Direct Binding ELISA Research Articles

Physicochemical and immunological studies on 4-hydroxynonenal modified HSA: Implications of protein damage by lipid peroxidation products in the etiopathogenesis of SLE

4-Hydroxynonenal (HNE) is the most abundant and toxic aldehyde generated by the oxidation of plasma membrane polyunsaturated fatty acids. Systemic lupus erythematosus (SLE), a chronic autoimmune disease, is primarily characterized by increased levels of autoantibodies, predominantly against ds-DNA. However, the initial antigenic stimulus for the disease etiopathogenesis has remained elusive. HNE has been extensively used as a biomarker of oxidative stress. It can form adduct with proteins, making them highly immunogenic. Increased levels of such aldehyde-protein adducts have been reported in various pathological states, including autoimmune disorders like SLE and arthritis. In the present study, HNE-mediated structural changes in human serum albumin (HSA) were characterized by UV, fluorescence, CD and FT-IR spectroscopy as well as by polyacrylamide gel electrophoresis. Furthermore, immunogenicity of native and HNE-modified HSA was probed in female rabbits. The HNE-modified HSA was highly immunogenic eliciting high titre immunogen specific antibodies. Binding of SLE anti-DNA antibodies was analyzed by direct binding and competition ELISA. The data show preferential binding of SLE autoantibodies to HNE-modified HSA as compared to native HSA or native DNA. Our results suggest that HNE modification generates neoepitopes on HSA causing enhanced autoantibodies production. The results point towards the possible role of HNE-modified HSA in SLE etiopathogenesis.

Suppression of Mycobacterium Tuberculosis Induced Reactive Oxygen Species andTumor Necrosis Factor-Alpha Activity in Human Monocytes of Systemic LupusErythematosus Patients by Reduced Glutathione

The etiology and pathogenesis of systemic lupus erythematosus remains unknown, evidence exists for the involvement of mycobacterial antigen. This study is aimed to determine the effect of Mycobacterium tuberculosis on clinical course of SLE patients and the role of ROS and TNF-α in the pathogenesis of tuberculosis associated SLE patients. This study was done on 100 patients divided into SLE group (n=30), TB group (n=30), SLE-TB group (n=30) and control group (n=10). All patients underwent clinical, biochemical and immunological evaluation by employing techniques such as SDS-PAGE, direct binding and competition ELISA, PBMC and cell culture. Fever, arthritis, skin rash, photosensitivity were more common in both SLE and SLE-TB group. Reduced glutathione showed amelioration of ROS and TNF-α induced action, which in turn, subsequently suppressed the immune-bindings observed in monocytes of TB and SLE patients cultured without glutathione. Data shows that SLE patients are more susceptible to developing Mycobacterium tuberculosis, as ROS and TNF-α in SLE patients could activate the replication of mycobacterial Ag85B (30 kDa) after bacilli infection.

A human monoclonal antibody Fab reactive to oxidized LDL and carbamylated LDL recognizes human and mouse atherosclerotic lesions

This study was undertaken to produce a Fab fragment of a human monoclonal antibody reactive to oxidized and carbamylated low-density lipoprotein (oxLDL and cLDL) using phage display technology. An analysis of DNA sequences of this Fab, termed plaque 15,16-46 Fab, revealed that the rearranged VH was highly mutated. Complementarity-determining regions of the VH showed a very high R/S ratio and contained many positively charged amino acids. In direct binding and competitive ELISA, the Fab reacted strongly with both MDA-LDL and Cu-oxLDL forms of oxLDL, and also showed high affinity for cLDL. Immunofluorescence and immunohistochemical analyses showed that this Fab positively stained atherosclerotic aortic plaques in ApoE−/− mice as well as those in patients with atherosclerosis. The Fab also showed positive staining in placental decidua from patients with preeclampsia. It is suggested that the plaque 15,16-46 Fab against oxLDL and cLDL might possibly be applicable for developing a diagnostic reagent for both human and rodent animal research to detect and characterize atherosclerotic disease progression in atherosclerotic lesions as well as exploring the pathogenesis of atherogenic diseases such as preeclampsia.

Glycated human DNA is a preferred antigen for anti-DNA antibodies in diabetic patients

Aims Glycation of proteins and DNA, results in the generation of free radicals causing structural modification of biomacromolecule. This leads to the generation of neo-antigenic epitopes having implication in diabetes mellitus. In this study, human placental DNA was glycated with fructose and its binding was probed with the serum antibodies from type 1 and 2 diabetes patients. Methods Glycation was carried out by incubating DNA (10 μg/ml) with fructose (25 mM) for 5 days at 37 °C. The induced structural changes in DNA were studied by spectroscopic techniques, thermal denaturation studies and agarose gel electrophoresis. Furthermore, binding characteristics of autoantibodies in diabetes (type 1 and 2) patients were assessed by direct binding and competitive ELISA. Results DNA glycation with fructose resulted in single strand breaks, hyperchromicity in UV spectrum and increased fluorescence intensity. Thermal denaturation studies demonstrated the unstacking of bases and early onset of duplex unwinding. Type 1 diabetes patients exhibited enhanced binding with glycated DNA as compared to native form, while for type 2 diabetes only those with secondary complications (Nephropathy) showed higher binding. Conclusions Glycation of DNA has resulted in structural perturbation causing generation of neo-antigenic epitopes that are better antigens for antibodies in diabetes patients.

Immunological functions of oxidized human immunoglobulin G in type 1diabetes mellitus: its potential role in diabetic smokers as a biomarker of elevated oxidative stress.

The role of oxidized immunoglobulin G in type 1 diabetic smokers has been investigated in the present study. Human immunoglobulin G (IgG) was modified by reactive oxygen species (ROS). The binding characteristics of circulating autoantibodies in type 1 diabetes patients against native and modified IgG were assessed by direct binding ELISA. High degree of specific binding by 68.5% of patients sera towards ROS-modified IgG was observed in comparison to its native analogue (p < 0.05). In addition, diabetic smokers (n = 28) were examined and the results were compared with diabetic non-smokers (n = 26). Circulating antibodies of diabetic smokers showed substantially stronger binding to modified IgG as compared with the antibodies present in diabetic non-smokers (p < 0.05). Normal human sera (n = 53) showed negligible binding with either antigen. Competitive inhibition ELISA reiterates the direct binding results. The increase in total serum protein carbonyl levels in the diabetic smokers was largely due to an increase in oxidized IgG. Diabetic smokers showed substantially higher carbonyl contents in sera as well as in purified IgG as compared with sera and IgG of diabetic non-smokers. Collectively, the oxidation of plasma proteins, especially IgG, might enhance oxidative stress in type 1 diabetes smokers.

Immunological Functions of Oxidized Human Immunoglobulin G in Type 1 Diabetes Mellitus: Its Potential Role in Diabetic Smokers as a Biomarker of Elevated Oxidative Stress

The role of oxidized immunoglobulin G in type 1 diabetic smokers has been investigated in the present study. Human immunoglobulin G (IgG) was modified by reactive oxygen species (ROS). The binding characteristics of circulating autoantibodies in type 1 diabetes patients against native and modified IgG were assessed by direct binding ELISA. High degree of specific binding by 68.5% of patients sera towards ROS-modified IgG was observed in comparison to its native analogue (p&lt; 0.05). In addition, diabetic smokers (n= 28) were examined and the results were compared with diabetic non-smokers (n= 26). Circulating antibodies of diabetic smokers showed substantially stronger binding to modified IgG as compared with the antibodies present in diabetic non-smokers (p&lt; 0.05). Normal human sera (n= 53) showed negligible binding with either antigen. Competitive inhibition ELISA reiterates the direct binding results. The increase in total serum protein carbonyl levels in the diabetic smokers was largely due to an increase in oxidized IgG. Diabetic smokers showed substantially higher carbonyl contents in sera as well as in purified IgG as compared with sera and IgG of diabetic non-smokers. Collectively, the oxidation of plasma proteins, especially IgG, might enhance oxidative stress in type 1 diabetes smokers.

Hydroxyl Radical Modification of Immunoglobulin G Generated Cross-Reactive Antibodies: Its Potential Role in Systemic Lupus Erythematosus

Objective:Role of reactive oxygen species (ROS) modified human Immunoglobulin G (IgG) in systemic lupus erythematosus (SLE) has been investigated.Methods:Human IgG was modified by hydroxyl-radicals. Immunogenicity of native and modified human IgG was probed by inducing polyclonal antibodies in rabbits. Cross-reactions of induced antibodies with nucleic acid, chromatin, different blood proteins and their ROS modified conformers were determined by competitive inhibition ELISA. The binding characteristics of circulating autoantibodies in SLE patients (n = 72) against native and modified IgG were screened by direct binding and competition ELISA and the results were compared with healthy age-matched controls (n = 39).Results:Induced antibodies against ROS-modified human IgG exhibited diverse antigen binding characteristics. Native DNA, native chromatin and their ROS-modified conformers were found to be effective inhibitors of induced antibody-immunogen interaction. Induced antibodies against native human IgG showed negligible binding to the above mentioned nucleic acid antigens. SLE sera (48.6%) showed strong binding to ROS-human IgG in comparison with its native analogue (P < 0.01). Normal human sera (NHS) showed negligible binding with either antigen (P > 0.05).Conclusion:ROS-induced modifications in human IgG present neo-epitopes, and make it a potential immunogen. The induced antibodies against ROS-modified human IgG resembled the diverse antigen-binding characteristics of naturally occurring SLE anti-DNA autoantibodies. ROS-modified IgG may be one of the factors for the induction of circulating SLE autoantibodies.

Immunochemical Studies on Catechol-Estrogen Modified Plasmid: Possible Role in Rheumatoid Arthritis

Increased concentrations of estrogen metabolites (catecholestrogens) have been found in rheumatoid arthritis (RA) but the exact patho-etiology remains elusive. The binding of antibodies from the sera of RA patients and control subjects to native and modified DNA was studied by direct binding and inhibition ELISA, quantitative precipitin titration. Experimentally induced antibodies were also checked to detect oxidative lesions in the DNA as well as for the estimation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in different fluids of RA. Anti-DNA IgG from RA sera, exhibited increased recognition of modified DNA than native DNA (nDNA; P < 0.001). The relative affinity of anti-DNA antibodies for modified and nDNA was in the order of 1.85 × 10(-7), 1.23 × 10(-7), and 1.2 × 10(-6). Samples of DNA from RA patients showed a significant inhibition in the induced antibody activity in comparison to DNA isolates from controls (P < 0.001). The concentration of 8-OHdG evaluated by induced antibody in RA patients was found to be significantly higher than controls ((P < 0.0001, P < 0.01, P < 0.05). High binding of modified DNA with the IgG from RA patient might explain possible antigenic role of 4-OHE(2)-modified DNA in the production of anti-DNA antibodies. In addition, the induced antibodies have been shown to represent an alternative immunochemical probe to detect oxidative lesions in DNA as well as for the estimation of 8-OHdG levels in different body fluid of RA patients, which may be used as marker in the diagnosis of the disease.

Detection of botulinum neurotoxin serotype B at sub mouse LD(50) levels by a sandwich immunoassay and its application to toxin detection in milk.

BackgroundBotulinum neurotoxin (BoNT), the causative agent of botulism, a serious neuroparylatic disease, is produced by the anaerobic bacterium Clostridium botulinum and consists of a family of seven serotypes (A-H). We previously reported production of high-affinity monoclonal antibodies to BoNT serotype A.Methods and FindingsRecombinant peptide fragments of the light chain, the transmembrane and receptor-binding domains of the heavy chain of botulinum neurotoxin type B (BoNT/B) were expressed in Escherichia coli as GST-fusion proteins and purified. These proteins were used to immunize BALB/cJ mice for the generation of monoclonal antibodies (mAbs). Antibody-producing hybridomas were detected using either a direct binding ELISA binding to plate-immobilized BoNT/B, or with a capture-capture ELISA whereby the capacity of the antibody to capture BoNT/B from solution was tested. A total of five mAbs were selected, two of which bound the toxin light chain and three bound the receptor-binding domain of BoNT/B heavy chain. MAb MCS6-27 was identified via capture-capture ELISA and was the only mAb able to bind BoNT/B in solution under physiological conditions. MAbs F24-1, F26-16, F27-33 and F29-40 were identified via direct binding ELISA, and were able to capture BoNT/B in solution only in the presence of 0.5–0.9 mM sodium dodecyl sulphate (SDS). MAb MCS6-27 and an anti-BoNT/B polyclonal antibody were incorporated into a sandwich ELISA that did not require SDS.ConclusionsWe report here the generation of monoclonal antibodies to serotype B and the subsequent development of a sensitive sandwich immunoassay. This immunoassay has a detection limit of 100 fg BoNT/B, fifty times more sensitive than the mouse bioassay detection limit of 5 pg BoNT/B. Additionally, this assay detected as little as 39 pg/mL of toxin in skim, 2% and whole milk.

CHROMATOGRAPHIC SEPARATION OF THYROID STIMULATING HORMONE FROM LUTEINIZING HORMONE

A new preparative scale procedure is described for the efficient purification of Thyroid stimulating hormone (TSH) from buffalo pituitary glands. The methodology involves the use of hydrophobic interaction chromatography for the separation of TSH from LH which is more abundant than TSH in the pituitary gland. The two hormones were isolated from the pituitary extract by ammonium sulphate precipitation and were then separated on a Phenyl-Sepharose column. The fractions obtained from Phenyl-Sepharose chromatography were assessed using a Uno-Q column on FPLC where enrichment of the TSH in the PS-2 fraction was evident. The final removal of the trace amounts of the LH was done by ion exchange chromatography in tandem on CM-Sephadex and DEAE-Sephacel columns. All the chromatography fractions were assessed in a heterologous direct binding ELISA using bTSH and buLH as the reference hormones and anti ovine TSH beta and anti bovine LH beta as the antisera. The purified fraction (DEAE-0.5 M peak) when checked exhibited no LH immuno reactivity in a western blot against anti bovine LH beta antiserum. Forty two milligrams of a 200 fold purified buTSH per kg wet glands was obtained.

Hydroxyl Radical Damaged Thymidine-5’-Monophophate Presents Unique Epitopes for Cancer Antibodies

Reactive oxygen species (ROS) plays an important role in carcinogenesis and hydroxyl radical (OH) contributes to the structural changes in DNA that characterize the cancer like phenotype. The role of hydroxyl radical (OH) damaged thymidine 5'-monophosphate (TMP) in cancer patients has been investigated in the present study. TMP was conjugated to bovine serum albumin (BSA), and then TMP-BSA conjugate was modified by hydroxyl radicals. Cancer patients (n = 99) were screened by direct binding ELISA for the detection of antibodies against native and ROS-modified conjugates and the results were compared with healthy age-matched controls (n = 29). High degree of specific binding by cancer serum antibodies towards ROS modified TMP-BSA conjugate, in comparison to unmodified conjugate (p&lt;0.05) was observed. Healthy individuals showed negligible binding with either antigen. Competitive inhibition ELISA reiterates the direct binding results. Protein-A affinity purified IgG from cancer patients further substantiated the enhanced recognition towards modified conjugate as compared to unmodified conjugate. The present study clearly shows the perturbation in TMP-BSA conjugate by hydroxyl radical presenting unique neo-epitopes on TMP that might from one of the factors in antigen driven induction of antibodies in cancer patients.

Polynucleotide specificity of anti-reactive oxygen species (ROS) DNA antibodies.

Hydrogen peroxide in the presence of short wavelength UV light was able to induce alterations in native DNA fragments of 300 bp (ROS-DNA), thereby rendering it immunogenic in experimental animals. The specificity of induced antibodies was investigated by direct binding and competition ELISA. Inhibition studies revealed nearly 89% inhibition in the antibody binding by the immunogen and recognition of native B-, A- and allied conformations presented by various synthetic polynucleotides. Gel retardation assay reiterated the formation of immune complexes between induced antibodies and native and ROS-DNA fragments. It was observed that naturally occurring anti-DNA autoantibodies from systemic lupus erythematosus (SLE) sera recognize ROS-DNA. The comparison of the specificities of anti-DNA autoantibodies from 10 SLE patients showed a 20-50-fold preference for ROS-DNA over native DNA. These results demonstrate that anti-DNA antibodies can be induced by ROS-DNA, and that some of the autoimmune DNA binding antibodies found in SLE may result from response to reactive oxygen species.

Hydroxyl radical damaged Immunoglobulin G in patients with rheumatoid arthritis: Biochemical and immunological studies

Objectives The role of hydroxyl radical ( OH) damaged Immunoglobulin G (IgG) in rheumatoid arthritis (RA) has been investigated. Design and methods The study was hypothesized that oxidative by-products, like OH-damage IgG, help to initiate autoimmunity in RA. To test this hypothesis, IgG was modified by OH. Immunogenicity of native and modified IgG was probed by inducing polyclonal antibodies in rabbits. Autoantibodies from 77 RA sera were screened by direct binding and competition ELISA. Results The OH caused extensive damage to IgG. The OH-IgG was found to be highly immunogenic in rabbits as compare to native IgG. High degree of specific binding by 72.7% RA sera autoantibodies towards OH-IgG was observed, in comparison to its native analogue ( p < 0.05). Conclusion The OH modification of IgG causes perturbations, resulting in the generation of neo-epitopes, and making it a potential immunogen. The IgG modified with the ·OH may be one of the factors for the induction of circulating RA autoantibodies.

Caprine (Capra hircus) Luteinizing Hormone: Purification and Chromatographic Investigation of its Different Isoforms

Luteinizing Hormone (LH) from goat pituitary glands has been purified and characterized with respect to its size and subunit nature. The purification at each step was monitored by protein estimation, SDS‐PAGE, and direct binding ELISA. The final product was found to be over 90 fold purified as compared to the starting pituitary extract, and the yield of the final purified LH was found to be 65.3 mg/kilogram of wet pituitary glands. Fractionation of the cLH into different charge isoforms by SP‐Sephadex ion exchanger has been observed. Chromatography on immobilized Con A lectin resulted in fractionation of the purified cLH into unbound (2%), loosely bound (85%), and firmly bound (13%) fractions indicating oligosaccharide heterogeneity. The purified hormone was capable of stimulating weight increase in the seminal vesicles in immature male rats, with a biopotency equivalent to the 2200 I.U. of hCG per mg of purified cLH. The FSH content of the purified cLH was found to be less than 0.0165% as indicated by in vivo Steelman‐Pohley assay.

Immunogenicity of mitochondrial DNA modified by hydroxyl radical

Mitochondria consume about 90 percent of oxygen used by the body, and are a particularly rich source of reactive oxygen species (ROS). In this research communication mitochondrial DNA (mtDNA) was isolated from fresh goat liver and modified in vitro by hydroxyl radical generated from UV irradiation (254 nm) of hydrogen peroxide. As a consequence of hydroxyl radical modification, mtDNA showed hyperchromicity and sensitivity to nuclease S1 digestion as compared to control mtDNA. Animals immunized with mtDNA and ROS-modified mtDNA induced antibodies as detected by direct binding and competition ELISA. The data suggest that immunogenicity of mtDNA got augmented after treatment with hydroxyl radical. IgG isolated from immune sera showed specificity for respective immunogen and cross-reaction with other nucleic acids. Binding of induced antibodies with array of antigens clearly indicates their polyspecific nature. Moreover, the polyspecificity exhibited by induced antibodies is unique in view of similar multiple antigen binding properties of naturally occurring anti-DNA antibodies derived from SLE patients.

Enhanced recognition of reactive oxygen species damaged human serum albumin by circulating systemic lupus erythematosus autoantibodies

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with autoantibodies as a near universal feature of the disease. Earlier investigations from our laboratory revealed increased oxidative damage in SLE patients. Therefore, we hypothesized that oxidative by-products, such as hydroxyl radical (√OH), could lead to neoantigens like √OH damaged human serum albumin (HSA), which could in turn initiate autoimmunity in SLE. In the present study, the binding characteristics of SLE autoantibodies with native and √OH damaged HSA were assessed. SLE patients (n = 74) were examined by direct binding ELISA and the results were compared with healthy age- and sex-matched controls (n = 44). High degree of specific binding by 52.7% of patients sera towards √OH damaged HSA, in comparison to its native analogue (p < 0.05) was observed. Normal human sera showed negligible binding with either antigen. Competitive ELISA and gel retardation assays reiterate the direct binding results. The increase in total serum protein carbonyl levels in the SLE patients was largely due to an increase in oxidized albumin. HSA of SLE patients (SLE-HSA) and normal subjects (normal-HSA) were purified. Spectroscopic analysis confirmed that the SLE-HSA samples contained higher levels of carbonyls than normal-HSA (p < 0.01). SLE-HSA was conformationally altered, with more exposure of its hydrophobic regions. Collectively, the oxidation of plasma proteins, especially HSA, might enhance oxidative stress in SLE patients.

Stability of Major Allergen Proteins in Extract Mixes Diluted in Human Serum Albumin (HSA), Normal Saline (NSP), or Glycerin (GLY)

RATIONALE: The stability of allergen proteins in extract mixes serially diluted in NSP, HSA, or GLY and stored refrigerated for 1 year or 1 month at room temperature was investigated. Major allergen levels in grass (Gr 5), mite (Group 1 and 2), cat (Fel d 1), birch (Bet v 1), olive (Ole e 1), English plantain (Pla l 1), mugwort/sage (Art v 1), and Bermuda (Cyn d 1) were measured.METHODS: Five mixes of extracts from ALK-Abelló were made and diluted 5 fold. Cockroach, Aspergillus, or mixed mold extract was added to a portion of the 1:5v/v dilution. Some vials were stored at room temperature. Major allergens were measured using very sensitive direct binding ELISA methods.RESULTS: Most allergens had at least 50% of their activity after 3 months in all diluents. At 6 months olive, English plantain, Bermuda, birch and mite allergens began to lose activity in dilution 1:125v/v in NSP but lost almost all major allergen in dilution 1:625v/v in NSP or GLY. All allergens except Bermuda were stable at 6 months in HSA and all except Bermuda, English plantain and birch were stable at 12 months. Cockroach extract degraded all the allergens except for mite proteins. Molds degraded the grass, olive and plantain allergens significantly. Allergens maintained >40% of their activity after 1 month at room temperature.CONCLUSIONS: Allergen proteins have different stability profiles depending on the protein, the diluent, and the concentration. Some proteins in dilute solutions without added albumin protein lost ELISA binding activity in glycerinated and aqueous diluents. RATIONALE: The stability of allergen proteins in extract mixes serially diluted in NSP, HSA, or GLY and stored refrigerated for 1 year or 1 month at room temperature was investigated. Major allergen levels in grass (Gr 5), mite (Group 1 and 2), cat (Fel d 1), birch (Bet v 1), olive (Ole e 1), English plantain (Pla l 1), mugwort/sage (Art v 1), and Bermuda (Cyn d 1) were measured. METHODS: Five mixes of extracts from ALK-Abelló were made and diluted 5 fold. Cockroach, Aspergillus, or mixed mold extract was added to a portion of the 1:5v/v dilution. Some vials were stored at room temperature. Major allergens were measured using very sensitive direct binding ELISA methods. RESULTS: Most allergens had at least 50% of their activity after 3 months in all diluents. At 6 months olive, English plantain, Bermuda, birch and mite allergens began to lose activity in dilution 1:125v/v in NSP but lost almost all major allergen in dilution 1:625v/v in NSP or GLY. All allergens except Bermuda were stable at 6 months in HSA and all except Bermuda, English plantain and birch were stable at 12 months. Cockroach extract degraded all the allergens except for mite proteins. Molds degraded the grass, olive and plantain allergens significantly. Allergens maintained >40% of their activity after 1 month at room temperature. CONCLUSIONS: Allergen proteins have different stability profiles depending on the protein, the diluent, and the concentration. Some proteins in dilute solutions without added albumin protein lost ELISA binding activity in glycerinated and aqueous diluents.

Characterization of chromatin modified with reactive oxygen species: Recognition by autoantibodies in cancer

Objectives To study the binding of chromatin modified with reactive oxygen species (ROS) with circulating autoantibodies present in cancer patients. Design and methods Chromatin isolated from goat liver was modified by superoxide radical plus singlet oxygen and hydroxyl radicals. Sera from 47 patients with various types of cancers were tested for binding to native and modified chromatin by direct binding and competition ELISA. Results Maximum modification of thymine (54%) was found in case of chromatin modified with hydroxyl radical whereas by the combined action of singlet oxygen and superoxide anion radical, guanine was modified most (72%). Autoantibodies in cancer sera recognized modified chromatin in preference to native chromatin. Band shift assay with affinity-purified IgG from sera of cancer patients reiterated the results obtained with serum samples. Conclusion Greater recognition of ROS-modified chromatin with the autoantibodies in cancer sera is indicative of reactive-oxygen-species-induced chromatin damage in cancer patients.

A Fatal Bleeding Disorder Due to Antibody Mediated Acquired Factor X and Prothrombin Deficiency.

Abnormalities in hemostasis are well described in patients with malignant disorders. While hemostatic activation resulting in thrombosis is most often described, acquired hemorrhagic disorders have also been reported. We report a case of a fatal hemorrhagic disorder in a lymphoma patient due to an acquired Factor X and prothrombin deficiency. The patient's plasma contained a non-inhibitory IgG antibody that cross-reacted with and cleared from the circulation both Factor X and Prothrombin. The patient was a 72 year old male who was first noted to have persistant bleeding after angioplasty in 1991. Over the subsequent 10 years he had repeated bleeding episodes and was noted to have a prolonged PT and a PTT which corrected with a 50–50 mix of normal plasma. In 2002 he was diagnosed with a monocytooid B cell lymphoma. In April 2002 the patient had an extensive hemostatic evaluation by one of the authors (DIF) which is included in Table 1.These studies again showed a prolonged PT and aPTT which corrected with a 50–50 mix. Factor II activity and antigen were significantly reduced. While Factor X activity was low normal, Factor X antigen was decreased compared to the normal controls. Patient IgG was then isolated by Stap A chromatography and anit-prothrombin antibodies directed were isolated by affinity chromatography on prothrombin-sepharose. Isolated antibodies were subtyped as an IgG subclass 4. The antibodies were assayed for their interaction with prothrombin by a direct binding ELISA and found to be calcium dependent since they did not bind in the presence of 5 mM EDTA. The interaction of the affinity isolated antibody with prothrombin, Factor X and Factor IX was assessed using the Western Blot method. Surprisingly, the antibody also bound strongly to Factor X and to a lesser degree to Factor IX. Western blot analysis of the Factor X and Factor IX preparations using a monoclonal anti-prothrombin antibody failed to demonstrate any prothrombin contamination of these proteins. A competition ELISA using prothrombin, Factor X and Factor IX showed that the antibody had a 3-fold greater affinity to Factor X then prothrombin. A Western blot using purified prothrombin fragment F1.2 confirmed that the antibody bound to a metal dependent conformational determinant on the amino-terminal Gla containing region of Factor II.In April 2003 the patient developed weakness in his left leg and was found to have a right parietotemporal subdural hematoma. Factor II activity was again only 34%. Intravenous IgG 1gm/kg on two separate days did not correct his coagulation studies. In June 2003 the patient developed a new large left sudural hematoma. He rapidly deteriorated and expired. We previosuly reported a lymphoma patient who developed acquired prothrombin deficiency associated with a non-inhibitory antibody (Cancer 2001; 91: 636), however, this is the first reported case of combined acquired Factor X and Prothrombin deficiency due to a cross-reactive non-inhibitory IgG antibody.Table1:Hemostatic AssaysPatientNormal RangePT (INR)13.4(1.3)4.1–10.2PT 50/5010.3aPTT(ratio)52.5(1.8)26.5–36.1aPTT 50/5029.7Thrombin Time17.714.8–18.4Fibrinogen(mg/dL)398155–369Factor V (Activity%)12960–180Factor II (Activity%)4270–150Factor II (Antigen mcg/ml)46.1102–126Factor X (Activity%)6560–150Factor X (Antigen mcg/ml)6.27.9–11.5Lupus AnticoagulantNegativeNegative

Reactive oxygen species damaged human serum albumin in patients with type 1 diabetes mellitus: Biochemical and immunological studies

The role of hydroxyl radical ( OH) damaged human serum albumin (HSA) in type 1 diabetes has been investigated in the present study. Hydroxyl radical induced modification on HSA has been studied by UV absorption spectroscopy, ANS fluorescence and carbonyl estimation. Hydroxyl radical modified HSA was found to be highly immunogenic in rabbits as compared to native HSA. The binding characteristics of circulating autoantibodies in type 1 diabetes patients against native and modified HSA were assessed. Diabetes patients ( n = 31) were examined by direct binding ELISA and the results were compared with healthy age-matched controls ( n = 22). High degree of specific binding by 54.8% of patients sera towards OH modified HSA, in comparison to its native analogue ( p < 0.05) was observed. Sera from those type 1 diabetes patients having smoking history, high aging with high degree of disease showed substantially stronger binding to OH modified HSA over native HSA in particular. Normal human sera showed negligible binding with either antigen. Competitive inhibition ELISA reiterates the direct binding results. Gel retardation assay further substantiated the enhanced recognition of modified HSA by circulating autoantibodies in diabetes patients. The increase in total serum protein carbonyl levels in the diabetes patients was largely due to an increase in oxidized albumin. HSA of diabetes mellitus patients (DM-HSA) and normal subjects (normal-HSA) were purified on a Sephacryl S-200 HR column. Spectroscopic analysis confirmed that the DM-HSA samples contained higher levels of carbonyls than normal-HSA ( p < 0.001). DM-HSA was conformationally altered, with more exposure of its hydrophobic regions. Collectively, the oxidation of plasma proteins, especially HSA, might enhance oxidative stress in type 1 diabetes mellitus patients.