Immunoglobulin Fraction Research Articles

Experimental Testing of a Size-Exclusion Chromatography Method Used for Evaluation of Molecular Parameters of Equine Anti-Ebola Immunoglobulin

Haemorrhagic fever caused by the Ebola virus is a highly hazardous infectious disease with a mortality rate of 50– 90 %. Heterologous immunoglobulins with a high virus-neutralizing titer are an important element of the WHO-endorsed set of measures for emergency prevention and treatment of the disease. Specific activity of these products is largely determined by their fractional composition, and, in particular, by molecular mass distribution (MMD). The size-exclusion-high-performance liquid chromatography (SEC-HPLC) has traditionally been used for determination of the MMD of the target protein in human immunoglobulin-based products. The use of this method for evaluation of molecular parameters of heterologous immunoglobulin requires confirmation of its specificity, accuracy and precision, and establishment of the chromatographic system suitability criteria in the context of a new test object.The aimof the study was to test the applicability of the SEC-HPLC method to the assessment of molecular parameters of anti-Ebola immunoglobulin derived from horse serum.Materials and methods: three batches of purified equine anti-Ebola immunoglobulin were used in the study. Normal equine and human immunoglobulins of the IgG isotype were used as reference standards. The HPLC test procedures described in the European Pharmacopoeia 9.6 and State Pharmacopoeia of the Russian Federation, 14th ed., were used for determination of monomers and other immunoglobulin fractions. An Agilent 1260 Infinity (Agilent, USA) HPLC system with a diode array detector and an Agilent Bio SEC-3 HPLC column were used for quality evaluation of the tested products.Results: the resolution factor between IgG monomer and dimer peaks (1.69 and 2.10), and the chromatographic column efficiency (>2000) make it possible to use the SEC-HPLC system for evaluation of molecular parameters of heterologous immunoglobulin. The study demonstrated reproducibility of the test procedure.Conclusions: the study confirmed the applicability of the SEC-HPLC procedure for evaluation of molecular parameters of anti-Ebola immunoglobulin derived from horse serum. It demonstrated the compliance of the purified immunoglobulin to the national and international quality requirements in terms of «Molecular parameters».

EFFECT OF CHLORELLA VULGARIS ALGAE SUPPLEMENTATION ON GROWTH PERFORMANCE OF SUCKLING FRIESIAN CALVES

Twenty newly born male and female Friesian calves with an average live body weight (LBW) of 30.40±0.36 kg were divided into five similar groups (2 male + 2 female in each group) and continued in a feeding trial until weaning to study the effect of supplementing the algae on suckling calves growth performance. Diet of cows and their suckling calves in G1 did not supplemented with algae and served as a control diet. Whereas, cows and their suckling calves were supplemented with algae and their media at levels of 2 ml (G2) and 4 ml (G3) / kg LBW, as low and high levels, respectively. While, cows in G4 and G5 were unsupplemented with algae, but their suckling calves were supplemented with Chlorella vulgaris algae and their media at the same above levels. Results showed that groups supplemented with high level of algae (G3 and G5) were recorded significantly (P<0.05) the highest digestibility of all nutrients and feeding values followed by groups supplemented with low level of algae (G2 and G4), while control group (G1) had the lowest values. The DMI were the same for different groups, while the intake of TDN and DCP were significantly (P<0.05) higher for G3 and G5 compared to those of control one (G1), but the low levels of algae rations (G2 and G4) were insignificant higher than those of control ration as well. Mostly, blood serum measurements (total protein, globulin and albumin) in addition of creatinine and liver enzymes activity (AST and ALT) were markedly improved versus to those of control group (G1). Moreover, the concentrations of immunoglobulin fractions (IgA, IgM and IgG) were increased significantly (P<0.05) with chlorella supplementation and increased with increasing the level of chlorella supplement. Weaning weight and average daily gain of suckling calves increased significantly (P<0.05) with chlorella algae supplementation as well as with increasing supplementation level. Chlorella supplementation improved significantly (P<0.05) feed conversion, being decreased the amounts of DM, TDN and DCP required per one kg live weight gain. Also, chlorella supplementation improved economic efficiency, which decreased feed cost per one kg live weight gain and increased the total and net revenue of live weight gain and economic efficiency.

DETERMINATION OF PARAPROTEIN IN SERUM AND URINE BY ELECTROPHORESIS FOR DIAGNOSING MULTIPLE MYELOMA (MM), EXPERIENCES FROM THE PHF UNIVERSITY CLINIC OF HEMATOLOGY FOR THE PERIOD FROM 2015 TO 2017

Introduction: Multiple myeloma is a clonal disorder characterized by the proliferation of mature B cells (plasma cells) in the bone marrow i.e. the appearance of monoclonal immunoglobulin fraction (M component, paraprotein) in the serum or occasionally only during electrophoresis, in the urine. Generally, multiple myeloma is the most common primary bone cancer which can be defined as a malignant tumor of the bone marrow. Regarding the benign form (MGUS, monoclonal gammopathy of undetermined significance), it should be noted that it is about 100 times more common than the myeloma. In order to diagnose this disease, serum and urine are examined by electrophoresis because this technique firstly enables the separation of the proteins and then the detection of the desired group of proteins, in the specific case, the paraprotein group. However, the presence of such proteins in serum or urine is not always a sign for multiple myeloma, so in order to diagnose this disease, additional tests need to be done. These tests include: determination of the number of blood cells, determination of the level of blood calcium, determination of the level of urea and creatinine, determination of the percentage of plasma cells in the bone marrow. Besides these tests, the usual additional test includes detection of β–2–microglobulin, which is another protein that can be produced by multiple myeloma cells.Aims of the study: Overview of the diagnosis of multiple myeloma by determination of the paraprotein in serum or urine by electrophoresis; Determination of the prevalence of multiple myeloma in patients on the territory of the Republic of N. Macedonia from 2015 to 2017; Analysis of the obtained data in relation to the gender and the age of the patients.Materials and methods: For the purpose of this study, data obtained from multiple myeloma patients, diagnosed and treated at the PHF University Clinic of Hematology, were analyzed. The data were collected over a period of three years (from 2015 to 2017). The study included patients over 40 years of age. The entire research was done in the laboratory of PHF University Clinic of Hematology.Results: According to the research, 19 patients with multiple myeloma were registered in 2015, 22 patients with multiple myeloma were registered in 2016 and 25 patients with multiple myeloma were registered in 2017. In terms of gender, this disease is more common in men and in terms of age, the same disease is most prevalent in patients between 70 and 90 years of age.Conclusion: The incidence of multiple myeloma is approximately 5 to 7 patients per 100,000 persons annually. The disease data obtained in this study are identical to those of the World Health Organization (WHO). In most patients who were diagnosed with multiple myeloma, renal dysfunction was also observed.

Reference change values of M-protein, free light chain and immunoglobulins in monoclonal gammopathy

ObjectivesClinical decisions in patients with monoclonal gammopathies may be highly imprecise because of variations of parameters used in diagnosis. In this study, we aimed to calculate the variation in M-protein, free light chains (FLCs), and immunoglobulins in respective patients. Design & methodsWe analyzed the data of clinically stable patients with monoclonal gammopathy (MG), which were monitored for 7-years to determine the biological variations and reference change values (RCV) of serum M-protein, monoclonal serum FLCs and immunoglobulin (Ig) concentrations. Patients that were included in the study had no change in diagnosis and showed <5 g/L change in serum M-protein during the monitoring. From the patients included at least 3 consecutive samples were analyzed within 8 months and 7 years of initial diagnosis. ResultsThe total coefficient of variations (CV) was calculated for M-protein and involved/uninvolved fractions of FLCs and immunoglobulins. From 38 patients and 456 samples that were included in the study, the total CVs were calculated for serial M-proteins (8.9%), serum involved FLCs (iFLC, 21.4%), involved Ig (i-Ig, 8.7%) and uninvolved Ig (u-Ig, 9.1%). Combining these CVs and the interassay analytical CVs, we calculated the biological CV for the serum M-protein (8.4%), serum iFLC concentration (21.1%), i-Ig (8.6%) and u-Ig (9.0%). A significant correlation was found in multiple myeloma patients between the κ/λ light chain ratio (rFLC) with i-Ig, the difference between i-Ig level and u-Ig level (d-Ig) and ratio Ig (r-Ig) (r = 0.790, 0.703 and 0.711, respectively). These correlations were not found in patients suffering from MG of undetermined significance and smoldering multiple myeloma. Conclusionsi-Ig determinations may be an alternative to M-protein for MGs. The variations in serum FLC measurements during MG monitoring were greater than those observed in serum M-proteins and therefore need to be more rigorously revised for recommendations.

Тhe technology of creation and quality testing of immunofluorescent probes with dye alexa-488 for analysis of celular populations by flow cytometry

Fluorescent probes based on monoclonal antibodies (MAb) are widely used in scientific and clinical research in the field of oncology, hematology, immunology, epidemiology. Objective: to create of fluorescent probes based on the MAb and the fluorescent dye Alexa-488 for the analysis of cellular populations by flow cytometry. Materials and methods. MAb to B lymphocyte antigen (clone ICO-180), fluorescent dye Alexa-488 were used in the work. MAb was isolated from ascitic fluid by combined purification of the immunoglobulin fraction with caprylic acid and salting out with ammonium sulfate. Gel filtration on a PD-10 column was used to purify the conjugates (immunofluorescent probes, IFP), the concentration and labeling density of the IFP were determined spectrophotometrically. The determination of the working titer of the IFP was performed using the antibody titration method proposed by C.C. Stewart. Results. The optimal time of incubation of MAb with a fluorophore was experimentally determined. The optimal conditions for labeling MAb of the IСO series with the dye are: a carbonate buffer with pH 8,3, the concentration of antibodies in the reaction mixture is 1 mg/ml, molar ratio of active dye – 10–100 mmol per 1 mmol of protein, the incubation time is 90 minutes, the temperature is 18–25 °C. We obtained a panel of conjugates of MAb with Alexa-488, differing in their different labeling densities. Evaluation of the biological activity of the resulting conjugates was carried out on peripheral blood cells of donors in the concentration range of MAb 0,5–100 μg/ml. Conclusion. The optimal conditions for labeling MAb of the IСO series with the dye are: a carbonate buffer with pH 8,3, the concentration of antibodies in the reaction mixture is 1 mg/ml, the incubation time is 90 minutes, the temperature is 18–25 °C. The optimum density of labeling is in the range 5–13,5 M:M, the optimal concentration of antibodies is in the range of 5–25 μg/ml.

Increased central adiposity is associated with pro-inflammatory immunoglobulin G N-glycans

BackgroundIncreased body fat may be associated with an increased risk of developing an underlying pro-inflammatory state, thus leading to greater risk of developing certain chronic conditions. Immunoglobulin G has the ability to exert both anti- and pro-inflammatory effects, and the N-glycosylation of the fragment crystallisable portion is involved in mediating this process. Body mass index, a rudimentary yet gold standard indication for body fat, has been shown to be associated with agalactosylated immunoglobulin G N-glycans. AimWe aimed to determine the association between increased body fat and the immunoglobulin G glycosylation features, comparing body mass index to other measures of body fat distribution. MethodsWe investigated a sample of 637 community-based 45–69 year olds, with mixed phenotypes, residing in Busselton, Western Australia. Body mass index and the waist-to-hip and waist-to-height ratios were calculated using anthropometry, while dual-energy x-ray absorptiometry was performed to gain an accurate measure of total and area specific body fat. Serum immunoglobulin GN-glycans were analysed by ultra-performance liquid chromatography. ResultsTwenty-two N-glycan peaks were found to be associated with at least one of the fat measures. While the previous association of body mass index to agalactosylated immunoglobulin G was replicated, measures of central adiposity explained the most variation in the immunoglobulin G glycome. ConclusionCentral adiposity is associated with an increased pro-inflammatory fraction of immunoglobulin G, suggesting that the android/gynoid ratio or waist-to-height ratio instead be considered when controlling for adiposity in immunoglobulin G glycome biomarker studies.

BIOLOGY OF THE ORAL CAVITY IN CHILDREN LIVING IN THE CITY OF CHIRCHIK

The study of microbiological and immunological parameters of the oral cavity among children living in the industrial city of Chirchik, the total number of anaerobes, aerobes, phagocytic activity of neutrophils, the level of lysozyme and the immunoglobulin A secretion fraction of the secretory fraction (sIgA) has been studied. To address this goal, we conducted microbiological and immunological studies in 57 children living in the town of Chirchik. All these children, depending on their age, were divided into 3 groups: Group 1 included 17 children aged 6-7 years. Group 2 consisted of 25 children aged 8-12 years. The third group consisted of 15 children aged 13-15. In children aged 6-7 years, it can be noted that the total number of the optional group of microbes is higher than in anaerobic. In this case, the quantitative indicators of the lactobacillus are especially affected among the anaerobes, so their number is Lg. 2.10 ± 0.1 cfu /ml. Alarming in this group of children is the sowing of pathogenic strains of staphylococcus. The remaining groups of microbes, although there are minor changes, but they are unreliable. At children in the age of 2 and 3 group the flora of the oral cavity in many respects stands close to normal indices. Although it should be noted a significant increase in the number of streptococci, especially strains of Str. mutans and mitis. In children in the age group 6-7 years, almost all indicators show immunodeficiency, so the lysozyme titer was 14 ± 0.41 mg /%, it was normally 19.180 ± 0.60 mg/%, the index of phagocytosis was 48.4 ± 1.45% significantly lower than the control indices, the level of secretory immunoglobulin of class A (sIgA) was 1.7 ± 0.1 g/l, which is much lower than normal values. It is interesting to note that in children older than 8-12 and 13-15 years, these indicators have improved significantly, although it should be noted that their immunodeficiency has worsened, only in terms of the fraction of secretory immunoglobulin A (sIgA). From the above, it follows: 1. According to the sanitary and hygienic and microbiological studies carried out, it is possible to state that the city of Chirchik is an ecologically unfavorable region. 2. The study of oral micro-ecology in children in Chirchik made it possible to establish that in children in the age group 6-7 years the most pronounced are dysbiotic conditions, the main feature of which is the decrease in the amount of lactobacillus, but the increase in the number of staphylococci and fungi of the genus Candida 3. When studying the local factors of oral protection in children living in Chirchik in the age aspect, it was found that immunodeficiency states are most pronounced in children of the I group. 4. The study of the state of colonization resistance of microbes in children living in the city of Chirchik established that the most pronounced violations in these issues were noted in the children of group I.

Overactivity of Alternative Pathway Convertases in Patients With Complement-Mediated Renal Diseases.

Overactivation of the alternative pathway of the complement system is associated with the renal diseases atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G). C3 nephritic factors (C3NeF) play an important role in C3G pathogenesis by stabilizing the key enzymatic complex of complement, the C3 convertase. However, the reliability of assays detecting these autoantibodies is limited. Therefore, in this study, we validated and optimized a prototype hemolytic method for robust detection and characterization of factors causing convertase overactivity in large patient cohorts. The assay assesses convertase activity directly in the physiological milieu of serum and therefore is not restricted to detection of stabilizing autoantibodies such as C3NeF but may also reveal genetic variants resulting in prolonged convertase activity. We first defined clear cutoff values based on convertase activity in healthy controls. Next, we evaluated 27 C3G patient samples and found 16 positive for prolonged convertase activity, indicating the presence of factors influencing convertase stability. In three patients, the overactive convertase profile was persistent over disease course while in another patient the increased stability normalized in remission. In all these four patients, the convertase-stabilizing activity resided in the purified immunoglobulin (Ig) fraction, demonstrating the autoantibody nature. By contrast, the Igs of a familial aHUS patient carrying the complement factor B mutation p.Lys323Glu did not reveal convertase stabilization. However, in serum prolonged convertase activity was observed and segregated with the mutation in both affected and unaffected family members. In conclusion, we present a robust and reliable method for the detection, characterization, and evaluation over time of factors prolonging convertase activity (C3NeF or certain mutations) in patient cohorts. This assay may provide new insights in disease pathogenesis and may contribute to the development of more personalized treatment strategies.

Neutralizing Anti-Cytokine Autoantibodies Against Interferon-α in Immunodysregulation Polyendocrinopathy Enteropathy X-Linked.

Anti-cytokine autoantibodies (ACAAs) have been described in a growing number of primary immunodeficiencies with autoimmune features, including autoimmune polyendocrine syndrome type I (APS-1), a prototypical disease of defective T cell-mediated central tolerance. Whether defects in peripheral tolerance lead to similar ACAAs is unknown. Immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) is caused by mutations in FOXP3, a master regulator of T regulatory cells (Treg), and consequently results in defective T cell-mediated peripheral tolerance. Unique autoantibodies have previously been described in IPEX. To test the hypothesis that ACAAs are present in IPEX, we designed and fabricated antigen microarrays. We discovered elevated levels of IgG ACAAs against interferon-α (IFN-α) in a cohort of IPEX patients. Serum from IPEX patients blocked IFN-α signaling in vitro and blocking activity was tightly correlated with ACAA titer. To show that blocking activity was mediated by IgG and not other serum factors, we purified IgG and showed that blocking activity was contained entirely in the immunoglobulin fraction. We also screened for ACAAs against IFN-α in a second geographically distinct cohort. In these samples, ACAAs against IFN-α were elevated in a post hoc analysis. In summary, we report the discovery of ACAAs against IFN-α in IPEX, an experiment of nature demonstrating the important role of peripheral T cell tolerance.

Hot topic: Holder pasteurization of human milk affects some bioactive proteins

The aim of this research was to investigate the effect of Holder pasteurization (HoP; 62.5°C, 30 min) on the protein profile and activities of glutathione peroxidase (GPx) and lysozyme (LZ) in human milk. Over 6 mo of lactation, human milk samples were analyzed before (raw) and after HoP for GPx and LZ activity and electrophoresis protein profile. Holder pasteurization reduced human milk lactoferrin, immunoglobulin fractions, and GPx activity. In addition, GPx activity, which is high in colostrum and transitional milk, was naturally reduced over the 6-mo lactation period. In contrast, HoP did not affect human milk LZ activity. Besides its critical cellular antioxidant role in protecting the organism from oxidative damage, GPx decreases the redox potential of milk, stimulating the growth of anaerobic microorganisms, such as the probiotic Bifidobacterium. Considering the role of lactoferrin in infant health, we conclude that an important part of its function has been inactivated by pasteurization. These compounds should be replaced by human milk banks after the HoP step to recover lost functionality. Otherwise, an alternative technology to HoP that better retains human milk properties should be used by milk banks to eliminate the risk of transmission of infectious agents.

Increased cerebrospinal fluid albumin and immunoglobulin A fractions forecast cortical atrophy and longitudinal functional deterioration in relapsing-remitting multiple sclerosis

Background: Currently, no unequivocal predictors of disease evolution exist in patients with multiple sclerosis (MS). Cortical atrophy measurements are, however, closely associated with cumulative disability. Objective: Here, we aim to forecast longitudinal magnetic resonance imaging (MRI)-driven cortical atrophy and clinical disability from cerebrospinal fluid (CSF) markers. Methods: We analyzed CSF fractions of albumin and immunoglobulins (Ig) A, G, and M and their CSF to serum quotients. Results: Widespread atrophy was highly associated with increased baseline CSF concentrations and quotients of albumin and IgA. Patients with increased CSFIgA and CSFIgM showed higher functional disability at follow-up. Conclusion: CSF markers of blood–brain barrier integrity and specific immune response forecast emerging gray matter pathology and disease progression in MS.

A study to Evaluate Immunoglobulins fraction by Serum Protein Electrophoresis for occurrence of Multiple Myeloma in a Tertiary Care Hospital

Background: Serum protein electrophoresis (SPE) is a diagnostic laboratory test that separates and quantifies several classes of serum proteins and that identifies and characterizes the monoclonal gammopathies (M-protein). Aims and Objective: This study was taken up with an aim to evaluate the relevance and significance of immunoglobulin fraction in discerning multiple myeloma and to assess the morbidity of cases in relation to its clinical diagnosis. Materials and Method: This study was carried out by analysing data with gamma globulins above biological reference range (0.6-1.6gm/dL) from the samples requested for serum protein electrophoresis by cellulose acetate method during Sept 2013 to Aug 2014 in St. John’s medical college biochemistry lab, Bengaluru (Total of 95 cases). Densitometrically estimated M proteins were further divided into 2 groups of below and above 3gm/dL according to the IMWG criteria for classification of Multiple myeloma. Result: Out of 95 cases, 55(57.9 %) were above 3gm/dL and the remaining 40(42.1%) were below 3gm/dL. Among those 95 cases 18 were confirmed with multiple myeloma. 13 out of 18 cases fall among the group where gamma globulin is > 3g/dL with 81% of them having gamma above 4g/dL. Five cases of multiple myeloma had also been confirmed with their gamma globulins falling below 3g/dL which is compelling. Conclusion: The significance of elevated gamma globulins value less than the prescribed criteria of IMWG for diagnosing Multiple myeloma gave us importance of clinical correlation before releasing report. Though the morbidity percentage (20%) of multiple myeloma among the group with gamma globulins less than 3gm/dL, is less but nevertheless significant.More number of studies with large sample numbers is required to understand the cases of multiple myeloma with their gamma globulins falling below the IMWG criteria cut off limit.

Production of pure fractions of immunoglobulin G subclass autoantibodies against tumor necrosis factor.

Autoantibodies directed against cytokines are important effector molecules regulating the biological activity of cytokines. There is experimental evidence indicating that autoantibodies belonging to different immunoglobulin G (IgG) subclasses may have different functional activity. The purpose of this work was to develop a protocol for the purification of fractions of IgG subclass antibodies directed against tumor necrosis factor (TNF). We developed a series of steps, including gel filtration, positive and negative affinity chromatography, and ultrafiltration, to achieve this goal. Our protocol purified IgG subclass autoantibodies directed against TNF from a human immunoglobulin preparation. The isolation of these anti-TNF autoantibodies will enable evaluation of the effect of TNF-specific antibodies on TNF biological activity. Our newly developed technique for purifying subclasses of anti-TNF autoantibodies may be important for both basic research on the functional activity of these autoantibodies and for clinical immunology.

The development of the technology aimed to obtain serum for diagnosis and treatment of the feline calicivirus

An effective scheme of immunization to obtain hyperimmune serum against FCV was developed. The antigen was being injected subcutaneously and intramuscular with simultaneous use of immunostimulator "Imunofan». An allocation of globulin fraction of proteins by desalting ammonium sulfate was performed. The fractions of pure immunoglobulin G with specific gravity 2 times higher than in the neat serum were being received

A nonchromatographic process for purification of secretory immunoglobulins from caprine whey.

Secretory immunoglobulins are an important antibody class being primarily responsible for immunoprotection of mucosal surfaces. A simple, non-chromatographic purification process for secretory immunoglobulins from caprine whey was developed. In the first process step whey was concentrated 30-40-fold on a 500kDa membrane, thereby increasing the purity from 3% to 15%. The second step consisted of a fractionated PEG precipitation, in which high molecular weight impurities were removed first and in the second stage the secretory immunoglobulins were precipitated, leaving a majority of the low molecular weight proteins in solution. The re-dissolved secretory immunoglobulin fraction had a purity of 43% which could then be increased to 72% by diafiltration at a volume exchange factor of 10. Further increase of purity was only possible at the expense of very high buffer consumption. If diafiltration was performed directly after ultrafiltration, followed by precipitation, the yield was higher but purity was only 54%. Overall, filtration performance was characterized by high concentration polarization, therefore process conditions were set to low trans-membrane pressure and moderate protein concentration. As such purity and to a lesser extent throughput were the major objectives rather than yield, since whey, as a by-product of the dairy industry, is a cheap raw material of almost unlimited supply. Ultra-/diafiltration performance was described well by correlations using dimensionless numbers. Compared with a theoretical model (Graetz/Leveque solution) the flux was slightly overestimated. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:642-653, 2017.

Conjugation of Peroxidase from Brassica oleracea gongylodes for Use as a Label- Prospect of a Novel Enzyme Tag for Immunoassay Systems

Peroxidase tagged proteins are being used successfully as immune-histological probes for the demonstration of tissue antigens, and in enzyme amplified immunoassay systems for the quantitative determination of soluble and insoluble antigens. The glycoprotein nature of peroxidases can be exploited for conjugation to proteins of interest. Peroxidase extracted from the bulbs of Brassica oleracea gongylodes was salted out at 40-80% ammonium sulfate saturation and activated by treatment with 1-Fluoro-2,4-dinitro benzene (FDNB) and periodate. Treatment with 0.08% FDNB and 12.5mM periodate was optimized for activation of the enzyme. The treated enzyme was found to conjugate successfully to immunoglobulin fractions harvested from egg yolk (IgY), human plasma and goat serum. Enzyme conjugated to IgY fraction showed improvement in its pH stability and temperature stability. The affinity of the enzyme for its substrate phenol did not alter to a significant extent upon activation and conjugation. The conjugates exhibited high affinity towards phenol, bromocresol purple and bromothymol blue in comparison to HRP conjugates prepared using the same protocol. Int. J. Appl. Sci. Biotechnol. Vol 5(1): 59-65

Clinical implications of serum N-glycan profiling as a diagnostic and prognostic biomarker in germ-cell tumors.

Serum biomarker monitoring is essential for management of germ‐cell tumors (GCT). However, not all GCT are positive for conventional tumor markers. We examined whether serum N‐glycan‐based biomarkers can be applied for detection and prognosis in patients with GCT. We performed a comprehensive N‐glycan structural analysis of sera from 54 untreated GCT patients and 103 age‐adjusted healthy volunteers using glycoblotting methods and mass spectrometry. Candidate N‐glycans were selected from those with the highest association; cutoff concentration values were established, and an N‐glycan score was created based on the number of positive N‐glycans present. The validity of this score for diagnosis and prognosis was analyzed using a receiver operating characteristic (ROC) curve. We identified five candidate N‐glycans significantly associated with GCT patients. The accuracy of the N‐glycan score for GCT was significant with an area‐under‐the‐curve (AUC) value of 0.87. Diagnostically, the N‐glycan score detected 10 of 12 (83%) patients with negative conventional tumor markers. Prognostically, the N‐glycan score comprised four candidate N‐glycans. The predictive value of the prognostic N‐glycan score was significant, with an AUC value of 0.89. A high value prognostic N‐glycan score was significantly associated with poor prognosis. Finally, to identify a potential carrier protein, immunoglobulin (Ig) fractions of sera were subjected to N‐glycan analysis and compared to whole sera. Candidate N‐glycans in Ig‐fractions were significantly decreased; therefore, the carrier protein for candidate N‐glycans is likely not an immunoglobulin. In summary, our newly developed N‐glycan score seems to be a practical diagnostic and prognostic method for GCT.

Methods for Posttranslational Induction of Polyreactivity of Antibodies.

An antibody molecule that recognizes multiple unrelated antigens is defined as polyreactive. Polyreactivity is an intrinsic characteristic of immune repertoires. Degenerated antigen binding diversifies the repertoire of specificities, thus contributing to immune defense and immune regulation. Immune repertoire contains also a fraction of immunoglobulins, which acquire polyreactivity only following contact with various protein-destabilizing or pro-oxidative substances. Posttranslational induction of the antibody polyreactivity may have important repercussion for laboratory practice, as well as in cases of pathological conditions accompanied by liberation of large quantities of pro-oxidative substances such as heme, labile iron, or reactive oxygen species. Antibodies with induced polyreactivity have been demonstrated to exert pathogen neutralization and immune regulatory potential in inflammatory conditions, suggesting that this phenomenon may be exploited for design of therapeutic strategies. In this article, we provide description of the basic procedures for uncovering of the cryptic polyreactivity of antibodies by heme, ferrous ions, and acid pH solution.

Application of native agarose gel electrophoresis of serum proteins in veterinary diagnostics

Abstract Electrophoretic techniques, used to separate mixtures of electrically charged particles, are widely used in science. One of these techniques, native protein electrophoresis in an agarose gel, is applied in human and veterinary medicine. Changes in the proportions of individual protein fractions correspond to significant changes in the physiology of the body. Although the pattern obtained by electrophoretic separation rarely indicates a specific disease, it provides valuable information for the differential diagnosis. Decades of research on the types of patterns obtained in the case of particular diseases have led to the accumulation of substantial knowledge. The paper presents the available information on this topic. Serum protein electrophoresis is recommended in cases of increased levels of total protein in order to reveal the nature of the process. The basic information which can be obtained from electrophoretic separation includes the immune status of the organism. Both increased antigenic stimulation and immunodeficiency are clearly visible in electropherograms. Moreover, the level of heterogeneity of the corresponding protein fractions can help to distinguish between infectious diseases and cancer - multiple myeloma - the latter producing a homogeneous immunoglobulin fraction. Analysis of other protein fractions helps to detect or confirm an ongoing inflammatory process and provides information regarding liver function. Even when the concentration of total protein is within the reference range, this analysis can be recommended as a basic laboratory test.

Phosphocholine-Specific Antibodies Improve T-Dependent Antibody Responses against OVA Encapsulated into Phosphatidylcholine-Containing Liposomes.

Liposomes containing phosphatidylcholine have been widely used as adjuvants. Recently, we demonstrated that B-1 cells produce dipalmitoyl-phosphatidylcholine (DPPC)-specific IgM upon immunization of BALB/c mice with DPPC-liposomes encapsulating ovalbumin (OVA). Although this preparation enhanced the OVA-specific humoral response, the contribution of anti-DPPC antibodies to this effect was unclear. Here, we demonstrate that these antibodies are secreted by B-1 cells independently of the presence of OVA in the formulation. We also confirm that these antibodies are specific for phosphocholine. The anti-OVA humoral response was partially restored in B-1 cells-deficient BALB/xid mice by immunization with the liposomes opsonized with the serum total immunoglobulin (Ig) fraction containing anti-phosphocholine antibodies, generated in wild-type animals. This result could be related to the increased phagocytosis by peritoneal macrophages of the particles opsonized with the serum total Ig or IgM fractions, both containing anti-phosphocholine antibodies. In conclusion, in the present work, it has been demonstrated that phosphocholine-specific antibodies improve T-dependent antibody responses against OVA carried by DPPC-liposomes.