotin-labelled antigen radioimmunoassay (BILA) for antibodies to membrane antigens useful for monoclonal antibody screening

Viable cells or protein extracts were labelled with N-hydroxysuccinimidobiotin and used as target antigens in a biotin-labelled antigen radioimmunoassay (BILA). The binding of the biotinylated antigens to capture antibodies coated on the bottoms of 96-well plastic plates were measured using 125I-labelled streptavidin as the detection step. The assay circumvents some of the problems associated with solid-phase RIA and permits screening of antibodies to undefined protein antigens present in very small amounts in complex protein solutions.     

The Pregnancy Depression Scale (PDS): a screening tool for depression in pregnancy

Depression in pregnancy can be underdiagnosed as a consequence of the symptoms being misattributed to “normal pregnancy.” There are currently no validated clinician-rated scales that assess for depression specifically during pregnancy. We sought to develop a brief, convenient screening tool to identify depression in pregnant women in the community setting. Prospective mood data using the 28-item Hamilton Depression Rating Scale (HDRS) were collected monthly in 196 pregnant women with a history of a major depressive disorder. These data were analyzed to delineate those HDRS items associated (elevated) with normal pregnancy vs. those indicative of a pregnant woman meeting diagnostic criteria for a major depressive episode. Endorsement of symptoms on seven items of the HDRS were highly predictive of having a major depressive episode during pregnancy. We present a well-validated, brief scale to screen pregnant women for clinical depression. Whether this study will generalize to women who do not have a history of major depression remains to be studied.     

Human leucocyte antigen (HLA) expression of primary trophoblast cells and placental cell lines,determined using single antigen beads to characterize allotype specificities of anti-HLA antibodies

Human trophoblast cells express an unusual repertoire of human leucocyte antigen (HLA) molecules which has been difficult to define. Close homology between and extreme polymorphism at the classical HLA class-I (HLA-I) loci has made it difficult to generate locus-specific monoclonal antibodies (mAbs). The problem of defining an antibody''s reactivity against the thousands of existing HLA-I allotypes has often made it impossible to determine the HLA bound by a mAb in biological samples from a normal outbred population. Here we have used commercially available beads coated with individual HLA-I to characterize experimentally the reactivity of nine mAb against 96 common HLA-I allotypes. In conjunction with donor HLA-I genotyping, we could then define the specific HLA molecules bound by these antibodies in normal individuals. We used this approach to analyse the HLA expression of primary trophoblast cells from normal pregnancies; the choriocarcinoma cells JEG-3 and JAR; and the placental cell lines HTR-8/SVneo, Swan-71 and TEV-1. We confirm that primary villous trophoblast cells are HLA null whereas extravillous trophoblast cells express HLA-C, HLA-G and HLA-E, but not HLA-A, HLA-B or HLA-DR molecules in normal pregnancy. Tumour-derived JEG-3 and JAR cells reflect extravillous and villous trophoblast HLA phenotypes, respectively, but the HLA repertoire of the in vitro derived placental cell lines is not representative of either in vivo trophoblast phenotype. This study raises questions regarding the validity of using the placental cell lines that are currently available as model systems for immunological interactions between fetal trophoblast and maternal leucocytes bearing receptors for HLA molecules.     

The significance of free and immune-complexed hydatid-specific antigen(s) as an immunodiagnostic tool for human hydatidosis.

Micro-enzyme-linked immunosorbent assay (Micro-ELISA) systems were developed and evaluated for the detection of circulating (free or immune-complexed) hydatid antigens in the sera of patients with hydatidosis, by employing monospecific antibodies to hydatid-specific antigens of 8-kDa and 116-kDa. Fifteen (75%) of 20 sera from patients with hydatidosis had both 8-kDa and 116-kDa antigens freely circulating in their sera while three and two samples, respectively, had only 8-kDa or 116-kDa antigen. All the surgically confirmed cases of hydatidosis had detectable levels of both 8-kDa and 116-kDa circulating immune complexes in glycine HCl-treated sera. However, none of the sera from control subjects (patients with cysticercosis, ascariasis, ancylostomiasis, hymenolepiasis, amoebic liver abscess or viral hepatitis) had any detectable level of either type of circulating specific antigen. These results suggest that the demonstration of either 8- or 116-kDa antigen(s) in free or immune-complex form could confirm the diagnosis of hydatidosis.     

The antigen spot test (AST): a highly sensitive assay for the detection of antibodies

A method is described for detection of antibodies by means of nitrocellulose or diazobenzyloxymethyl (DBM) paper on which various antigens have been spotted. The sensitivity of this antigen spot test (AST) is comparable with that of RIA and ELISA. The method requires only nanogram amounts of antigen. Since a variety of antigens can be spotted on a single piece of nitrocellulose or DBM paper, this antigen spot test is especially useful for specificity controls on antibodies.     

The properties of isolated serum and urinary antibodies to a single antigen

Purified antibodies to tetanus toxoid have been separated from serum γG-globulins and from low molecular weight fragments of urinary γ-globulins (Fu fragments).

Isolated Fu antibodies have been shown to be of low molecular weight and appear to be bivalent; they agglutinate antigen-coated red cells, fix complement and contain an average of five disulphide bonds, one of which is more labile than the remaining four. The antibody activity of the molecule is destroyed by reduction of this labile disulphide bond. These results are discussed in the light of current concepts of antibody structure.

     

Comprehensive assessment for serum treatment for single antigen test for detection of HLA antibodies

The single antigen test is widely used in the field of transplantation to determine the specificity of HLA antibodies. It will be beneficial to standardize the procedure of the single antigen test among HLA laboratories. It is not uncommon that single antigen testing on native sera fails to detect antibodies with very high concentrations. It has been shown that cleavage products of activated complement components may mask strongly binding antibodies in single antigen testing. To overcome inhibition by the activated complement products, sera are pretreated with ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), or heat inactivation before single antigen testing. However, no studies have been published to systemically compare the impact of these treatments on single antigen testing. The aim of this study is to understand the different effects these treatments may have on single antigen test results. We found that mean fluorescence intensity (MFI) obtained from sera treated with EDTA and heat inactivation were nearly identical, while DTT treatment was less potent to remove the inhibition. In addition, sera dilution did not further increase MFI of antibodies after EDTA treatment. Our results provide guidance to choose a pretreatment reagent for single antigen testing, and to compare studies obtained from laboratories using different treatments.     

Detection of human antibodies to hepatitis B surface antigen (HBsAg) by an enzyme-immunoassay for HBsAg.

We studied the feasibility of detecting antibody to hepatitis B surface antigen (anti-HBs) by an inhibition assay using the reagents of an enzyme-immunoassay for HBsAg (Hepanostika). Several modifications of the basic assay were investigated. Sensitivity was greatest when the test sample was incubated with a predetermined amount of HBsAg before the usual procedure of HBsAg detection. The presence of anti-HBs in the test sample was shown by a reduction of the solid-phase bound enzyme label. Results were obtained with a dilution series of serum samples containing anti-HBs, the anti-HBs Reference Panel of the American Bureau of Biologics, sera of hepatitis B patients, and sera of two individuals passively immunised with anti-HBs. The enzyme-immunoassay method showed at least the same sensitivity as passive haemagglutination. It was less sensitive than a commercially available radioimmunoassay (Ausab). There are no indications that non-specific reactions occur frequently. This study also revealed that the antigenaemia of acute hepatitis-B patients can be interrupted by a transient seroconversion.     

Significant IgG subclass heterogeneity in HLA-specific antibodies: Implications for pathogenicity,prognosis, and the rejection response

IgG subclasses differ in their ability to fix complement and bind Fc receptors. This study describes a detailed analysis of the distribution of HLA-specific IgG subclasses in order to define how this varies in sensitised waiting-list patients. We found significant variation in the level, presence and combinations of each HLA-specific IgG subclass between and within individuals and this is influenced by the type of sensitising event. Graft failure in particular provokes higher levels of IgG1 (vs transfusion, p = 0.071 and pregnancy, p = 0.042), IgG2 (vs transfusion, p = 0.001 and pregnancy, p = 0.016), and IgG4 (vs transfusion, p = 0.052). Both graft failure and pregnancy tend to stimulate multiple IgG subclass responses against HLA, whereas transfusion stimulated antibodies are dominated by responses limited to IgG1 (p = 0.033) and have a low incidence of IgG4 (p = 0.046). In marked contrast, IgG4 characterised nearly all HLA DQ-specific antibodies stimulated by graft rejection (p = 0.006). Such widely varying IgG subclass heterogeneity is likely to be due to underlying immunological processes dependent on the route of sensitisation. This diversity, which implies functional variation, may help explain why HLA-specific antibodies are an obstacle to transplantation in some circumstances but not others. The subclass association with rejection has potential as a biomarker for chronic rejection.     

Use of an enzyme-linked immunosorbent assay (ELISA) for screening of hybridoma antibodies against cell surface antigens

A micro-ELISA for screening of antibodies from hybridoma cultures against surface antigens of human melanoma is described. The technique employs alkaline phosphatase-conjugated protein A and target cells attached to poly-l-lysine-coated microtiter plates. The micro-ELISA is equally sensitive as the radioimmunoassay. Mild glutaraldehyde treatment of cells did not lead to an appreciable loss of antigen activity. The fixed cells can be stored at 4°C for at least 6 weeks. It is concluded that the ELISA is superior to the radioimmunoassay in the following aspects: (1) exclusion of radioactive hazards, (2) speed of performance, and (3) lower costs.     

The GIX antigen of murine leukemia virus: an analysis with monoclonal antibodies

The G_IX antigen of murine retrovirus gp70 is a marker for virus replication in various cell types, as well as for T-cell differentiation and leukemogenesis in particular mouse strains. The serological definition of G_IX depends on the cytotoxicity of a particular rat antiserum for thymocytes from strain 129 mice. We have found that two rat monoclonal antibodies, elicited by immunization with AKR ecotropic virus or AKR leukemia cells, co-type closely with G_IX in several assay systems. Like G_IX, the epitopes identified by the monoclonal antibodies are amplified on thymocytes during leukemogenesis in AKR mice. These antibodies are cytotoxic for G_IX⁺ leukemia cells and for fibroblasts infected with G_IX⁺ viral serotypes. Though absorbed by G_IX⁺ thymocytes of various strains, the antibodies are not cytotoxic for these cells. We ascribe the latter result to lower representation of antigenic sites on normal thymocytes, and postulate that more than one epitope may participate in classical G_IX-mediated cytotoxicity. The monoclonal antibodies do not react with viral env products synthesized in the presence of the inhibitor of glycosylation, tunicamycin. Reactivity with the antibodies appears to require a stable configurational change in the env precursor protein coinciding with glycosylation, rather than direct participation of carbohydrate in the antigenic site. Thus subsequent enzymatic removal of carbohydrate chains in vitro does not alter reactivity with the antibodies.     

Monoclonal antibodies embedded in their hybridoma cells: an immunodiagnostic concept

Aldehyde fixation of hybridoma cells during active production of monoclonal antibodies (MAbs) resulted in the formation of an insoluble phase because a large number of immunologically active antibody molecules were immobilized on the fixed hybridoma cells. Such MAb preparations specific to large protein molecules and small haptens were used for the development of a homogeneous immunoassay concept.     

The Fas antigen (CD95) on human lymphoid cells: epitope analysis with ten antibodies

The expression of CD95 antigen was examined on adult and cord blood lymphocytes using a highly sensitive immunofluorescence/flow cytometric procedure. CD95 was expressed by the majority of circulating blood T cells in adults, and by a smaller proportion of CD4+ and CD8+ T cells in cord blood. The majority of circulating B cells did not react with seven CD95 antibodies, but three antibodies did stain B cells. In tonsil sections, CD95 was expressed throughout the tissue, but germinal centres showed generally stronger staining than the surrounding follicular mantle and interfollicular areas. This was confirmed by flow cytometry, which showed expression preferentially on B cells with a germinal centre phenotype. Because different antibodies stained different proportions of B cells, CD95 epitopes were examined by inhibition, additive binding and protease susceptibility studies using a panel of ten CD95 antibodies. B cells apparently reacting selectively with CD95 antibodies were sorted and CD95 mRNA was reverse transcribed to cDNA and analyzed, in order to confirm the presence of CD95 in cells which reacted selectively and to explore the possible existence of CD95 isoforms. The major cDNA band was identical in the two populations. Inhibition of N-glycosylation suggested that the epitopes detected differentially could not be accounted for by differential N-glycosylation.     

Human monoclonal antibodies reactive with cell surface antigens on human leukemia cell lines: many antibodies are (auto)antibodies

Primary Epstein Barr Virus (EBV) transformants from peripheral blood mononuclear cells (PBM) established in macrocultures were screened for the secretion of antibodies reactive with cell surface antigens on one or another of two indicator human leukemic cell lines and fused with the HMMA2.11TG/O human fusion partner. Human monoclonal antibodies (HuMAbs) were readily obtained. Relative oligoclonality of the primary EBV macrocultures was documented by the number of antibody secreting hybridomas (1-100%). The method permitted preselection for fusion of transformants producing antibodies of certain specificities and/or class. Fourteen HuMAbs, primarily of the IgM class, have been obtained. Those IgM HuMAbs obtained from patients with active diseases, e.g. Acute Lymphoblastic Leukemia (3 HuMAbs), and HIV infection (4 HuMAbs), were found to have a relatively broad spectrum of reactivity with cell lines of various hematopoietic lineages and normal cells, although several show selective reactivity with T cell lineage tumors or a selected population of cells. HuMAbs from normal donors of both the IgG and IgM class were obtained. The IgM HuMAbs from one volunteer reacted primarily with autologous and allogeneic macrophages (autologous PBM from the other patients were not available) as well as a diverse number of hematopoietic cell lines. From others, the IgG HuMAbs demonstrated a more restricted spectrum of reactivity, while the IgM HuMAbs reacted with both autologous and allogeneic normal cells. Thus, the B cell repertoire contains cells capable of secreting cell surface reactive antibodies and many of these antibodies express characteristics of autoantibodies. Those that did not react with autologous or allogeneic PBM may react with other autoantigens which have been expressed on the malignant cells used as screening targets or may represent true antitumor antibodies.     

Establishment of human cell lines producing anti-D monoclonal antibodies: identification of Rhesus D antigen

Two cell lines producing monoclonal antibodies have been established from peripheral blood of a negative Rhesus blood donor which has been immunized with positive Rhesus red blood cells. Two monoclonal antibodies Co II 8.8 and Co II 7.12 have been selected. Both are IgG1 antibodies, but recognize different epitopes on the Rhesus D antigen, apparently associated with different subunits of the D antigen. Thus the Co II 8.8, like the positive serum, immunoprecipitates an antigen of a relative molecular weight of 33 kDa, while the Co II 7.12 recognizes an antigen of Mr 42 kDa.     

Anti-soluble liver antigen (SLA) antibodies in chronic HCV infection

Hepatitis C infection is associated with autoimmune disorders, such as the production of autoantibodies. Anti-LKM1 and anti-LC1, immunomarkers of type 2 autoimmune hepatitis, have been previously associated with a HCV infection. Anti-Soluble-Liver-Antigen autoantibodies (SLA) are specifically associated with type 1 and type 2 autoimmune hepatitis and more closely related to patients who relapse after steroid therapy. The recent molecular cloning of the soluble liver antigen provides the opportunity to develop more specific tests for the detection of antibodies against it. The aim of this work is to characterize anti-soluble-liver autoantibodies in sera from patients chronically infected by HCV. A recombinant cDNA from activated Jurkat cells coding for the full length tRNP(Ser)Sec/SLA antigen was obtained. ELISA, Western Blot and immunoprecipitation tests were developed and used to search for linear and conformational epitopes recognized by anti-SLA antibodies in sera from patients chronically infected by HCV. Anti-soluble liver antigen antibodies were found in sera from 10.4% of HCV-infected patients. The prevalence was significantly increased to 27% when anti-LKM1 was also present. Most anti-SLA reactivity was directed against conformational epitopes on the antigen. The means titers by ELISA were lower than those obtained in type 2 AIH. The result of autoantibody isotyping showed a subclass restriction to IgG1 and also IgG4. This study shows the presence of anti-SLA antibodies in approximately 10% of HCV infected patients. The prevalence of SLA autoantibodies in HCV infected patients increases when LKM1 autoantibodies are also present. The relationship between the prevalence of this characteristic autoimmune hepatitis autoantibody and the implication of an autoimmune phenomenon in the liver injury of patients chronically infected by HCV needs further investigation.