Problems and pitfalls with measurement of antibody affinity using solid phase binding in the ELISA

Current methods of estimation of antibody affinity constants using ELISA assume homogeneous binding of antibody to the solid phase, despite many reports in the literature that this is not true. I have derived theoretical antibody binding curves for solid phase antigen assuming homogeneous antibody binding. I have compared these curves with a set of experimental binding curves of monoclonal antibodies to the serum protein fibronectin. The results conclusively show that while some monoclonal antibodies behave as predicted by theory, others show departures from homogeneous binding which can be explained by various surface effects. I have discussed how these surface effects can cause errors in estimates of either liquid phase or solid phase affinities using the ELISA, and have demonstrated the limitations of methods of affinity ranking.     

Different anticoagulant and immunological properties of anti-prothrombin antibodies in patients with antiphospholipid antibodies

Lupus anticoagulant (LA) antibodies are acquired inhibitors of coagulation belonging-together with anticardiolipid (aCL) antibodies-to the family of antiphospholipid antibodies. Since LA antibodies affect coagulation reactions via recognition of the complex of lipid-bound prothrombin, they may be better named anti-prothrombin antibodies. We studied their immunological properties in the plasma of 59 patients with antiphospholipid antibodies by means of specific ELISA systems that allowed the characterization of the interaction of these antibodies with human prothrombin and anionic phospholipids. The mode of presentation of prothrombin was found to greatly influence the reactivity of anti-prothrombin antibodies. In fact, when plain polystyrene plates were used to immobilize prothrombin, virtually no binding was observed. Conversely, when prothrombin was coated on high-activated PVC ELISA plates, 34 samples (58%) contained antibodies that recognize human prothrombin in solid phase. In particular, IgG antibodies were found in 21 plasmas and IgM in 22; both IgG and IgM isotypes were present in 9 of these cases. A higher prevalence was observed in the ELISA for the detection of the antibodies directed at the calcium-mediated complex of phosphatidylserine (PS)-bound prothrombin: 53 samples (90%), preadsorbed with cardiolipin liposomes to remove aCL antibodies, showed the presence of IgG and/or IgM anti-prothrombin antibodies. When the results were analyzed according to the immunoglobulin isotypes, 44 (75%) and 39 (66%) samples were found to contain IgG and IgM anti-prothrombin antibodies, respectively. Both IgG and IgM were present in the plasma of 30 patients. Only half of these samples reacted also with PVC-bound prothrombin. Apparently, the higher rate of positivity of the ELISA for the detection of antibodies to the complex of PS-bound prothrombin was not due to differences in the amount of antigen available in the 2 systems, as judged by binding experiments performed with a rabbit polyclonal anti-human prothrombin antiserum. Finally, the anticoagulant properties of 14 total IgG preparations (12 of them contained anti-prothrombin antibodies positive in both ELISA systems, whereas the other 2 cases reacted either with PVC-bound prothrombin only or with PS-bound prothrombin only) were evaluated by diluted Russell's Viper Venom Time and by diluted activated Partial Thromboplastin Time. To rule out the beta 2-glycoprotein I (beta 2-GPI)-dependent anticoagulant effect of the aCL antibodies contained in the preparations, the coagulation tests were performed in beta 2-GPI deficient plasma. Six preparations failed to show anticoagulant activity in both assay systems, suggesting that 2 types of IgG anti-prothrombin antibodies exist, that differ with respect to their anticoagulant properties. These findings suggest that anti-prothrombin antibodies resemble aCL antibodies with respect to the behaviour in "in vitro" coagulation reactions and underline the wide heterogeneity of antiphospholipid antibodies.     

Immobilized hirudin and hirudin-based peptides used for the purification of recombinant human thrombin prepared from recombinant human prothrombin

A single step solid phase radioimmunoassay (SS-SPRIA) has been developed for human chorionic gonadotropin (hCG) using monoclonal antibodies (MAb) from culture media adsorbed immunochemically on plastic tubes. The assays have been found to be very simple in terms of operation and do not demand purification of MAbs. Several MAbs which do not show any displacement in liquid phase RIA and ELISA provide a satisfactory SS-SPRIA. Our investigations revealed that the assumption regarding the stability of the primary Mab-Ag complex during incubation and washing steps in ELISAs is not strictly valid for dissociable MAbs. A comparison of different assay systems suggests that the single step SPRIA offers additional advantages over conventionally used multistep ELISA procedures and provides a quantitative probe for the analysis of epitope-paratope interactions.     

Antibodies to beta 2-glycoprotein-I: urea resistance, binding specificity, and association with thrombosis

The aim of the present study was to evaluate the urea resistance and binding characteristics of anti-beta 2-glycoprotein I (anti-beta 2GPI) antibodies using standard anticardiolipin (aCL) and anti-beta 2GPI enzyme immunosorbent assays (ELISAs). Sera from patients with antiphospholipid syndrome (APS) (n = 22) and non-APS (n = 24), positive in a standard aCL ELISA, were tested in an anti-beta 2GPI ELISA performed in polystyrene-irradiated ELISA plates. Urea resistance aCL and anti-beta 2GPI ELISAs were performed by measuring the ability of antibodies to recognize antigen in the presence of 2 M urea. The serum dilution after urea treatment (D) expressed as a percentage of the serum dilution without urea treatment (D(o)) corresponding to the same optical density was defined as residual activity (RA = 100 D/D(o)). The higher the RA, the higher the resistance of the antibodies to urea. APS compared to non-APS sera had higher aCL binding (absorbance values ranging between 0.180 and 1.400; median, 0.717 vs 0.120-1.273; median, 0.250, respectively; P < 0.004). Six APS patients' sera had low aCL levels but they expressed RA > or = 30%. Anti-beta 2GPI antibodies were detected in 15 of 22 APS vs 3 of 24 non-APS patients (P < 0.03); RA > or = 30% was detected in 15 of 22 APS vs 1 of 23 non-APS patients (P < 0.004). Using a CL affinity column, antibodies were purified from three APS anti-beta 2GPI negative and three non-APS anti-beta 2GPI-positive patients and tested in a aCL ELISA, using highly purified bovine serum albumin (BSA) as a blocking agent (modified ELISA); reactivity was not detected in two APS and one non-APS sera. On the contrary, the reactivity of the purified antibodies was high when beta 2GPI was incubated with CL in the ELISA plates; thus some anti-beta 2GPI negative sera from APS patients recognized the CL/beta 2GPI complex, rather than CL or beta 2GPI alone. In conclusion, anti-beta 2GPI antibodies are common in the APS patients, but a number of such patients recognize the CL/beta 2GPI complex and not CL or beta 2GPI. Antibodies to either beta 2GPI or the CL/beta 2GPI complex derived from APS sera present a high resistance to urea. Anti-beta 2GPI antibodies of low urea resistance exist in a minority of non-APS patients with autoimmune disease.     

Increased immunoglobulin binding to cerebral endothelium in patients with antiphospholipid antibodies

BACKGROUND AND PURPOSE: There is a strong link between antiphospholipid antibodies and stroke. The mechanism of action of antiphospholipid antibodies is unknown. Most theories of pathogenesis center around platelet or endothelial cell dysfunction. Our aim was to determine if there were immunoglobulins in the sera of patients with antiphospholipid antibodies that bind human brain microvascular endothelial cells. METHODS: We studied sera from three groups of subjects: patients with antiphospholipid antibodies and stroke (group 1), healthy control subjects (group 2), and patients with stroke but without antiphospholipid antibodies (group 3). We isolated human brain microvascular endothelial cells from temporal lobectomy specimens and used a cellular enzyme-linked immunosorbent assay (ELISA) to measure immunoglobulin binding to endothelial cells derived from human brain and from human umbilical vein. We used a chromium release assay to measure cytotoxicity. RESULTS: Patients with antiphospholipid antibodies and stroke had significantly higher immunoglobulin binding to human brain microvascular endothelial cells than subjects in the other groups ([ELISA index+standard deviation], 63 +/- 37 [group 1] versus 7 +/- 7 [group 2] versus 7 +/- 7 [group 3], P < .001). There was, however, poor correlation between binding to brain endothelial cells and binding to cardiolipin. The binding to brain microvascular cells was not specific to brain endothelium, as similar results were found in an ELISA using human umbilical vein cells. There was no evidence of complement-mediated brain endothelial cell cytotoxicity. CONCLUSIONS: Patients with stroke and antiphospholipid antibodies frequently have human brain microvascular endothelial-reactive antibodies in their serum. These antibodies are distinct from those to cardiolipin. We found no evidence that these antibodies are cytotoxic.     

Characterization of monoclonal antibodies that recognize the thermolabile toxin of Vibrio cholerae

The obtention of two monoclonal antibodies which recognize a single epitope present in the subunit B of the thermolabile toxin of Vibrio cholerae and another which shows a cross-reaction with those produced by certain enteropathogenic toxins, is reported. The standardization of a solid phase indirect immunoenzymatic assay (ELISA) for the primary screening and selection of hybrids was performed; in addition, the isotype was determined.     

Excretion-secretion antigens from adult Dirofilaria immitis in the diagnosis of human filariasis by solid phase immunoenzyme assay

The solid phase enzymatic immuno-assay (ELISA) was normalized for detecting antibodies against. Filaria using excretion-secretion antigens (ESA) from Dirofilaria immitis adults in a group of asymptomatic and microfilaraemic patients infected by different species of filariae (loa loa, Wuchereria bancrofti, Onchocerca volvulus, Mansonella ozzardi and Mansonella perstans), and in another group of symptomatic and aminorofilaraemic patients, temporary residents in an area with endemic loiasis. The ESA-ELISA specificity permitted the distinction between filariasic and non-filariasic patients.     

American Heart Association Report on the Public Access Defibrillation Conference, December 8-10, 1994. American Heart Association Taskforce on Automatic External Defibrillation

Experimental conditions (pH 6.5, 24 h reaction, peptide:biotin ratio 1:5) were defined for preferential incorporation of the biotin molecule in the N-terminal alpha-amino group of peptides. This strategy could be helpful in numerous applications when an entire peptide chain must remain accessible for antibody or receptor binding. We illustrate this advantage in a solid-phase enzyme immunoassay designed to detect antibodies specific for bovine beta-lactoglobulin present in rabbit or human sera. This test involves synthetic peptides biotinylated in different positions and immobilized on a solid phase. The use of biotin/streptavidin interactions permitted more efficient detection of specific anti-peptide antibodies than solid phases prepared using conventional passive-adsorption techniques. The highest levels of antibody binding were measured when biotinylation occurred at the N-terminal extremity of immobilized peptides.     

Mass-sensing, multianalyte microarray immunoassay with imaging detection

Miniaturization of ligand binding assays may reduce costs by decreasing reagent consumption, but it is less apparent that miniaturized assays can simultaneously exceed the sensitivity of macroscopic techniques by analyte "harvesting" to exploit the total analyte mass available in a sample. Capture reagents (avidin or antibodies) immobilized in 200-microm diameter zones are shown to substantially deplete analyte from a liquid sample during a 1-3-h incubation, and the assays that result sense the total analyte mass in a sample rather than its concentration. Detection of as few as 10(5) molecules of analyte per zone is possible by fluorescence imaging in situ on the solid phase using a near-infrared dye label. Single and multianalyte mass-sensing sandwich array assays of the IgG subclasses show the sensitivity and specificity of ELISA methods but use less than 1/100 the capture antibody required by the 96-well plate format.     

Development of male contraceptive vaccine--a perspective

Anti-disialoganglioside (GD2) monoclonal antibodies (MAbs) have been used in vivo for immunolocalization and in phase I and II trials to target disseminated neuroblastoma, the most common extracranial solid tumor in children. However, the efficacy of these first-generation MAbs is likely to be improved by using engineered anti-GD2 antibodies. The generation of single-chain antibody fragments (scFv) could be very helpful as these molecules can be further modified to produce recombinant molecules with pre-defined properties such as immunotoxins, chimeric, or bispecific antibodies. Thus, a scFv directed against GD2 (scFv 7A4) was cloned, sequenced, and expressed. Its binding properties were characterized and compared to that of the parental MAb 7A4. Nucleotide sequence analysis of the scFv 7A4 indicated that its VH region belongs to the V region IIID subgroup and the V kappa to the V region II subgroup. The scFv 7A4 bound to GD2+ neuroblastoma cell lines but not to GD2- cell lines or to GD2- cells isolated from peripheral blood. ELISA and thin-layer chromatography (TLC) indicated that it retained the anti-GD2 specificity, and exhibited a slight cross-reaction with GD3 as the parental MAb. This scFv makes it possible to develop new useful reagents through genetic engineering for adjuvant tumor therapy.     

Inhibition of binding of anti-PLA1 antibodies to platelets with monoclonal antibody LK-4. Evidence for multiple PLA1 receptor sites on platelet GPIIIa

The PLA1 epitope on platelet GPIIIa has a sulfhydryl-dependent conformation and is dependent on a leucine 33/proline33 polymorphism. Monoclonal antibody LK-4 differentiates PLA1/PLA1 from PLA2/PLA2 platelet lysates on solid phase enzyme-linked immunosorbent assay (ELISA), as well as immunoblot. To determine whether LK-4 reacts at or near the binding site(s) for human anti-PLA1, nine such antibodies (Abs) (six neonatal; three posttransfusion) were examined in the presence and absence of LK-4 for binding to platelets, as well as rGPIIIa 1-66, a recombinant glutathione S-transferase fusion peptide. All nine human Abs bound to rGPIIIa 1-66, as well as platelets, in a saturation-dependent manner, employing both solid phase ELISA, as well as flow cytometry. Binding of all nine Abs to rGPIIIa 1-66 or platelets was inhibited by LK-4. IC50's for inhibition of binding of anti-PLA1 to rGPIIIa 1-66 varied from 8 to 160 micrograms/mL (5 x 10(-8)- 1 x 10(-6) mol/L). However, IC50's for LK-4 inhibition of binding to platelets was strikingly different. Six of the nine Abs had IC50's of 1 to 10 micrograms/mL (8-fold to 16-fold greater inhibition than with rGPIIIa 1-66), whereas three neonatal Abs had IC50's of 380 to 1,013 micrograms/mL (6-fold to 48-fold less inhibition than with rGPIIIa 1-66). Similar results were noted with intact GPIIIa, rGPIIIa 1-66 blocked the binding of anti-PLA1 Abs to platelets and served to segregate the nine patients into two groups: a sensitive group of anti-PLA1 Abs from six patients in which binding to platelets was progressively inhibited by increasing concentrations of rGPIIIa 1-66 with inhibition at 1 micrograms/mL of 18% and inhibition at 256 micrograms/mL of 78%; a second resistant group of three anti-PLA1 Abs from three patients in which inhibition was first noted at 16 micrograms/mL of 4% with 35% inhibition at 256 micrograms/mL. Thus, LK-4 binds to GPIIIa at the 1-66 N-terminal region, inhibits binding of anti-PLA1 Ab to platelets, and segregates, anti-PLA1 Abs into two groups. These data are compatible with two or more receptor sites for anti-PLA1 Ab: one that is present on rGPIIIa 1-66 and sensitive to LK-4 inhibition, another that is present on rGPIIIa 1-66, as well as other site(s) on platelet GPIIIa and insensitive to inhibition.     

Do patients with antiphospholipid syndrome have autoantibodies to beta 2-glycoprotein I?

High positive anticardiolipin antibody tests have been associated with recurrent thrombosis and pregnancy loss. Although these antibodies were believed to bind negatively charged phospholipids, recent reports have suggested that a serum protein, beta 2-glycoprotein I (beta 2-GPI), may be the true antigen for these antibodies. To resolve this issue, we compared binding of 75 anticardiolipin-positive and 71 anticardiolipin-negative serum samples from patients with rheumatic diseases to beta 2-GPI by using an enzyme-linked immunosorbent assay (ELISA). Serum samples from 30 healthy blood donors and 10 laboratory personnel were used as normal controls. We found no difference in binding between the three groups of serum samples. In addition, when binding to beta 2-GPI coated plates was compared with binding to ELISA plates without beta 2-GPI (blank), no difference was observed. Finally, binding of anticardiolipin-positive serum samples to plates coated with cardiolipin-beta 2-GPI mixture varied directly with the cardiolipin concentrations. Based on these findings, we conclude that anticardiolipin-positive serum samples do not bind beta 2-GPI.     

Generation of polyclonal antibodies against nisin: immunization strategies and immunoassay development

Murine polyclonal antibodies reactive to the lantibiotic bacteriocin nisin A (nisA) have been produced by immunization with nisA-cholera toxin and nisA-keyhole limpet hemocyanin (nisA-KLH) conjugates. Mice immunized with nisA-cholera toxin developed nisA-specific antibodies with low relative affinities and poor sensitivities, while the immunization of mice with nisA-KLH conjugates resulted in the production of nisA-specific antibodies with high relative affinities and much-increased sensitivities. nisA antibodies could also be readily mass produced in less than 8 weeks in ascites fluid by using the nisA-KLH conjugate. A competitive direct enzyme-linked immunosorbent assay (ELISA) whereby nisA-horseradish peroxidase and free nisA competed for antibody binding was devised. The detection limit for nisA in the competitive direct ELISA with the nisA-KLH-generated antibodies was from 5 to 100 ng/ml, while the amount of free nisA required for 50% antibody binding inhibition ranged from 0.3 to 5 micrograms /ml. Both antisera and ascites polyclonal antibodies cross-reacted with nisZ either in the supernatant of a producer strain or with the pure lantibiotic but did not cross-react at all with non-lantibiotic-type bacteriocins. These polyclonal antibodies should find a wide usage from nisA ELISA analysis in foods and other matrices.     

Total simultaneous repair of coarctation and intracardiac pathology in adult patients

OBJECTIVE: To determine whether neutralizing antibodies (NABs) to interferon beta (IFNbeta)-1a (Avonex) and IFNbeta-1b (Betaseron) cross-react. BACKGROUND: A total of 38% of MS patients treated with IFNbeta-1b and 22% of those treated with IFNbeta-1a were reported to develop NABs, which could reduce the clinical efficacy of the drug. METHODS: Blood from 10 MS patients was collected before and at 3 and 6 months after initiating treatment with IFNbeta-1a. ELISA was performed to detect binding antibodies to IFNbeta-1a. Sera from patients who tested positive for binding antibodies to IFNbeta-1a were then screened for NABs to IFNbeta-1a in a biologic assay based on neutralization of antiviral activity. These serum samples were subsequently tested for cross-reactivity with IFNbeta-1b both in the ELISA and the biologic assay. In the second part of the study, sera from patients who participated in the phase III IFNbeta-1b trial at the University of Maryland were examined for cross-reactivity with IFNbeta-1a in the ELISA and the biologic assay. RESULTS: Of the 10 patients treated with IFNbeta-1a, three developed binding as well as NABs to IFNbeta-1a 6 months after treatment, and these antibodies cross-reacted with IFNbeta-1b both in the binding and the biologic assay. Similarly, sera from six patients with NABs to IFNbeta-1b showed cross-reactivity with IFNbeta-1a in the binding assay. Three of these six serum samples tested for neutralizing activity against IFNbeta-1a demonstrated the presence of NABs to IFNbeta-1a. CONCLUSIONS: NABs to IFNbeta-1a (Avonex) and IFNbeta-1b (Betaseron) cross-react, both in the binding and the biologic assays. This suggests that switching to alternate IFNbeta preparation in patients who develop NABs may not be clinically beneficial. Studies examining cross-reactivity between NABs to IFNbeta-1a and IFNbeta-1b in a large number of patients are indicated.     

Peripheral neuroepithelioma of the kidney

The protective anti-beta abortus monoclonal antibody ISS/32 (Ab1) was used as an immunogen to induce anti-idiotypic antibodies (Ab2) in rabbits. The purpose was to produce and characterize anti-idiotypic antibodies that share conformational similarity with the corresponding bacterial epitope recognized by Ab1. The rabbit anti-IdAb so induced was isolated and affinity-purified. Its specificity for the paratope of Ab1 was determined by evaluating its ability to compete with B. abortus for binding to Ab1 in a competitive ELISA assay. The anti-idiotypic ISS/32 antibodies were able to compete with B. abortus for binding to Ab1 in a dose-dependent manner. Hence, the data indicated that the rabbit anti-Id ISS/32 antibodies reacted with or near the antigen-binding site of Ab1.     

Clinical evaluation of a capture ELISA for detection of proteinase-3 antineutrophil cytoplasmic antibody

Detection of antineutrophil cytoplasmic antibodies (ANCA) has become a useful tool in the diagnosis of Wegener's granulomatosis and microscopic polyangiitis. However, the results obtained with indirect immunofluorescence (IIF) and by ELISA for ANCA demonstration do not always correlate. A possible explanation for this finding could be that proteins are denatured during the process of antigen purification or during coating onto the solid phase. To avoid this possibility, a monoclonal antibody to PR3 that is precoated on the plate can be used. In the present study we have used the monoclonal antibody (MoAb) 4A3 for the capture of PR3 in an ELISA, and a clinical evaluation of the diagnostic properties of the new capture ELISA has been made. The sensitivity of the capture PR3-ANCA ELISA was 85% in a material of c-ANCA positive sera. A specificity of 90% was obtained in analyses from patients having various forms of glomerulonephritis. There was a significantly higher diagnostic sensitivity of the capture PR3-ANCA ELISA (85%) compared to c-ANCA by IIF (58%) in patients with Wegener's granulomatosis with renal involvement. Capture PR3-ANCA and direct ELISA for MPO-ANCA together gave a diagnostic sensitivity of 98%, versus 75% using IIF. In conclusion, the capture PR3-ANCA ELISA seems to be a valuable tool in the diagnosis of Wegener's granulomatosis with renal involvement. Preliminary data suggest that the technique may have an advantage over direct ELISA for PR3-ANCA, as well as in the follow-up of c-/PR3-ANCA associated vasculitides. However, further prospective studies are needed to clarify this premise.     

Hematuria in the child. Investigation plan in pediatric practice

BACKGROUND: Grass pollen extracts are complex mixtures consisting of different major allergenic and non-allergenic components. Phl p 4 is an important allergen, because more than 75% of grass pollen allergic patients produce specific IgE antibodies against group 4 allergens. OBJECTIVE: This study was designed to investigate the specificity of monoclonal antibodies (MoAbs) produced against Phl p 4 and to verify the presence of group 4-like proteins in different grass pollen. Furthermore the usefulness of MoAbs for quantification of group 4 allergens was studied. METHODS: Group 4 analogues were investigated by immunoblotting and ELISA inhibition using three MoAbs. The specificity of antibodies was studied using isolated group 1 and group 5 allergens. Quantification of group 4 allergen was achieved by a two-site solid-phase ELISA. Phl p 4 was purified from whole pollen extract by chromatographic or electrophoretic techniques and used as standard. RESULTS: The MoAbs studied bound strongly to proteins from timothy grass pollen extract at a mw of 55 kDa and a pI of 9.0-9.3. Phl p 4 homologes with similar mw were detected in Dactylis glomerata, Festuca pratensis, Holcus lanatus, Poa pratensis, Lolium perenne. Epitope mapping showed that all three MoAb recognized unrelated regions on Phl p 4. A two-site binding ELISA using MoAbs was developed for determination of Phl p 4 in Phleum pratense extracts. The method was able to evaluate group 4 in mass units with a working range between 150 and 2000 ng/mL. The absolute amounts of group 4 in extracts of several grasses varied considerably but was always-less than 1% of the total protein. CONCLUSION: Group 4 homologes are present in the various grass extracts but to different extents. The group 4 ELISA could be very useful as a additional tool for providing information concerning the composition of grass pollen extracts.     

Upregulation of cathepsin D expression in the dedifferentiating salamander limb regenerates and enhancement of its expression by retinoic acid

The antiphospholipid antibodies (aPL) present in "antiphospholipid-protein syndrome and autoimmune disorders" are associated with thromboembolic episodes, such as venous and/or arterial thrombosis and fetal loss. Patients with antiphospholipid antibodies have, by definition, laboratory abnormalities in either coagulation assays or various solid phase immunoassays ELISA or radioimmunoassays (RIA). These assay systems were initially thought to detect antibodies against phospholipids. The problem was complicated when it was reported that phospholipid is not the sole antigen but only a part of it, the other contribution being due to b2-glycoprotein I (b2-GP I). More findings, demonstrate that the aPL are in fact anti-b2-GP I antibodies directed against a epitope which is expressed when b2-GP I is bound to anionic phospholipid or another suitable surface. Recent studies have demonstrated that antibodies related to lupus anticoagulant (LA) induce an anticoagulant activity in b2-GP I. Some of these LA require binding to phospholipid. However, not all LA require b2-GP I as a cofactor. Human prothrombin is an antigen for some LA IgG's. Finally, a subclassification of phospholipid-dependent coagulation test anticoagulants is described, there appear to be several subclasses of LA, and the clinical and laboratory criteria required to establish the diagnosis of antiphospholipid-protein syndrome is emphasised.     

Vitamin C improves endothelium-dependent vasodilation in forearm resistance vessels of humans with hypercholesterolemia

The development of new techniques for the detection of ovarian antibodies has challenged early concepts about the rarity of ovarian antibodies in idiopathic premature ovarian failure (POF), but few attempts have been made to compare results between assays. We have sought to define the prevalence of ovarian autoimmunity in a group of 30 idiopathic POF patients compared to a group of 12 patients with POF plus an associated autoimmune disease and a group of 38 controls, using an enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IFL). Ovarian antibodies were detected in 27% of idiopathic POF patients by ELISA (not significantly different compared to POF patients with associated autoimmune disease; P < 0.0003 compared to controls) but only 7% of these patients were positive by IFL. In a further, pre-selected group of individuals, all positive for ovarian antibodies by IFL, 53% had measurable antibodies by ELISA. Some overlap was therefore demonstrated between the two techniques but many POF patients had ovarian antibodies detectable by only one method. Immunoblotting studies revealed that no consistent pattern of binding could be demonstrated for these patients. These results call into question the specificity of ovarian antibodies as a marker for autoimmune POF.     

Acute inflammatory demyelinating polyradiculopathy in children: clinical and electrodiagnostic studies

Diabetes mellitus is associated with an increased risk of atherosclerosis. The oxidation of low-density lipoproteins (LDL) is considered a key event in the initiation of atherosclerosis. To investigate LDL oxidation in vivo we measured autoantibodies to oxidised LDL (oxLDL) in 94 patients with insulin-dependent diabetes mellitus (IDDM), compared to 27 age-matched, healthy control subjects. Patients and control subjects were screened for autoantibodies using a solid phase ELISA, comparing the binding to oxLDL with that to native LDL (nLDL). In patients with IDDM the oxLDL/nLDL antibody ratio was significantly higher than in control subjects (means+/-SEM: 2.24+/-0.26 vs 1.17+/-0.17, p < 0.03). Antibody-negative patients had a longer diabetes duration (13.5+/-1.3 vs 9.1+/-1.1 years, p < 0.01) and higher actual and mean HbA1c levels compared to antibody-positive patients (8.8+/-0.2 vs 7.9+/-0.2%, p < 0.005 and 8.3+/-0.2 vs 7.7+/-0.2%, p < 0.03; respectively). In patients with a high microangiopathy score, the antibody ratio was lower than in patients without complications (1.04+/-0.10 vs 2.40+/-0.29, p < 0.01). OxLDL specific immune complexes were found exclusively in antibody-negative as compared to antibody-positive patients (18.3 vs 0 %; p < 0.01). Our data demonstrate an inverse relationship between free oxLDL antibodies and the severity of the disease. This apparent paradox can be explained in part by our demonstration of oxLDL immune complexes, masking free antibodies.