Serum Fractions Research Articles

Why working with porcine circulating serum amyloid A is a pig of a job

Serum amyloid A (SAA) is a major acute phase protein in most species, and is widely employed as a health marker. Systemic SAA isoforms (SAA1, and SAA2) are apolipoproteins synthesized by the liver which associate with high density lipoproteins (HDL). Local SAA (SAA3) isoforms are synthesized in other tissues and are present in colostrums, mastitic milk and mammary dry secretions. Of systemic SAA the bulk is monomeric and bound to HDL, and a small proportion is found in serum in a multimeric form with a buried HDL binding site. In most species, systemic SAA could easily be studied by purifying it from serum of diseased individuals by hydrophobic interaction chromatography methods. For years, we were not able to isolate systemic pig SAA using the latter methods, and found that the bulk of pig SAA did not reside in the HDL-rich serum fractions but in the soluble protein fraction mainly as a multimeric protein.Based on these surprising results, we analysed in silico the theoretical properties and predicted the secondary structure of pig SAA by using the published pig primary SAA amino acid sequence. Results of the analysis confirmed that systemic pig SAA had the highest homology with local SAA3 which in other species is the isoform associated with non-hepatic production in tissues such as mammary gland and intestinal epithelium. Furthermore, the primary sequence of the pig SAA N-terminal HDL binding site did differ considerably from SAA1/2. Secondary structure analysis of the predicted alpha–helical structure of this HDL binding site showed a considerable reduction in hydrophobicity compared to SAA1/2. Based on these results, it is argued that systemic acute phase SAA in the pig has the structural properties of locally produced SAA (SAA3). It is proposed that in pig SAA multimers the charged N-terminal sequence is buried, which would explain their different properties.It is concluded that pig systemic SAA is unique compared to other species, which raises questions about the proposed importance of acute phase SAA in HDL metabolism during inflammation in this species.

Exercise promotes bone marrow cell survival and recipient reconstitution post-bone marrow transplantation, which is associated with increased survival

Bone marrow transplantation (BMT) is associated with a high risk of mortality, partially because of the harmful effects of the preconditioning myeloablative regimens. We have recently demonstrated increased bone marrow cell survival and proliferation in response to exercise training, which may be attributable to increased quality of the niche. The purpose of the present study was to determine the extent to which exercise preconditioning of recipients could increase the success of BMT. Recipient mice remained sedentary (SED) or were exercise-trained (EX) on a treadmill (3 d/wk for 8 weeks) before reconstitution with green fluorescent protein (GFP)-labeled donor marrow. Recipient survival, both donor-derived and total (donor- and recipient-derived) blood reconstitution were measured by flow cytometry. The first and fourth day after BMT apoptosis, cellularity and donor cell homing were determined in the recipients' bone marrow cavity by flow cytometry. Whereas only 25% of SED mice survived, 82% of EX recipients survived the BMT. Homing of donor-derived marrow cells to the recipients' marrow cavity acutely after BMT was not altered in EX, but EX mice displayed decreased levels (10%; p < 0.05) of activated caspase-3/-7 one day after BMT, leading to a maintenance of marrow cellularity in mice preconditioned with exercise. The acute inhibition of marrow cell apoptosis in EX mice resulted in increased total blood cell reconstitution at 1 and 3.5 months after BMT in EX mice (42% and 43%, respectively; both p < 0.05). Short- and long-term donor-derived engraftment was not different between EX and SED recipients. Exercise training increases recipient survival after BMT with increased total blood cell reconstitution.

Capture, Enrichment, and Mass Spectrometric Detection of Low‐Molecular‐Weight Biomarkers with Nanoporous Silicon Microparticles

Mining the disease information contained in the low-molecular-weight range of a proteomic profile is becoming of increasing interest in cancer research. This work evaluates the ability of nanoporous silicon microparticles (NPSMPs) to capture, enrich, protect, and detect low-molecular-weight peptides (LMWPs) sieved from a pool of highly abundant plasma proteins. The average pore size and porosity of NPSMPs are controlled by the electrochemical preparation conditions, and the critical parameters for admission or exclusion of protein with a definite molecular weight are determined by reflectometric-interference Fourier transform spectroscopy (RIFTS). Sodium dodecyl sulfate polyacrylamide-gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis of the proteins captured by the NPSMPs show that the chemical nature of the NPSMPs surface and the solution pH also play vital roles in determining the affinity of NPSMPs for target analytes. It is found that carboxyl-terminated porous microparticles with a porosity of 26% (pore diameter around 9.0 nm) specifically fractionate, enrich and protect LMWPs sieved from either simulated samples or human serum samples. Moreover, NPSMPs containing captured peptides can be directly spotted onto a MALDI plate. When placed in a conventional MALDI matrix, laser irradiation of the particles results in the release of the target peptides confined in the nanopores, which are then ionized and detected in the MALDI experiment. As a proof-of-principle test case, mass spectra of NPSMPs prepared using serum from colorectal cancer patients and from control patients can be clearly distinguished by statistical analysis. The work demonstrates the utility of the method for discovery of biomarkers in the untapped LMWP fraction of human serum, which can be of significant value in the early diagnosis and management of diseases.

Validation of free flow electrophoresis as a novel plasma and serum processing and fractionation method in biobanking.

Free flow electrophoresis (FFE) is a fractionation method, based on isoelectric focusing (IEF). We validate the reproducibility of the method and show that it can be applied by biobanks in order to fractionate fluid biospecimens efficiently and reproducibly and to facilitate downstream proteomic applications. We also propose a simple method allowing researchers to assess the reproducibility of each FFE run.

Serum long chain n‐3 fatty acids (EPA and DHA) in the pregnant mother are independent of risk of type 1 diabetes in the offspring

This article aims to study whether higher proportions of the long chain n-3 fatty acids eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) in the phospholipid fraction of serum samples in pregnancy were associated with a lower risk of childhood onset type 1 diabetes in the offspring. In a prospective cohort of nearly 30 000 pregnant women who gave birth in Norway during 1992-1994, we analysed serum samples from 89 women whose child developed type 1 diabetes and was included in the nationwide Norwegian Childhood Diabetes Registry and 125 randomly selected women whose child did not develop type 1 diabetes before 15 years of age. Specific fatty acids were expressed as the proportion of total fatty acids (g/100 g) in the phospholipid fraction in serum analysed using solid phase extraction and gas chromatography with flame ionization detection. There was no significant association between EPA or DHA in maternal serum and risk of type 1 diabetes in the offspring. Odds ratio (OR) for upper versus lower quartile of EPA was 0.75 [95% confidence interval (CI) 0.34-1.65], test for trend p = 0.4, and for DHA OR = 0.71 (95% CI 0.33-1.53), test for trend p = 0.6. No significant association was found for the sum of n-3 fatty acids, or for n-6/n-3 ratio in the mother with risk of type 1 diabetes in the offspring. Our data did not support the hypothesis that higher proportions of maternal EPA or DHA during pregnancy are associated with a lower risk of type 1 diabetes in the offspring.

PP008. Lipotoxicity and preeclampsia: A probable correlation (preliminary results)

The impact of preeclampsia (PE) of pregnancy is seen as a global concern. Despite their importance, PE does not have well-established causes. Several studies point to obesity as a predisposing factor for PE [1]. In a systematic review Duckitt and Harrington showed that in women with BMI⩾30 the relative risk of developing PE is equal to 2.1 [2]. Bianco et al. demonstrated that the chance of patients with BMI⩾35 developing PE is more than 4 times [3]. Furthermore, it is believed that the intracellular accumulation of triglycerides cause mitochondrial dysfunction, more electron transport and generation of ROS (Reactive Oxygen Species) [4], causing a harmful effect on the endothelium. To compare the incidence of potential lipotoxicity markers among normal pregnant women and with pre-eclampsia. A case-control study, including pregnant women from October 2011 to January 2012 at Hospital Guilherme Álvaro in Santos/Brazil. The study group consisted of 27 mothers with pre-eclampsia, according to the criteria of NHBPEP (2000) and the control group consisted of 27 mothers with normal pregnancy, who gave birth vaginally, at term without hypertension. The exclusion criteria were diagnosis of collagen diseases, smoking, diabetes, twin pregnancy and fetal malformations. Blood samples were collected immediately after delivery and the results were analyzed in conjunction with maternal data, according to the following variables: age, body mass index (BMI), serum cholesterol total and fractions, glucose, triglyceride and creative protein. Analysis of SF data was performed by calculating the odds ratio adopting an hypothesis rejection level of 0.05. The PE group had BMI>30 (or), serum triglycerides>150 (or) and LDL cholesterol>100 (or) more frequently than normal mothers, and as regards statistical significance comparing the age of the women, glucose level, HDS cholesterol, there was no significant difference between the groups. In pregnancy, lipotoxicity reduces the ability of trophoblastic invasion, causing maternal systemic endothelial and placental dysfunction causing alterations in metabolic functions. All such changes maintain a close relationship with the pathophysiology of PE. Monitoring pregestational period of obese patients or those with lipid disorders is part of the medical arsenal against PE by monitoring and advising on the best time to conceive.

P2X7 Receptor-mediated Scavenger Activity of Mononuclear Phagocytes toward Non-opsonized Particles and Apoptotic Cells Is Inhibited by Serum Glycoproteins but Remains Active in Cerebrospinal Fluid

Rapid phagocytosis of non-opsonized particles including apoptotic cells is an important process that involves direct recognition of the target by multiple scavenger receptors including P2X7 on the phagocyte surface. Using a real-time phagocytosis assay, we studied the effect of serum proteins on this phagocytic process. Inclusion of 1-5% serum completely abolished phagocytosis of non-opsonized YG beads by human monocytes. Inhibition was reversed by pretreatment of serum with 1-10 mM tetraethylenepentamine, a copper/zinc chelator. Inhibitory proteins from the serum were determined as negatively charged glycoproteins (pI < 6) with molecular masses between 100 and 300 kDa. A glycoprotein-rich inhibitory fraction of serum not only abolished YG bead uptake but also inhibited phagocytosis of apoptotic lymphocytes or neuronal cells by human monocyte-derived macrophages. Three copper- and/or zinc-containing serum glycoproteins, ceruloplasmin, serum amyloid P-component, and amyloid precursor protein, were identified, and the purified proteins were shown to inhibit the phagocytosis of beads by monocytes as well as phagocytosis of apoptotic neuronal cells by macrophages. Human adult cerebrospinal fluid, which contains very little glycoprotein, had no inhibitory effect on phagocytosis of either beads or apoptotic cells. These data suggest for the first time that metal-interacting glycoproteins present within serum are able to inhibit the scavenger activity of mononuclear phagocytes toward insoluble debris and apoptotic cells.

Evaluation of the radioprotective action of anserine along with zinc in albino rats exposed to gamma-radiation

Administration of dietary antioxidants has been suggested to protect against the subsequent liver tissue damage. The present data to explore the hepatoprotective and antioxidant effect of anserine nitrate and /or zinc chloride against γ-irradiation induced hepatotoxicity. Healthy male albino rats were exposed to γ-irradiation from Co60 gamma cell 3500 at dose level (5.7 Gy) at a dose rate 2.67 rad/sec after 24 h and 14 days on the liver and to determine both prophylactic and therapeutic role of intraperitoneally administrated. Exposure to γ- irradiation induced a significant increase in levels of ALP, ALT and AST, while levels of glucose, total proteins, albumin, triglycerides, cholesterol, LDL-C, HDL-C, total, direct and indirect bilirubin and serum fractions were significantly decreased except for total lipids level which was almost not affected. Administration of anserine and/or zinc prior or after radiation exposure was found to offer protection against γ-irradiation induced hepatocellular damage and oxidative stress in rats, probably by exerting a protective effect against hepatocellular necrosis via its free radical scavenging and membrane stabilizing ability.

Combination of media, biomaterials and extracellular matrix proteins to enhance the differentiation of neural stem/precursor cells into neurons

The purpose of this study was to induce the differentiation of neural stem/precursor cells (NSPC) more towards neurons than glial cells by the combination of media, biomaterials and extracellular matrix (ECM) proteins. Considering the role of serum, 10% fetal bovine serum or its fractions were added to DMEM/F12 medium to examine the effect of the differentiation-promoting potential on cultured NSPC isolated from embryonic rat cerebral cortex. The NSPC were cultured for 7days, after which differentiation was assayed using immunocytochemistry for lineage specific markers. It was demonstrated that molecules promoting neuron differentiation were present in serum with molecular weight <100kDa, which could dominate the differentiation of NSPC principally into neurons in the presence of basic fibroblast growth factor. In contrast, NSPC were induced to differentiate predominantly into glial cell phenotypes in the presence of whole serum components. Based on medium containing serum fraction, semi-quantification showed that the MAP2-positive percentage of the immunoreactive ratio within migrated cells could be promoted over 85% by combining poly(ethylene-co-vinyl alcohol) biomaterial and fibronectin matrix protein. These results are very encouraging, since an environment favorable for neuronal differentiation should be useful in the development of strategies for controlling the behavior of NSPC in neuroscience research.

Abstract P310: The Distribution of Total- and HDL Cholesterol and Triglycerides is Significantly Different across Quartiles of Vitamin D in Chest-Pain Patients with a Suspected Acute Coronary Syndrome

Background: The purpose of this study was to assess the distribution of serum lipids including total cholesterol, HDL cholesterol and triglycerides across quartiles of vitamin D in blood samples collected from 982 chest-pain patients with a suspected acute coronary syndrome (ACS) from Salta, Northern-Argentina. Methods: Vitamin D status was obtained by determination of 25-hydroxy-vitamin D (25(OH)D) in serum. Blood samples for measurement of 25(OH)D and circulating lipid fractions in serum were obtained immediately following admission. Results: In the total population the concentration of total cholesterol, HDL cholesterol and triglycerides was found to differ significantly across quartiles of 25(OH)D, p = 0.016, p = 0.01 and p = 0.000, respectively. See Figure 1, 2 and 3, respectively. In the Troponin T (TnT) positive population (TnT &gt; 0.01 ng/mL), only total cholesterol and triglycerides differed significantly across the 25(OH)D quartiles, p = 0.000 and p = 0.003, respectively, whereas HDL cholesterol alone differed in the TnT negative group, p = 0.025. Conclusion: Concentrations of the main lipid fractions are related to the vitamin D status in patients admitted with chest pain and suspected ACS.

Soluble Serum CD81 Is Elevated in Patients with Chronic Hepatitis C and Correlates with Alanine Aminotransferase Serum Activity

AimCellular CD81 is a well characterized hepatitis C virus (HCV) entry factor, while the relevance of soluble exosomal CD81 in HCV pathogenesis is poorly defined. We performed a case-control study to investigate whether soluble CD81 in the exosomal serum fraction is associated with HCV replication and inflammatory activity.Patients and MethodsFour cohorts were investigated, patients with chronic hepatitis C (n = 37), patients with chronic HCV infection and persistently normal ALT levels (n = 24), patients with long term sustained virologic response (SVR, n = 7), and healthy volunteers (n = 23). Concentration of soluble CD81 was assessed semi-quantitatively after differential centrifugation ranging from 200 g to 100,000 g in the fifth centrifugation fraction by immunoblotting and densitometry.ResultsSoluble CD81 was increased in patients with chronic hepatitis C compared to healthy subjects (p = 0.03) and cured patients (p = 0.017). Patients with chronic HCV infection and persistently normal ALT levels and patients with long term SVR had similar soluble CD81 levels as healthy controls (p>0.2). Overall, soluble CD81 levels were associated with ALT levels (r = 0.334, p = 0.016) and severe liver fibrosis (p = 0.027).ConclusionCD81 is increased in the exosomal serum fraction in patients with chronic hepatitis C and appears to be associated with inflammatory activity and severity of fibrosis.

Evaluation of a PCR Test for Detection of Treponema pallidum in Swabs and Blood

Syphilis diagnosis is based on clinical observation, serological analysis, and dark-field microscopy (DFM) detection of Treponema pallidum subsp. pallidum, the etiological agent of syphilis, in skin ulcers. We performed a nested PCR (nPCR) assay specifically amplifying the tpp47 gene of T. pallidum from swab and blood specimens. We studied a cohort of 294 patients with suspected syphilis and 35 healthy volunteers. Eighty-seven of the 294 patients had primary syphilis, 103 had secondary syphilis, 40 had latent syphilis, and 64 were found not to have syphilis. The T. pallidum nPCR results for swab specimens were highly concordant with syphilis diagnosis, with a sensitivity of 82% and a specificity of 95%. Reasonable agreement was observed between the results obtained with the nPCR and DFM methods (kappa = 0.53). No agreement was found between the nPCR detection of T. pallidum in blood and the diagnosis of syphilis, with sensitivities of 29, 18, 14.7, and 24% and specificities of 96, 92, 93, and 97% for peripheral blood mononuclear cell (PBMC), plasma, serum, and whole-blood fractions, respectively. HIV status did not affect the frequency of T. pallidum detection in any of the specimens tested. Swab specimens from mucosal or skin lesions seemed to be more useful than blood for the efficient detection of the T. pallidum genome and, thus, for the diagnosis of syphilis.

Search for breast cancer biomarkers in fractionated serum samples by protein profiling with SELDI-TOF MS.

Many high-abundant acute phase reactants have been previously detected as potential breast cancer biomar-kers. However, they are unlikely to be specific for breast cancer. Cancer-specific biomarkers are thought to be among the lower abundant proteins. We aimed to detect lower abundant discriminating proteins by performing serum fractionation by strong anion exchange chromatography preceding protein profiling with SELDI-TOF MS. In a pilot study, we tested the different fractions resulting from fractionation, on several array types. Fraction 3 on IMAC30 and Fraction 6 on Q10 yielded the most discriminative proteins and were used for serum protein profiling of 73 incident breast cancer cases and 73 matched controls. Eight peaks showed statistically significantly different intensities between cases and controls (P⧁0.05), and had less than 10% chance to be a false-positive finding. Seven of these were tentatively identified as apolipoprotein C-II (m/z 8,909), oxidized apolipoprotein C-II (m/z 8,925), apolipoprotein C-III (m/z 8,746), fragment of coagulation factor XIIIa (m/z 3,959), heterodimer of apolipoprotein A-I and apolipoprotein A-II (m/z 45,435), hemoglobin B-chain (m/z 15,915), and post-translational modified hemoglobin (m/z 15,346). By extensive serum fractionation, we detected many more proteins than in previous studies without fractionation. However, discriminating proteins were still high abundant. Results indicate that either lower abundant proteins are less distinctive, or more rigorous fractionation and selective protein depletion, or a more sensitive assay, are needed to detect lower abundant discriminative proteins.

A multiplex lectin-channel monitoring method for human serum glycoproteins by quantitative mass spectrometry

A mass profiling method and multiple reaction monitoring (MRM)-based quantitative approach were used to analyze multiple lectin-captured fractions of human serum using different lectins such as aleuria aurantia lectin (AAL), phytohemagglutinin-L(4) (L-PHA), concanavalin A (Con A), and Datura stramonium agglutinin (DSA) to quantitatively monitor protein glycosylation diversity. Each fraction, prepared by multiple lectin-fractionation and tryptic digestion, was analyzed by 1-D LC-MS/MS. Semi-quantitative profiling showed that the list of glycoproteins identified from each lectin-captured fraction is significantly different according to the used lectin. Thus, it was confirmed that the multiplex lectin-channel monitoring (LCM) using multiple lectins is useful for investigating protein glycosylation diversity in a proteome sample. Based on the semi-quantitative mass profiling, target proteins showing lectin-specificity among each lectin-captured fraction were selected and analyzed by the MRM-based method in triplicate using each lectin-captured fraction (average CV 7.9%). The MRM-based analysis for each lectin-captured fraction was similar to those obtained by the profiling experiments. The abundance of each target protein measured varied dramatically, based on the lectin-specificity. The multiplex LCM approach using MRM-based analyses is useful for quantitatively monitoring target protein glycoforms selectively fractionated by multiple lectins. Thus through multiplex LCM rather than single, we could inquire minutely into protein glycosylation states.

Effect of Lysine Clonixinate on the Pharmacokinetics and Anticoagulant Activity of Phenprocoumon

The effect of the non-steroidal anti-inflammatory drug lysine clonixinate ([2-(3-chloro-o-toluidino)nicotinic acid]-L-lysinate, CAS 55837-30-4) on the pharmacokinetics and anticoagulant activity of phenprocoumon (4-hydroxy-3-(1-phenylpropyl)-coumarin, CAS 435-97-2) was investigated in an open, randomised, two-fold, cross-over study in 12 healthy male volunteers. These subjects received a single dose of 18 mg phenprocoumon without or with concomitant treatment with lysine clonixinate (125 mg five times a day for 3 days before and 13 days after ingestion of a single dose of phenprocoumon). Pharmacokinetic parameters of phenprocoumon following oral administration were: CL/f: 0.779 +/- 0.157 ml/min, half-life of elimination: 147.2 +/- 19.9 h; free fraction in serum: 0.51 +/- 0.20%. These parameters were not significantly altered by concomitant treatment with lysine clonixinate. Prothrombin time increased from 13.3 +/- 1.3 s (at time 0) to 17.7 +/- 2.7 s following phenprocoumon and from 13.3 +/- 1.2 s to 18.0 +/- 2.2 s following combined administration. Prothrombin time returned to the pretreatment values 240 h after administration of phenprocoumon. The integrated effect (AUEC0-288 h) was identical following both treatments (4.303 +/- 461 and 4.303 +/- 312 s x h for phenprocoumon alone and phenprocoumon with lysine clonixinate, respectively). Thus, lysine clonixinate administered in therapeutic doses does not affect the pharmacokinetics and anticoagulant activity of phenproxoumon.

Effect of antibiotics on immunophysiological status and their taste attractiveness for rainbow trout Parasalmo (=Oncorhynchus) mykiss (Salmoniformes, Salmonidae)

Effect of threeand fivefold intramuscular inject ions of antibiotics oxytetracyclin hydrochloride and benzylpenicillin sodium (20 mg/kg) on immunophysiological status of juveniles of rainbow trout Paras� almo (=Oncorhynchus) mykiss (pathomorphological evaluation of internal organs, determination of the num� ber of different types of leucocytes in the blood, and electrophoretic investigation of protein profile of the blood serum) was investigated. It was determined that both antibiotics cause neutropenia, removal of albumin fraction in the blood serum, and appearance of lipoid assemblages and different pathologies of internal organs. Taste attractiveness of oxytetracyclin hydrochloride, benzylpenicillin sodium, cefazolinakos, and neomycin sulfate at the concentration in artificial food pellets of 1, 10, and 100 mg/ml was evaluated. It was determined that neomycin sulfate has more preferable taste for fishes, cefazolinakos is less attractive, and reaction of fishes to oxytetracyclin hydrochloride and benzylpenicillin sodium depends on their concentra� tion. The presence of two strongly different behavioral stereotypes of testing of food taste was confirmed.

Overton's Rule Helps To Estimate the Penetration of Anti-Infectives into Patients' Cerebrospinal Fluid

In 1900, Ernst Overton found that the entry of anilin dyes through the cell membranes of living cells depended on the lipophilicity of the dyes. The brain is surrounded by barriers consisting of lipid layers that possess several inward and outward active transport systems. In the absence of meningeal inflammation, the cerebrospinal fluid (CSF) penetration of anti-infectives in humans estimated by the ratio of the area under the concentration-time curve (AUC) in CSF (AUC(CSF)) to that in serum (AUC(CSF)/AUC(S)) correlated positively with the lipid-water partition coefficient at pH 7.0 (log D) (Spearman's rank correlation coefficient r(S) = 0.40; P = 0.01) and negatively with the molecular mass (MM) (r(S) = -0.33; P = 0.04). The ratio of AUC(CSF) to the AUC of the fraction in serum that was not bound (AUC(CSF)/AUC(S,free)) strongly correlated with log D (r(S) = 0.67; P < 0.0001). In the presence of meningeal inflammation, AUC(CSF)/AUC(S) also correlated positively with log D (r(S) = 0.46; P = 0.002) and negatively with the MM (r(S) = -0.37; P = 0.01). The correlation of AUC(CSF)/AUC(S,free) with log D (r(S) = 0.66; P < 0.0001) was as strong as in the absence of meningeal inflammation. Despite these clear correlations, Overton's rule was able to explain only part of the differences in CSF penetration of the individual compounds. The site of CSF withdrawal (lumbar versus ventricular CSF), age of the patients, underlying diseases, active transport, and alterations in the pharmacokinetics by comedications also appeared to strongly influence the CSF penetration of the drugs studied.

Neutralization and purification of thyroid stimulating antibody (TSAb) and thyroid blocking antibody (TBAb) by heterophilic antibody to animal IgG in Graves' disease

There are several reports that sera from Graves' patients contain heterophilic antibody (Ab) to animal IgG such as human anti-mouse antibody (HAMA). We examined the binding of TSAb and TBAb with heterophilic Ab. The binding of animal IgG with patient's IgG was examined by the inhibition of animal IgG on the binding of labeled bovine (b) IgG with patient's IgG. The binding to labeled bIgG was detected in the serum of 5 patients (2.7 %) among 185 patients with Graves' disease. The binding of the labeled bIgG with patient's IgG was inhibited by animal serum or the crude IgG (45% ammonium sulfate fraction of serum)(such as dog, horse, bovine, porcine, goat, ovine, rabbit, guinea-pig, rat, mouse) except human, monkey and chick. This heterophilic Ab which had cross-reaction with mammalian IgG (except human, monkey) was used as human anti-animal IgG Ab. TBII and TSAb activity of TSAb-positive serum, and TBII activity of TBAb-positive serum were neutralized by incubation with this Ab-bound column. Partial purification of TSAb- or TBAb- IgG from Protein A-purified TSAb- or TBAb-IgG was possible using this Ab-bound column. TBII and TSAb activity of TSAb-IgG and TBII activity of TBAb-IgG were neutralized by incubation with rabbit anti-human (h) IgG Ab (having cross-reaction with animal IgG). Further purification of Protein A-purified TSAb-IgG or TBAb-IgG by rabbit anti-hIgG Ab-bound column was impossible. The binding of TSAb and TBAb with heterophlic Ab means that TSAb-and TBAb-specific IgG have immunological similarity with mammalian species IgG compared to human IgG.

A unique two-way approach for the validation of total antioxidant capacity of serum samples

The human body is constantly exposed to a large variety of reactive oxygen species that are implicated in many pathophysiological conditions (atherosclerosis, cancer, neurodegenerative diseases etc.). Monitoring the antioxidant status of biological fluids could be used as an early warning sign 'biomarker' of possible disease onset. However, although several methods have been developed, questionable sensitivity, unreliability and non-reproducibility hamper all, making it difficult to have an internationally accepted standardized method. This study presents and demonstrates the remarkable ability of a newly developed antioxidant capacity assay method based on tetramethoxy azobismethylene quinone (TMAMQ) to measure the total antioxidant capacity of serum samples using three complimentary approaches. Using an UV-Vis spectroscopy and oxygen sensor, the reduction of TMAMQ by serum antioxidants was compared to either the formation of syringaldazine or consumption of oxygen. After adding a fraction of human serum, 4·01 μM TMAMQ was reduced to syringaldazine from a stock of 11·74 μM TMAMQ. Subsequent addition of laccase resulted in the oxidation of the formed syringaldazine back to TMAMQ resulting in an increase in TMAMQ concentration to 11·71 μM (re-establishing almost the same initial concentration of TMAMQ) while consuming 1·04 μM molecular oxygen. The reduction of TMAMQ by serum samples is directly proportional to the consumption of oxygen and the formation of syringaldazine. This means that either the formation of syringaldazine or oxygen consumption can be used to validate or confirm data obtained through monitoring TMAMQ reduction.

Competitive Selection from Single Domain Antibody Libraries Allows Isolation of High-Affinity Antihapten Antibodies That Are Not Favored in the llama Immune Response

Single-domain antibodies (sdAbs) found in camelids lack a light chain, and their antigen-binding site sits completely in the heavy-chain variable domain (VHH). Their simplicity, thermostability, and ease in expression have made VHHs highly attractive. Although this has been successfully exploited for macromolecular antigens, their application to the detection of small molecules is still limited to a very few reports, mostly describing low-affinity VHHs. Using triclocarban (TCC) as a model hapten, we found that conventional antibodies, IgG1 fraction, reacted with free TCC with a higher relative affinity (IC(50) 51.0 ng/mL) than did the sdAbs (IgG2 and IgG3, 497 and 370 ng/mL, respectively). A VHH library was prepared, and by elution of phage with limiting concentrations of TCC and competitive selection of binders, we were able to isolate high-affinity clones, K(D) 0.98-1.37 nM (SPR), which allowed development of a competitive assay for TCC with an IC(50) = 3.5 ng/mL (11 nM). This represents a 100-fold improvement with regard to the performance of the sdAb serum fraction, and it is 100-fold better than the IC(50) attained with other antihapten VHHs reported thus far. Despite the modest overall antihapten sdAbs response in llamas, a small subpopulation of high-affinity VHHs is generated that can be isolated by careful design of the selection process.