Patients with Insulin-dependent Diabetes but not those with Non-insulin-dependent Diabetes have Anti-sulfatide Antibodies as Determined with a New ELISA Assay

Background: In sera from newly diagnosed insulin-dependent diabetes mellitus patients (IDDM type 1) autoantibodies occur against different antigen determinants often shared with neural tissues. The role of these autoantibodies in the disease process is not yet clarified but they can be used as a diagnostic tool in the detection of IDDM patients. Methods: We have analysed the occurrence of sulfatide autoantibodies in serum from patients with type 1 diabetes (n = 20), individuals with pre-type 1 diabetes (n = 6), patients with type 2 diabetes (n = 32) and controls (n = 43). The method used for the determination of the autoantibodies was a newly developed microtitre-ELISA assay utilizing a complex of sulfatide-albumin as the ligand. Results: The new assay procedure for serum sulfatide autoantibodies showed good reproducibility. The total (day-to-day) imprecision based on analyses of three different serum samples with positive titres varied between 11 and 14% during an assay period of 6 months. None of the controls (0/43) had positive titres of sulfatide antibodies. Of the patients with type 1 diabetes, 85% displayed positive titres of anti-sulfatide antibodies while none of the type 2 patients did so. All individuals with pre-type 1 diabetes had positive titres of sulfatide antibodies. Conclusions: We conclude that sulfatide autoantibodies in serum can be reproducibly assayed by the newly developed microtitre-ELISA procedure. Elevated titres of sulfatide autoantibodies are a constant finding in newly diagnosed type 1 patients.     

Allergen specific IgGd antibodies in dogs with atopic dermatitis as determined by the enzyme linked immunosorbent assay (ELISA).

Allergen specific IgGd antibodies were detected by the enzyme linked immunosorbent assay (ELISA) in 89% of the 62 atopic dogs studied. Antibodies were found most frequently against house dust (47%), human dander (50%), grass pollens (58%) and spring tree pollens (43%). These antibodies were also found in 11 of 20 dogs with atopic symptoms but without immediate skin test reactivity to inhalant allergens. Agreement between the presence of skin reactivity and allergen specific IgGd titres ranged from one of 14 for cat dander to 22 of 34 for house dust. Among dogs with atopic symptoms but without skin test reactivity and specific IgGd titres to the respective allergens, the agreement varied between 28 of 54 for human dander and 67 of 68 for cat dander. In view of the value of the dog as an experimental model of atopic disease in man, further studies of the pathophysiological significance of IgGd antibodies are warranted. In addition, reconsideration of the diagnostic criteria for canine atopic dermatitis, as done by Hanifin & Rajka (1980) in man, seems indicated.     

Mycoplasmal antibodies as determined with an enzyme-linked immunosorbent assay, in tubal factor infertility

A total of 81 infertile women, who had been referred for diagnostic loparoscopy, were tested for the presence of antibodies to Mycoplasma hominis and T-mycoplasma. Out of 81, 30 had tubal adhesions and 51 had unilateral/bilateral tubal blockage. Antibodies to M. hominis were found in 21/30 (70%) and 14/51 (27.45%) women, antibodies to T-mycoplasma in 12/20 (40% and 39/51 (76.47%) women with tubal disorder. In a control group of 40 pregnant women, antibodies to the same two organisms occurred in 10% and 32.5%. Antibodies to M. hominis and T-mycoplasma were significantly (P < 0.001) more common in women with tubal disorder. Our results confirm the important role of M. hominis and T-mycoplasma in the aetiology of tubal infertility.     

Seroprevalence of hantavirus antibodies in Germany as determined by a new recombinant enzyme immunoassay

In order to elucidate the epidemiological importance of hemorrhagic fever with renal syndrome in Germany, the prevalence of antibodies against hantaviruses was determined in 13,358 sera from residents of various geographic regions, 1,284 sera from occupational risk groups and 287 sera from chronic hemodialysis patients. Serological investigations were performed using a highly specific transferable solid phase enzyme immunoassay based on the recombinant nucleocapsid proteins of a Hantaan and a Puumala serotype strain. The overall antibody prevalence was found to be 1.68 %. In the serum panels from western and southern Germany, it was determined to be 1.83 % on average in contrast to only 0.8 % in the panel from eastern Germany. An endemic focus revealing an antibody prevalence of 3.12 % was detected in a low-mountain area called Suebian Alb, which is located in the federal state of Baden-Württemberg. Occupational risk groups and a group of chronic hemodialysis patients showed a significantly elevated antibody prevalence ranging from 3.3 % to 10 %. The Puumala serotype was found to be the prevailing virus, but the percentage of sera predominantly recognizing the Hantaan nucleocapsid protein increased towards the south and the east and was significantly elevated in dialysis patients.     

Measurement of anti-DNA antibodies by ELISA: a comparative study with Crithidia and a Farr assay

One hundred and twenty six sera from 116 patients with systemic lupus erythematosus (SLE) and from 51 control patients were assayed for the presence of anti-DNA antibodies, using a commercial enzyme linked immunosorbent assay (ELISA). Fifty three sera (42%) from SLE patients were positive and a further 13 sera (10%) fell in the 'equivocal' positive range. Three control sera were positive. In a standard 14C DNA Farr assay, 67 sera (53%) from SLE patients were positive. One control serum was weakly positive. There was a good linear correlation between absorption in the ELISA and the 14C DNA binding result (r = 0.73). Results in the ELISA and Farr assays were concordant in 96 of the 126 SLE sera, and 47 of 51 control sera. Sequential sera from a further 6 patients with fluctuating clinical activity of SLE showed similar patterns of change of anti-DNA antibodies in both assays. The ELISA was more sensitive than the Crithidia luciliae immunofluorescence assay which detected 44 positive sera (35%) in the SLE group. These results suggest that this ELISA assay may be a useful alternative to the Crithidia assay or an effective screen prior to testing in the more technically difficult and time consuming Farr assay for the measurement of anti-DNA antibodies.     

HLA-encoded susceptibility to insulin-dependent diabetes mellitus is determined by DR and DQ genes as well as their linkage disequilibria in a Chinese population

HLA-DRB1 and -DQB1 genes were analyzed in 98 Chinese IDDM patients and 205 control subjects from Taiwan. The DRB1^*0301-DQB1^*0201 haplotype conferred strong susceptibility (RR = 7.7, p_c < 10−5). DRB1^*0405 also conferred susceptibility (RR = 3.1, p_c < 0.0005) whereas DRB1^*0403 (RR = 0.7) and DRB1^*0406 (RR = 0.2) conferred protection. Indeed, the relative risk for the DRB1^*0405-DQB1^*0302 haplotype (RR = 33.7, p_c < 0.002) was 48 and 168 times higher than those conferred by the DRB1^*0403-DQB1^*0302 and DRB1^*0406-DQB1^*0302 haplotypes, respectively, suggesting that the protection conferred by DRB1^*0403 and 0406 is dominant over DQB1^*0302. The strong linkage disequilibrium observed between DQB1^*0302 and DRB1^*0403(0406) can thus explain the surprising finding that the frequency of DQB1^*0302 was not significantly increased in the Chinese IDDM patients (RR = 0.9). Because the DRB1^*0405-DQB1^*0302 haplotype (RR = 33.7) conferred higher susceptibility than the DRB1^*0405-DQB1^*0401 (RR = 2.5) or DRB1^*0405-DQB1^*0301 (RR = 2.1) haplotypes, DQB 1^*0302 is indeed a susceptibility factor, while both DQB1^*0301 and DQB1^*0401 may confer protection against IDDM. The increased frequency of the protective DQB1^*0401 allele in patients compared to controls is due to linkage disequilibrium between DRB1^*0405 and DQB1^*0401. Interestingly, the previously demonstrated protective effect of DQB1^*0602 was not very strong in the Chinese (RR = 0.4). Our results suggested that HLA-encoded susceptibility to IDDM is determined by the combined effects of all DR and DQ molecules present in an individual. Therefore, the genotypic combinations of DR and DQ genes as well as their linkage disequilibria can influence IDDM susceptibility. At least four DR and DQ molecules conferring high susceptibility (DRB1^*0301, DRB1^*0405, and DQ/β0301/0201 and 0301/0302) occur at high frequency in the Chinese population. However, linkage disequilibria between highly susceptible DR and protective DQ or vice versa (e.g., DRB1^*0405-DQB1^*0301(0401] and DRB1^*0403[0406]-DQB1^*0302) are probably responsible for the lower incidence of IDDM in the Chinese.     

Proinsulin autoantibodies: association with type I diabetes but not with islet cell antibodies, insulin autoantibodies or HLA-DR type

Antibodies reacting with proinsulin but not with insulin determinants have been observed recently in Type I diabetes. We describe here that ELISA-determined proinsulin autoantibodies (IgG-PAA) also occur in first-degree relatives of IDDM patients (38/513, 7.4% vs 1.9% in controls, P less than 0.025). In contrast to insulin autoantibodies (IgG-IAA) and islet cell antibodies (ICA) no association with HLA type was found. Furthermore, IgG-PAA occur independently of IgG-IAA and ICA. We conclude that the humoral autoimmune response to proinsulin determinants is under separate genetic control.     

Understanding why people with type 1 diabetes do not attend for specialist advice: a qualitative analysis of the views of people with insulin-dependent diabetes who do not attend diabetes clinic

Attendance at diabetes clinic is associated with improved medical outcome, however, significant numbers of people with type 1 diabetes choose not to attend. In order to understand the reasons underlying this decision, qualitative interviews were carried out with 12 long-term non-attenders. Three distinct groups emerged differing in terms of their cognitive and emotional responses to diabetes and their coping strategies: (1) the 'High fear' group; (2) the 'Patient as expert' group; and (3) the 'Low motivation' group. These differences should be recognized and suitable approaches developed to ensure that all people with diabetes are able to accept appropriate specialist support.     

Impaired function of antibodies to pneumococcal surface protein a but not to capsular polysaccharide in mexican american adults with type 2 diabetes mellitus

The goal of the study was to determine baseline protective titers of antibodies to Streptococcus pneumoniae surface protein A (PspA) and capsular polysaccharide in individuals with and individuals without type 2 diabetes mellitus. A total of 561 individuals (131 individuals with diabetes and 491 without) were screened for antibodies to PspA using a standard enzyme-linked immunosorbent assay (ELISA). A subset of participants with antibodies to PspA were retested using a WHO ELISA to determine titers of antibodies to capsular polysaccharide (CPS) (serotypes 4, 6B, 9V, 14, 18C, 19A, 19F, and 23F). Functional activity of antibodies was measured by assessing their ability to enhance complement (C3) deposition on pneumococci and promote killing of opsonized pneumococci. Titers of antibodies to protein antigens (PspA) were significantly lower in individuals with diabetes than controls without diabetes (P = 0.01), and antibodies showed a significantly reduced complement deposition ability (P = 0.02). Both antibody titers and complement deposition were negatively associated with hyperglycemia. Conversely, titers of antibodies to capsular polysaccharides were either comparable between the two groups or were significantly higher in individuals with diabetes, as was observed for CPS 14 (P = 0.05). The plasma specimens from individuals with diabetes also demonstrated a higher opsonophagocytic index against CPS serotype 14. Although we demonstrate comparable protective titers of antibodies to CPS in individuals with and individuals without diabetes, those with diabetes had lower PspA titers and poor opsonic activity strongly associated with hyperglycemia. These results suggest a link between diabetes and impairment of antibody response.     

A new enzyme-linked fluorescence assay (ELFA) for use with peroxidase-antibody conjugates: a comparison with ELISA for the quantitation of IgM antibodies to hepatitis B core antigen

A new enzyme-linked fluorescence assay (ELFA) suitable for use with peroxidase-antibody conjugates is described. The substrate for the assay is p-hydroxyphenylacetic acid, the fluorescent product of which is stable and unaffected by light. The assay compared favourably with a standard ELISA for the quantitation of IgM antibodies to hepatitis B core antigen.     

The antibody response of mice to allografts as determined by a plaque assay with allogeneic target cells

Mouse lymphoid cells producing lytic antibody against alloantigens have been detected in vitro with the help of a plaque assay using allogeneic tumor cells as indicator cells. In C3H mice injected with 30 × 10⁶ DBA/2 tumor cells, alloantibody plaque forming cells appeared in the spleen by day 3 and increased in number until day 9.     

Hen egg yolk as a source of antiviral antibodies in the enzymelinked immunosorbent assay (ELISA): A comparison of two plant viruses

A method of raising antibodies against plant viruses in hen egg yolk is described. Laying hens were immunized with citrus tristeza virus (CTV) or tobacco mosaic virus-avocado isolate (TMV-A). Antiviral antibodies in the yolks of sequentially laid eggs as well as in the serum were titrated by the (heterologous) antiglobulin double antibody sandwich form of the enzyme-linked immunosorbent assay (HADAS-ELISA). Antibodies first appeared in yolk 7 days after injection and peak levels were attained on day 9–11; these levels persisted for about 6–12 days. Non-specific yolk antibodies were removed by absorption with an extract of uninfected plant tissue. Using the HADAS-ELISA technique we found that yolk titres were equal to, or higher than those in serum. The benefits of using laying hens over conventional laboratory animals as a source of antiviral antibody are discussed.     

Patients with Type 2 diabetes mellitus have a worse functional outcome post knee arthroplasty: a matched cohort study

Knee arthroplasty provides not only pain relief but also an improvement in function and range of movement. Limited joint mobility is a common complication of diabetes mellitus. We therefore examined functional outcome post total knee arthroplasty in a cohort of subjects with (n=367) and a cohort matched for age, sex, BMI and functional movement at baseline, without diabetes mellitus (n=367). Participants were examined at baseline (pre-operatively), 1, 5 and 10 years post TKA. There was no significant difference in fixed flexion, maximal flexion or total range of movement between the two groups at baseline. By 1 year the group with diabetes had a significantly lower maximal flexion (p<0.001), total range of movement (p<0.001) and Knee Society Score (p=0.034). Similar results were observed at years 5 (except for the KSS) and 10 post procedure. At 5 years post arthroplasty a significant increase was observed in fixed flexion (p=0.026) in the diabetic group. Ten years post arthroplasty yielded similar results. This study demonstrates that the pre-operative presence of diabetes mellitus leads to a worse outcome post knee arthroplasty, although no significant difference was demonstrable in KSS at 5 years (p=0.35) suggesting patient satisfaction remains high during this period.     

Improvement in glycemic control following a diabetes education intervention is associated with change in diabetes distress but not change in depressive symptoms

In diabetes patients, depression is correlated with diabetes-specific emotional distress, and observational studies have suggested that diabetes distress may have a greater impact on diabetes outcomes than depression itself. To examine the relative effects of change in depressive symptoms and change in diabetes distress on change in glycemic control, we conducted a diabetes self-management education intervention in 234 type 2 diabetes (T2DM) patients, and measured glycemic control (HbA1c), depressive symptoms (CES-D), and diabetes distress (PAID) at baseline and 6 months. In multiple linear regression, change in depressive symptoms was not associated with change in HbA1c (P = 0.23). Change in diabetes distress was significantly associated with change in HbA1c (P < 0.01), such that a 10-point decrease in diabetes distress (which corresponds to the average change in distress in this study population) was associated with a 0.25% reduction in HbA1c. Change in diabetes distress, and not change in depressive symptoms, was associated with both short- and long-term change in glycemic control for patients with poorly controlled T2DM.     

Hydroxyapatite-coated nylon beads as a new reagent to develop a particle agglutination assay system for detecting Japanese encephalitis virus-specific human antibodies.

BACKGROUND: Detection of Japanese encephalitis virus (JEV)-specific antibodies is done today by hemagglutination-inhibition assay (HIA), neutralization assay (NTA) and enzyme-linked immunosorbent assay (ELISA). These conventional assays are often difficult to perform in diagnostic laboratories with insufficient resources. An alternative antibody detection kit, which is simple, preservable and inexpensive, is needed for extended use in rural areas of Asia. OBJECTIVES: (i) Characterization of a new antigen carrier, hydroxyapatite-coated nylon (Ha-Ny) beads, and (ii) evaluation of the JEV antigen-coated Ha-Ny beads as a reagent to detect anti-JEV antibodies in human serum samples. STUDY DESIGN: We examined the Ha-Ny beads for hydroxyapatite content, precipitation efficiency and protein adsorption ability. We then developed a particle agglutination assay system using the JEV antigen-coated Ha-Ny beads, and tried out the newly developed assay system with reference serum samples. RESULTS: The beads had the ability to adsorb 0.44 mg of lysozyme per gram. Sedimentation speed was 10.2 cm/30 min in phosphate buffered saline (PBS), pH 7.0. Binding of the JEV antigen on Ha-Ny beads was confirmed by scanning electron microscopy (SEM) and ELISA. Eighteen confirmed-human serum samples were tested by the newly developed particle agglutination assay system. The results were consistent with those from HIA, NTA and ELISA. CONCLUSION: The Ha-Ny beads can be applicable to the development of a new JEV antibody-detection kit, which does not require specific laboratory facilities.     

N-terminal region of ZW10 serves not only as a determinant for localization but also as a link with dynein function

ZW10 interacts with dynamitin, a subunit of the dynein accessory complex dynactin, and functions in termination of the spindle checkpoint during mitosis and in membrane transport between the endoplasmic reticulum (ER) and Golgi apparatus during interphase. Its associations with kinetochores and ER membranes are mediated by Zwint-1 and RINT-1, respectively. A previous yeast two-hybrid study showed that the C-terminal region of ZW10 interacts with dynamitin, and part of this region has been used as an inhibitor of ZW10 function. In the present study, we reinvestigated the interaction between ZW10 and dynamitin, and showed that the N-terminal region of ZW10 is the major binding site for dynamitin and, like full-length ZW10, could potentially move along microtubules to the centrosomal area in a dynein-dynactin-dependent manner. Competitive binding experiments demonstrated that dynamitin and RINT-1 occupy the same N-terminal region of ZW10 in a mutually exclusive fashion. Consistent with this, over-expression of RINT-1 interfered with the dynein-dynactin-mediated movement of ZW10 to the centrosomal area. Given that the N-terminal region of ZW10 also interacts with Zwint-1, this region may be important for switching partners; one partner is a determinant for localization (kinetochore and ER) and the other links ZW10 to dynein function.