Direct Binding ELISA Research Articles

Generation of Autoantibodies in Metal-catalyzed Oxidatively Damaged DNA in Various Cancer Subjects.

Free radicals exist as unstable and highly reactive substances, occurring both in and outside the body. Free radicals are labeled as electron-hungry molecules formed from metabolism and endogenous burning of oxygen. They are transported in cells, upsetting the arrangement of molecules and instigating cellular injury. Hydroxyl radical (•OH) is one of the highly reactive free radicals, which damages the biomolecules in its close vicinity. In the present study, DNA was modified by the hydroxyl radical generated via the Fenton reaction. The •OH-oxidized/-modified DNA (Ox-DNA) was characterized by UV-visible and fluorescence spectroscopy. Thermal denaturation was performed to reveal the susceptibility of modified DNA toward heat. The role of Ox-DNA was also established in probing the presence of autoantibodies against Ox-DNA in the sera of cancer patients by direct binding ELISA. The specificity of autoantibodies was also checked by inhibition ELISA. In biophysical characterization, an increase in hyperchromicity and relative reduction of fluorescence intensity for Ox-DNA was reported compared to the native DNA analog. A thermal denaturation study revealed that Ox-DNA was highly susceptible to heat in comparison to the native conformers. The direct binding ELISA showed the prevalence of autoantibodies from cancer patient sera separated for immunoassay detection against the Ox-DNA. The generated autoantibodies against the Ox-DNA were detected as highly specific against bladder, head, neck, and lung cancer, which was further confirmed by the inhibition ELISA for the serum and IgG antibodies. The generated neoepitopes on DNA molecules are recognized as nonself by the immune system, which leads to the formation of autoantibodies in cancer patients. Therefore, our study confirmed that oxidative stress plays a role in the structural perturbation of DNA and makes it immunogenic.

Generation of autoantibodies against glycated fibrinogen: Role in diabetic nephropathy and retinopathy

The process of glycation, characterized by the non-enzymatic reaction between sugars and free amino groups on biomolecules, is a key contributor to the development and progression of both microvascular and macrovascular complications associated with diabetes, particularly due to persistent hyperglycemia. This glycation process gives rise to advanced glycation end products (AGEs), which play a central role in the pathophysiology of diabetes complications, including nephropathy. The d-ribose-mediated glycation of fibrinogen plays a central role in the pathogenesis of diabetes nephropathy (DN) and retinopathy (DR) by the generation and accumulation of advanced glycation end products (AGEs). Glycated fibrinogen with d-ribose (Rb-gly-Fb) induces structural changes that trigger an autoimmune response by generating and exposing neoepitopes on fibrinogen molecules. The present research is designed to investigate the prevalence of autoantibodies against Rb-gly-Fb in individuals with type 2 diabetes mellitus (T2DM), DN & DR. Direct binding ELISA was used to test the binding affinity of autoantibodies from patients' sera against Rb-gly-Fb and competitive ELISA was used to confirm the direct binding findings by checking the bindings of isolated IgG against Rb-gly-Fb and its native conformer. In comparison to healthy subjects, 32% of T2DM, 67% of DN and 57.85% of DR patients' samples demonstrated a strong binding affinity towards Rb-gly-Fb. Both native and Rb-gly-Fb binding by healthy subjects (HS) sera were non-significant (p > 0.05). Furthermore, the early, intermediate, and end products of glycation have been assessed through biochemical and physicochemical analysis. The biochemical markers in the patient groups were also significant (p < 0.05) in comparison to the HS group. This study not only establishes the prevalence of autoantibodies against d-ribose glycated fibrinogen in DN but also highlights the potential of glycated fibrinogen as a biomarker for the detection of DN and/or DR. These insights may open new avenues for research into novel therapeutic strategies and the prevention of diabetes-related nephropathy and retinopathy.

Increased inflammatory cytokines and oxidative stress enhanced antibody production in breast and prostate cancer patients with COVID-19 related depression.

Cancer management is highly dependent on the immune status of the patient. During the COVID-19 pandemic, a large number of people suffered from anxiety and depression, especially cancer patients. The effect of depression on breast cancer (BC) and prostate cancer (PC) patients, during the pandemic has been analyzed in this study. Levels of proinflammatory cytokines (IFN-γ, TNF-α, and IL-6) and oxidative stress markers malondialdehyde (MDA) and carbonyl content (CC) were estimated in patients' serum samples. Serum antibodies against in vitro hydroxyl radical (•OH) modified pDNA (•OH-pDNA-Abs) were estimated using direct binding and inhibition ELISA. Cancer patients showed increased levels of proinflammatory cytokines (IFN-γ, TNF-α, and IL-6) and oxidative stress markers (MDA and CC levels), which were further significantly enhanced in cancer patients with depression compared to normal healthy (NH) individuals. Increased levels of •OH-pDNA-Abs were detected in breast cancer (0.506 ± 0.063) and prostate cancer (0.441 ± 0.066) patients compared to NH subjects. Serum antibodies were found to be significantly elevated in BC patients with depression (BCD) (0.698 ± 0.078) and prostate cancer patients with depression (PCD) (0.636 ± 0.058). Inhibition ELISA also exhibited significantly high percent inhibition in BCD (68.8% ± 7.8%) and PCD (62.9% ± 8.3%) subjects compared to BC (48.9% ± 8.1%), and PC (43.4% ± 7.5%) subjects. Cancer is characterized by enhanced oxidative stress and increased inflammation, which may be exaggerated with COVID-19 related depression. High oxidative stress and compromised antioxidant homeostasis exerts alterations in DNA, leading to formation of neo-antigens, subsequently leading to the generation of antibodies. COVID-19 pandemic related depression needs to be addressed globally for improved cancer patient care and cancer disease management.

Increased Levels of Autoantibodies against ROS-Modified Proteins in Depressed Individuals with Decrease in Antibodies against SARS-CoV-2 Antigen (S1-RBD)

Coronavirus 2019 (COVID-19) disease management is highly dependent on the immune status of the infected individual. An increase in the incidence of depression has been observed during the ongoing COVID-19 pandemic. Autoantibodies against in vitro reactive oxygen species (ROS) modified BSA and Lys as well as antibodies against receptor binding domain subunit S1 (S1-RBD) (S1-RBD-Abs) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were estimated using direct binding and competition ELISA. Serum samples were also tested for fasting blood glucose (FBG), malondialdehyde (MDA), carbonyl content (CC), interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Significant structural changes were observed in ROS modified BSA and Lys. Female depressed subjects who were also smokers (F-D-S) showed the highest levels of oxidative stress (MDA and CC levels). Similarly, increased levels of autoantibodies against ROS modified proteins were detected in F-D-S subjects, in males who were depressed and in smokers (M-D-S) compared to the other subjects from the rest of the groups. However, contrary to this observation, levels of S1-RBD-Abs were found to be lowest in the F-D-S and M-D-S groups. During the pandemic, large numbers of individuals have experienced depression, which may induce excessive oxidative stress, causing modifications in circulatory proteins. Thus, the formation of neo-antigens is induced, which lead to the generation of autoantibodies. The concomitant effect of increased autoantibodies with elevated levels of IFN-γ and TNF-α possibly tilt the immune balance toward autoantibody generation rather than the formation of S1-RBD-Abs. Thus, it is important to identify individuals who are at risk of depression to determine immune status and facilitate the better management of COVID-19.

Autoantibodies Against ROS-Human Serum Albumin-A Potent Immunological marker in Depressed Individuals with Smoking History

Abstract Background: Depression is one of the significant problems in adults that accounts for up to five percent of cases worldwide. Methods: Volunteers were divided into eight groups, and their serum samples were tested for FBG, carbonyl contents, IFN-γ and TNF-α. Reactive oxygen species (ROS) modified human serum albumin (HSA) (ROSHSA) was used as an antigen and levels of serum autoantibodies were estimated by direct binding and inhibition ELISA in all subjects. Results: Significant biophysical structural modifications were observed in ROS-HSA with increased carbonyl contents compared to native-HSA (N-HSA). Significantly high levels of carbonyl content (2.68 ± 0.33 nmol/mg protein; p &gt; 0.001) and pro-inflammatory cytokines IFN-γ (7.4 ± 0.61 pg/ml; p &gt; 0.001) and TNF-α (1.47 ± 0.23 pg/ml; p &gt; 0.001) were detected in serum samples from F-D-S. Similarly, a high level of autoantibodies against ROS-HSA was observed in females who were depressed and smokers (F-D-S) group (0.89 ± 0.07; p &gt; 0.001) compared to males who were both depressed and smokers (M-D-S) (0.66 ± 0.049). Furthermore, inhibition ELISA results exhibited high recognition of serum autoantibodies from F-D-S subjects (78.6 ± 5.7 mean maximum percentage inhibition MMPI) compared to M-D-S (58.8 ± 5.2 MMPI) subjects. Conclusion: Incoherence, long term unchecked chronic psychological stress may cause oxidation of blood proteins, which subsequently result in structural alterations of biomolecules, thus generating new-epitopes, capable of inducing autoantibodies specific for ROS-modified proteins. These autoantibodies may be a potential marker for subjects suffering from depression to understand the state of immune imbalance.

D-ribose-mediated glycation of fibrinogen: Role in the induction of adaptive immune response

A nonenzymatic reaction between reducing sugars and amino groups of proteins results in the formation of advanced glycation end products, which are linked to a number of chronic progressive diseases with macro- and microvascular complications. In this research, we sought to ascertain the immunological response to d-ibose-glycated fibrinogen. New Zealand White female rabbits were immunized with native and d-ribose-glycated (Rb-gly-Fb) fibrinogen and used for studying the immunological response. Serum from these rabbits analyzed using direct binding and competitive inhibition ELISA was found to contain a high titer of antibodies against Rb-gly-Fb; Rb-gly-Fb was much more immunogenic than its native form. The IgG against Rb-gly-Fb (Rb-gly-Fb-IgG) was highly specific against the immunogenic protein. Moreover, histopathology and immunofluorescence studies revealed the deposition of the Rb-gly-Fb-IgG immune complex in the glomerular basement membrane of the kidneys of immunized rabbits. Furthermore, immunization with Rb-gly-Fb increased the expression of genes encoding proinflammatory cytokines, tumour necrosis factor α, interleukin-6, interleukin-1β, and interferon-gamma, which is indicative of increased inflammation and the antigenic role of Rb-gly-Fb in provoking an immune response.

34920 Clinical immunogenicity of daxibotulinumtoxinA for injection (DAXI) in glabellar lines including subjects with multiple exposures: Pooled data from the SAKURA phase 3 trials

DAXI is a novel botulinum toxin type A (BoNTA) containing purified 150-kD core neurotoxin (daxibotulinumtoxinA) and proprietary stabilizing peptide (RTP004). As with any biologic, it was important to assess DAXI’s immunogenic potential. Neutralizing antibodies to daxibotulinumtoxinA and binding antibodies to daxibotulinumtoxinA or RTP004 were assessed in three phase 3 glabellar line studies (two placebo-controlled, single-dose studies [SAKURA 1 and 2] and an open-label study [SAKURA 3] of ≤3 treatments of 40U DAXI). Binding antibodies were detected by validated direct-binding ELISA in a multi-tiered (screening, confirmatory, and titration) assay. Subjects positive for daxibotulinumtoxinA-binding antibodies were evaluated for neutralizing antibodies (mouse protection assay). Overall, 2786 subjects received DAXI (n = 882, ≥2 treatments) and 2823 subjects were exposed to RTP004 as DAXI (n = 2786) or placebo (n = 203) (n = 914, ≥2 exposures). Of these, 2737 and 2772 were evaluable for daxibotulinumtoxinA and RTP004 antibodies, respectively. At baseline, 12/2737 (0.4%) subjects had binding antibodies to BoNTA and 66/2772 subjects (2.4%) had detectable RTP004 antibody titers. Treatment-emergent daxibotulinumtoxinA-binding antibodies were detected in 20 subjects (0.7%), and treatment-boosted binding antibodies in 1 subject (<0.1%). No subject developed daxibotulinumtoxinA-neutralizing antibodies. Treatment-induced anti-RTP004 binding antibodies were detected in 35 (1.3%) subjects. In most subjects, binding antibodies were transient. No subject had binding antibodies to both daxibotulinumtoxinA and RTP004. Transient binding antibodies to daxibotulinumtoxinA or RTP004 were found to not impact clinical efficacy, safety, or duration of action. No subjects developed neutralizing antibodies to daxibotulinumtoxinA. The findings suggest that DAXI administration results in a low incidence of antibody formation.

IDF21-0074 Role of methylglyoxal modified low density lipoprotein in type 2 diabetes mellitus and its role in periodontal disease

Background: Glycation, a physiological and pathological process mainly affects proteins, nucleic acids and lipids. Methylglyoxal (MGO) along with free radicals is known to contribute to the pathogenesis of several diseases, in disorders particularly having enhanced production of acetone and amino-acetone such as diabetes mellitus (DM). The accumulation of MGO induces β-cells dysfunction, insulin resistance as well as impairment of endothelial functions in DM patients. Also, the increased level of MGO intensifies ROS generation and AGEs formation which further accelerates the co-morbidities associated with type 2 diabetes mellitus (T2DM). There is an increased susceptibility and severity of periodontal diseases in case of patients with diabetes and vice-versa. Aim: The likely role of native low density lipoprotein (LDL) and glycated LDL in type 2 diabetes mellitus (T2DM) and periodontitis (PD) as well as in T2DM+PD patients has been evaluated. Method: Sera from T2DM+PD T2DM and PD patients (n=80), were screened for the presence of antibodies against MGO-LDL and native LDL. Besides, the conformational changes in LDL isolated from T2DM+PD, T2DM and PD patients (possibly due to glycoxidaton) were also studied by UV absorption, fluorescence and CD spectroscopy. ThT, Comet assay, SEM and TEM were also done. Cellular toxicity of the isolated LDL from both T2DM+PD, T2DM and PD patients was demonstrated by comet assay. Biochemical parameters like increase in carbonyl content, TBARS, TNF α and IL1β indicate LDL oxidation and increase in pro-inflammatory cytokines. Results: MGO-LDL (in comparison to parallel control) proved to be a potent antigen showing very high binding with T2DM+PD, T2DM and PD sera as observed in direct binding ELISA. The stronger binding of T2DM+PD, T2DM and PD auto-antibodies to MGO-LDL suggests the importance of this modification in the onset and pathogenesis of these diseases. Inhibition ELISA results showed very high percent inhibition of antibodies from T2DM+PD, T2DM and PD patients by MGO-LDL in comparison to native LDL pointing towards the possible role of glycoxidation of LDL in these patients. The modified LDL might have further generated high levels of circulating auto-antibodies in the sera of these patients. Further, to ascertain the specificity of auto-antibodies from T2DM+PD, T2DM and PD patients, competitive inhibition ELISA studies were done on IgG isolated from the sera of these patients. The samples of isolated IgG from T2DM+PD (T2DM+PD-IgG), T2DM (T2DM-IgG) and PD (PD-IgG) showed very strong recognition of MGO-LDL in comparison to the native counterpart. The specificity and affinity of T2DM+PD, T2DM and PD auto-antibodies towards MGO-LDL was further confirmed by gel retardation assay. The gel mobility results confirmed specificity of MGO-LDL towards auto-antibodies from T2DM and PD patients. Furthermore, high recognition of experimentally induced anti-MGO-LDL antibodies by T2DM+PD-LDL, T2DM-LDL and PD-LDL compared to NHS-LDL confirmed that the observed structural damage in T2DM+PD-LDL, T2DM-LDL and PD-LDL is due to glycoxidation. Discussion: The neo-epitopes on LDL created by its exposure to MGO appear to be responsible for induction and elevated levels of these antibodies, and thus MGO-LDL may be considered as potential antigenic candidate for auto-immune response in diabetes and periodontitis patients with a potential for biomarker development.

Glycation end-products specific auto-antibodies in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease, which is highly inflammatory. Compared to a healthy control group, SLE patients exhibit a higher concentration of advanced glycation end products (AGEs) and a lower concentration of receptors for AGEs (RAGE) in serum, however, the exact aetiology is still unclear. In the present study, non-enzymatic glycation induced modification of human serum albumin (HSA) has been studied by biophysical techniques. Glycated HSA (G-HSA) was used as an antigen, and serum autoantibody levels were estimated in SLE and normal humans (NH) against it, using direct binding ELISA and competitive inhibition ELISA. Compared to N-HSA, remarkable structural modifications were observed in G-HSA. Modified HSA also showed increased pentosidine fluorescence (213.7 ± 13.4 AU). Glycation of HSA induced a conversion of α-helix and random coil to β-sheet and β-turns. Serum immuno assays results exhibited significantly (p < 0.001) higher binding of G-HSA with serum autoantibodies from SLE patients when compared with native HSA (N-HSA). Furthermore, competitive ELISA results showed significantly (p < 0.001) high percent inhibition of serum IgG from SLE patients with modified antigen. Chronic inflammation with excessive oxidative stress in SLE patients could be a possible reason for structural alterations in blood proteins, generating highly immunogenic unique new-epitopes. These in turn induce the generation of specific autoantibodies against G-HSA that may serve as a potential biomarker for SLE pathogenesis.

Biophysical and Immunological Characterization of Carbamylated-DNA: Serum Autoantibodies of Diabetic Nephropathy Patients Exhibit Strong Binding with Carbamylated-DNA

Isocyanates (ICN) have drawn considerable attention in recent past as they can react with a variety of nucleophiles including DNA, proteins and cause damage(s). However, pathophysiological implications resulting from the occupational and accidental exposures of ICN are yet too elusive. It may be produced in high concentrations in the conditions of chronic renal failure and chronic inflammatory diseases (diabetes mellitus, rheumatoid arthritis, etc.). In this study, mammalian dsDNA was modified with ICN and characterized by UV, fluorescence, FT-IR and thermal melting methods. ICN mainly reacts with exocyclic nitrogen of DNA bases. Antibodies against carbamylated-DNA were raised in rabbits and characterized by direct binding and inhibition ELISA. The presence of anti-carbamylated-DNA autoantibodies in the sera of diabetic nephropathy patients was evaluated by ELISA. Carbamylated-DNA exhibited hyperchromicity at 260 nm and depicted increase in ethidium bromide-assisted fluorescence as compared to the native DNA. Furthermore, the appearance of prominent new peaks in FT-IR profile at different wave numbers suggested carbamoylation-induced changes in the vibration of sugar-phosphate backbone, base(s) etc. Carbamylated-DNA was highly immunogenic as compared to the native DNA. Experimental induction of antibodies against carbamylated-DNA and presence of autoantibodies against carbamylated-DNA in the sera of diabetic nephropathic patients point towards the significance of carbamylated-DNA in diabetic nephropathy.

Functional Similarity of Proposed Biosimilar ABP 798 with Rituximab

▪BackgroundRegulatory guidelines governing the development of biosimilars require that proposed biosimilars undergo comprehensive structural and functional characterization prior to the initiation of clinical investigation. ABP 798 is being developed as a biosimilar to rituximab. The reference product is approved in the US for treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemia, severe rheumatoid arthritis, granulomatosis with polyangiitis, and microscopic polyangiitis. Development of biosimilars is challenging as even small variations in structure have the potential to alter the function of the molecule and thus may impact efficacy, safety, and/or bioavailability. Here we compare ABP 798 and rituximab reference product with respect to binding properties (CD20, C1q, FcRn, and Fcγ receptors), antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and induction of apoptosis. The reference product was sourced from the US (rituximab US) and the EU (rituximab EU).MethodsBinding of ABP 798 and rituximab to the CD20 antigen was characterized using a cell-based CD20 binding assay utilizing the CD20 expressing WIL2-S human B-lymphoblastoid cell line. A direct binding ELISA was used to assess the binding of the Fc domain of ABP 798 to C1q. Binding of the Fc moiety of ABP 798 and rituximab to FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa (158V and 158F) were evaluated in AlphaLISAÒ competitive binding assays. Binding to FcRn was evaluated by an AlphaScreenÒ competitive binding assay. ADCC activity was evaluated in a functional cell-based assay, with WIL2-S cells used as target cells and NK92-MI cells, stably transfected with human CD16 (FcγRIIIa [158V]), used as effector cells. CDC activity was evaluated in a functional cell-based assay using the WIL2-S cell line and baby rabbit complement. Induction of apoptosis was assessed by measuring activation of caspase 3/7 in SU-DHL-4 cells, a CD20 expressing human B cell lymphoma cell line.ResultsRelative binding (%) as compared to an ABP 798 reference standard was similar between ABP 798 and rituximab (Table 1).The dose response profiles and relative activity for ADCC and CDC were also similar (ADCC relative cytotoxicity: ABP 798, 88%; rituximab US, 85%; rituximab EU, 86%; CDC relative cytotoxicity: ABP 798, 103%; rituximab US, 102%; rituximab EU, 104%). The dose-response profile for induction of caspase 3/7 was similar between ABP 798 and rituximab as well.ConclusionsThe results presented here demonstrate that ABP 798 is similar to rituximab sourced from both US and EU in terms of biological activity across the range of tested binding and functional assays. The functional assays tested the major and minor mechanisms of action related to clinical efficacy across approved rituximab indications and these results provide a foundation for further clinical development of ABP 798. [Display omitted] DisclosuresMcBride:amgen: Employment; amgen: Other: stockholder. Maher:amgen: Employment; amgen: Other: stockholder. Sweet:amgen: Employment; amgen: Other: stockholder. Foltz:amgen: Employment; amgen: Other: stockholder. Kuhns:amgen: Other: stockholder; amgen: Employment.

Comparison of the SARS-CoV-2 Surrogate Virus Neutralization Test (sVNT) Assay and Direct Binding ELISA (S-IgG) with the Cytopathic Effect Assay (CPE) in Analyzing the Neutralization Antibody of Vaccination People

The safety and effectiveness of the COVID19 vaccine are the key in vaccine development. Due to the difficulty and dangerous of virus neutralization experiment, many replacement assays were developed for testing neutralization antibody.

Hydroxyl radical induced structural perturbations make insulin highly immunogenic and generate an auto-immune response in type 2 diabetes mellitus

Reactive oxygen species (ROS) cause oxidative damage to proteins and generate deleterious by-products which induce a breakdown of immune tolerance and produce antibodies against host macromolecules with implication in human diseases. This study characterizes the hydroxyl radical (OH) modifications of insulin, evaluates its cytotoxicity and immunogenicity, and probes its role in type 2 diabetes (T2DM) autoimmunity. The results demonstrate susceptibility of insulin to modifications induced by OH, causing exposure of its chromophoric aromatic amino acid residues, quenching of tyrosine fluorescence intensity, loss of α-helix and gain in β content. Modification causes re-arrangement of native interactions of the aromatic residues in insulin. It enhanced the carbonyl content in insulin, exposed its hydrophobic patches and generated non-fibrillar, amorphous type of aggregates that are cytotoxic in nature. Native insulin induced low titre antibodies in immunized rabbits, whereas OH modified insulin generated a strong immune response. Competitive ELISA studies showed high specificity of antibodies generated against OH modified insulin towards the modified protein. Cross reaction studies showed the presence of common antigenic determinants on various oxidised proteins. Since T2DM patients show increased ROS production, oxidation of insulin is expected to occur, which might amplify autoimmune reactions against insulin. True to the assumption, direct binding ELISA showed the presence of anti-OH insulin circulating antibodies in T2DM patients which are specific for the oxidized insulin. In conclusion, insulin loses structural integrity to OH, forms cytotoxic amorphous aggregates, turns highly immunogenic and elicits humoral response in T2DM patients.

Methylglyoxal mediated glycation leads to neo-epitopes generation in fibrinogen: Role in the induction of adaptive immune response

In diabetes mellitus, hyperglycemia mediated non-enzymatic glycosylation of proteins results in the pathogenesis of diabetes-associated secondary complications via the generation of advanced glycation end products (AGEs). The focus of this study is to reveal the immunological aspects of methylglyoxal (MG) mediated glycation of fibrinogen protein. The induced immunogenicity of modified fibrinogen is analyzed by direct binding and inhibition ELISA. Direct binding ELISA confirmed that MG glycated fibrinogen (MG-Fib) is highly immunogenic and induces a high titer of antibodies in comparison to its native analog. Cross-reactivity and antigen-binding specificity of induced antibodies were confirmed by inhibition ELISA. The enhanced affinity of immunoglobulin G (IgG) from immunized rabbits' sera and MG glycated fibrinogen is probably the aftermath of neo-epitopes generation in the native structure of protein upon modification. Thus, we deduce that under the glycative stress, MG-mediated structural alterations in fibrinogen could induce the generation of antibodies which might serve as a potential biomarker in diabetes mellitus and its associated secondary disorders.

The Neoepitopes on Methylglyoxal- (MG-) Glycated Fibrinogen Generate Autoimmune Response: Its Role in Diabetes, Atherosclerosis, and Diabetic Atherosclerosis Subjects.

Objectives In diabetes mellitus, hyperglycemia-mediated nonenzymatic glycosylation of fibrinogen protein plays a crucial role in the pathogenesis of micro- and macrovascular complications especially atherosclerosis via the generation of advanced glycation end products (AGEs). Methylglyoxal (MG) induces glycation of fibrinogen, resulting in structural alterations that lead to autoimmune response via the generation of neoepitopes on protein molecules. The present study was designed to probe the prevalence of autoantibodies against MG-glycated fibrinogen (MG-Fib) in type 2 diabetes mellitus (T2DM), atherosclerosis (ATH), and diabetic atherosclerosis (T2DM-ATH) patients. Design and Methods. The binding affinity of autoantibodies in patients' sera (T2DM, n = 100; ATH, n = 100; and T2DM-ATH, n = 100) and isolated immunoglobulin G (IgG) against native fibrinogen (N-Fib) and MG-Fib to healthy subjects (HS, n = 50) was accessed by direct binding ELISA. The results of direct binding were further validated by competitive/inhibition ELISA. Moreover, AGE detection, ketoamines, protein carbonyls, hydroxymethylfurfural (HMF), thiobarbituric acid reactive substances (TBARS), and carboxymethyllysine (CML) concentrations in patients' sera were also determined. Furthermore, free lysine and free arginine residues were also estimated. Results The high binding affinity was observed in 54% of T2DM, 33% of ATH, and 65% of T2DM-ATH patients' samples with respect to healthy subjects against MG-Fib antigen in comparison to N-Fib (p < 0.05 to p < 0.0001). HS sera showed nonsignificant binding (p > 0.05) with N-Fib and MG-Fib. Other biochemical parameters were also found to be significant (p < 0.05) in the patient groups with respect to the HS group. Conclusions These findings in the future might pave a way to authenticate fibrinogen as a biomarker for the early detection of diabetes-associated micro- and macrovascular complications.

Endometrial cancer patients have high affinity antibodies for estrogen metabolite-receptor aggregate: A potential biomarker for EC.

Elevated levels of 16α-hydroxyestrone (16α-OHE1 ) have been described in endometrial cancer (EC) and estrogen receptors (ER) expressed in endometrial tissue, but research on their combined role is lacking. We aimed to investigate the affinity and binding specificity of EC antibodies against the 16α-OHE1 -ERα aggregate in the serum of EC patients. Specificities of EC antibodies were also evaluated according to various clinical characteristics found in these cancer patients. The binding specificity and affinity of EC antibodies against 16α-OHE1 -ERα in the serum of 120 EC patients were evaluated by direct binding and competition ELISA and quantitative precipitation titration. Binding of EC antibodies was also determined according to various clinical characteristics in EC patients through competition ELISA. Antibodies from EC patients demonstrated high recognition of 16α-OHE1 -ERα compared to ERα (P < 0.05) or 16α-OHE1 (P < 0.001). The relative affinity of EC IgG was 1.49 × 10-7 M, 1.34 × 10-6 M and 1.13 × 10-6 M for 16α-OHE1 -ERα, ERα and 16α-OHE1 , respectively. Several factors, such as obesity, postmenopausal status, use of hormonal therapy, ER and progesterone receptor (PR) status, low 2-OHE1 /16α-OHE1 ratio, chemotherapy and hypertension, augment the production of antibodies against 16α-OHE1 -ERα in EC patients. 16α-OHE1 -ERα is a high-affinity antigen for EC antibodies in the serum of EC patients and might function as a biomarker for this disease. Furthermore, several factors enhanced the production of antibodies against 16α-OHE1 -ERα in the sera of these EC patients.

The neoepitopes on methylglyoxal (MG) glycated LDL create autoimmune response; autoimmunity detection in T2DM patients with varying disease duration

AimsNon-enzymatic reaction of biomolecules leads to the formation of advanced glycation end products (AGEs). AGEs plays significant role in the pathophysiology of type 2 diabetes mellitus. Methylglyoxal (MG) is a highly reactive carbonyl compound which causes formation of early (ketoamines), intermediate (dicarbonyls) and advanced glycation end products (AGEs). Glycation also results in the generation of free radicals causing structural perturbations which leads to the generation of neoantigenic epitopes on LDL molecules. The aim of the present study was to investigate whether the modification of LDL results in auto-antibodies generation in type 2 diabetes patients'. MethodsThe binding affinity of circulating autoantibodies in patients against native and MG modified LDL were assessed as compared with healthy and age-matched controls (n = 50) and T2DM patients with disease duration (DD) 5–15 yrs (n = 80) and DD > 15 yrs (n = 50) were examined by direct binding ELISA. KeyfindingsThe high affinity binding were observed in 50% of T2DM with DD 5–15 and 62% of T2DM with DD > 15 of patient's sera antibodies to MG-LDL antigen, in comparison to its native analog (P < 0.05). NHS sera showed negligible binding with both native and glycated LDL. Competitive inhibition ELISA results exhibit greater affinity sera IgG than the direct binding ELISA results. The increase in glycation intermediate and ends product were also observed in T2DM patient's sera and NHS sera. SignificanceThere might be the generation of neoantigenic epitopes on LDL macromoleucle which results in generation of antibodies in T2DM. The prevalence of antibodies was dependent on disease duration.

Preferential recognition of epitopes on peroxynitrite-modified alpha-2-macroglobulin by circulating autoantibodies in rheumatoid arthritis patients

Autoimmune responses against post-translationally modified antigens are a hallmark of several autoimmune diseases. In this work, we have studied the changes in alpha-2-macroglobulin (α2M) upon modification by peroxynitrite. Furthermore, we have evaluated the immunogenicity of modified α2M in experimental rabbits and rheumatoid arthritis (RA) patients. Peroxynitrite-modified α2M showed disturbed microenvironment and altered aromatic residues under UV and fluorescence studies. Aggregation, reduction in β-sheet content, production of nitrotyrosine and shift in amide I and II bands were observed in the modified α2M by polyacrylamide gel electrophoresis besides CD and FTIR spectroscopic analysis. The exposure of hydrophobic clusters and changes in contact positions were observed in ANS and ThT binding assays. Immunological studies using ELISA showed peroxynitrite-modified α2M as highly immunogenic producing high titre of specific antibodies in immunized rabbits. Cross-reactivity studies revealed the polyspecificity of the elicited antibodies. Direct binding ELISA and competitive inhibition studies confirmed the presence of circulating antibodies in the sera of RA patients having high specificity towards the peroxynitrite-modified α2M as compared to the native α2M. Sera from healthy (normal) human subjects showed lower binding with the native and modified protein. This study confirms that peroxynitrite induces structural modifications in α2M and makes it immunogenic. The presence of neo-antigenic determinants on modified α2M with enhanced binding for circulating autoantibodies in RA patients could offer new possibilities for diagnosis and etiopathology of the disease. Communicated by Ramaswamy H. Sarma

Predictors of high molecular weight adiponectin in patients with colorectal cancer

Non-enzymatic reaction of biomolecules leads to the formation of advanced glycation end products (AGEs). AGEs plays significant role in the pathophysiology of type 2 diabetes mellitus. Methylglyoxal (MG) is a highly reactive carbonyl compound which causes formation of early (ketoamines), intermediate (dicarbonyls) and advanced glycation end products (AGEs). Glycation also results in the generation of free radicals causing structural perturbations which leads to the generation of neoantigenic epitopes on LDL molecules. The aim of the present study was to investigate whether the modification of LDL results in auto-antibodies generation in type 2 diabetes patients'.The binding affinity of circulating autoantibodies in patients against native and MG modified LDL were assessed as compared with healthy and age-matched controls (n = 50) and T2DM patients with disease duration (DD) 5–15 yrs (n = 80) and DD > 15 yrs (n = 50) were examined by direct binding ELISA.The high affinity binding were observed in 50% of T2DM with DD 5–15 and 62% of T2DM with DD > 15 of patient's sera antibodies to MG-LDL antigen, in comparison to its native analog (P < 0.05). NHS sera showed negligible binding with both native and glycated LDL. Competitive inhibition ELISA results exhibit greater affinity sera IgG than the direct binding ELISA results. The increase in glycation intermediate and ends product were also observed in T2DM patient's sera and NHS sera.There might be the generation of neoantigenic epitopes on LDL macromoleucle which results in generation of antibodies in T2DM. The prevalence of antibodies was dependent on disease duration.

Prevalence of auto-antibodies against D-ribose-glycated-hemoglobin in diabetes mellitus.

Glycation of biological macromolecules, due to hyperglycemia, promotes the formation of advanced glycation end products (AGEs). It is accelerated in diabetic patients and is responsible for the pathophysiology and progression of diabetes. Previous reports have shown that amount of AGEs formation and glycation-induced structural damage is higher in hemoglobin (Hb) than other proteins present in blood. In our previous study, we have shown structural changes in Hb by D-ribose which may result into the generation of immunogenic neo-epitopes. Thus, we hypothesized that D-ribose induced structural perturbations in Hb, could result in the formation of neo-epitopes which may provoke an auto-immune response and may also be involved in the immuno-pathogenesis of diabetes type-2 associated complications. Therefore, in the current study, we analyzed the prevalence of autoantibodies in diabetic patient's sera against D-ribose glycated-Hb by direct binding and competitive ELISA. Direct binding ELISA confirmed that autoantibodies in diabetic patients exhibit significantly high binding with D-ribose glycated-Hb as compared to its native form. The antigen binding specificity of these antibodies was also screened by competitive inhibition ELISA. We also used D-glucose glycated-Hb as a positive control to detect the presence of auto-antibodies by direct binding and inhibiton ELISA. We found that D-glucose glycated-Hb binds with T2DM samples but the affinity to binding is lower than D-ribose glycated-Hb. The overall findings of this study suggest the prevalence of circulating autoantibodies against D-ribose glycated-Hb in diabetic patients and thus, the level of these autoantibodies may be used as biomarker for progression of diabetes.