Chain-length dependent interfacial immunoreaction kinetics on self-assembled monolayers revealed by surface-enhanced infrared absorption spectroscopy.

Self-assembled monolayer (SAM) has been extensively applied as ideal interface layer for construction of biosensors. Its chain length and end functional groups determine the physical and chemical properties of the modified surfaces, which will affect the performance of constructed biosensors. Herein, we studied the influence of chain length of n-alkanethiols SAMs on the immunoreaction kinetics employing attenuated total reflection surface-enhanced infrared absorption spectroscopy (ATR-SEIRAS). Antibody (rabbit immunoglobulin) is assembled on carboxyl terminated SAMs of n-alkanethiols with different chain lengths (n = 3, 6, 11, 16). The whole fabrication steps of the immunoassay can be monitored in situ by the ATR-SEIRAS. From the time-dependent SEIRA spectra, the interfacial immunoreaction kinetics between the immobilized antibody and antigen (goat anti-rabbit immunoglobulin) can be evaluated. We found that the immunoreaction became faster with increasing the chain length of SAMs. This chain length dependent kinetics might be attributed to different orientations of the assembled antibody caused by different packing densities of SAMs. The present research offers a sensing platform to evaluate immunoassay kinetics and provides fundamentals for construction of immunoassay with high performance.

The current state of food allergy therapeutics.

The prevalence of IgE mediated food allergy is an increasing public health concern. The current standard of treatment is strict avoidance of the offending food(s). There are no FDA approved treatments for food allergy. This review will provide an overview of strategies currently under investigation for the treatment of food allergy. The main focus of research has been directed at various forms of immunotherapy, including oral, sublingual and epicutaneous delivery routes. While oral immunotherapy (OIT) has shown the greatest promise for efficacy in terms of amount of protein that can be ingested, it has also demonstrated less tolerability and a less favorable safety profile as compared to sublingual immunotherapy (SLIT) and epicutaneous immunotherapy (EPIT), which offers the least protection but has the best safety and tolerability profile. Investigation is also underway for modified antigens that may be used for immunotherapy and for adjuncts that may help facilitate immunotherapy, including biologics such as anti-IgE therapy, and also probiotics. There are also a number of preclinical concepts that are being evaluated to manipulate the antigens and/or the immune system that may one day be translatable to patients.

Microfluidic immunosensor for rapid and highly-sensitive salivary cortisol quantification.

This paper presents a novel poly(dimethylsiloxane) (PDMS) microfluidic immunosensor that integrates a complementary metal-oxide-semiconductor (CMOS) optical detection system for a rapid and highly-sensitive quantification of salivary cortisol. The simple and non-invasive method of saliva sampling provides an interesting alternative to the blood, allowing a fast sampling at short intervals, relevant for many clinical diagnostic applications. The developed approach is based on the covalent immobilization of a coating antibody (Ab), a polyclonal anti-IgG, onto a treated PDMS surface. The coating Ab binds the capture Ab, an IgG specific for cortisol, allowing its correct orientation. Horseradish peroxidase (HRP)-labelled cortisol is added to compete with the cortisol in the sample, for the capture Ab binding sites. The HRP-labelled cortisol, bonded to the capture Ab, is measured through the HRP enzyme and the tetramethylbenzidine (TMB) substrate reaction. The cortisol quantification is performed by colorimetric detection of HRP-labelled cortisol, through optical absorption at 450nm, using a CMOS silicon photodiode as the photodetector. Under the developed optimized conditions presented here, e.g., microfluidic channels geometry, immobilization method and immunoassay conditions, the immunosensor shows a linear range of detection between 0.01-20ng/mL, a limit of detection (LOD) of 18pg/mL and an analysis time of 35min, featuring a great potential for point-of-care applications requiring continuous monitoring of the salivary cortisol levels during a circadian cycle.

Therapeutic anti-IgE monoclonal antibody single chain variable fragment (scFv) safety and immunomodulatory effects after one time injection in four dogs.

BACKGROUND: The therapeutic monoclonal antibody omalizumab that is specific for IgE has proven to be an effective addition to the treatment of allergic disease in humans. HYPOTHESIS/OBJECTIVES: The aims of this study were to demonstrate the safety and immunomodulating effects of a single injection of a monoclonal antibody single chain variable fragments (scFv) specific for canine IgE in normal dogs. ANIMALS: Three normal dogs were bled for EDTA whole blood samples for 112 days post-injection (dpi). A fourth dog was monitored for 28 days. METHODS: Anti-IgE scFv was pegylated to minimize scFv dimerization. Four normal dogs were injected once subcutaneously with anti-IgE scFv at 1 mg/kg. Flow cytometry was performed on whole blood. Plasma levels of IgE were measured by ELISA. RESULTS: None of the four dogs showed signs of anaphylaxis. All dogs demonstrated decreases in IgE(+) cells in lymphocyte-gated events by 14 dpi. Dogs C and D returned to pre-injection levels by 21 days, whereas dogs A and B remained below pre-injection levels until Day 112. Similar differences were seen in IgE-bearing granulocyte-gated cells. Free plasma IgE decreased below pre-injection levels by 47% in Dog A and by 52% in Dog B at 112 days. Dogs C and D did not change by more than 32% from preinjection levels. CONCLUSION: A single injection of monomeric, pegylated scFv with high affinity for dog IgE was demonstrated to be safe. Marked reduction in IgE-bearing lymphocytes and granulocytes accompanied by reduced "free" plasma IgE level in two of four dogs is analogous to omalizumab in humans.

The Molecular Engineering of an Anti-Idiotypic Antibody for Pharmacokinetic Analysis of a Fully Human Anti-Infective.

Anti-idiotype monoclonal antibodies represent a class of reagents that are potentially optimal for analyzing the pharmacokinetics of fully human, anti-infective antibodies that have been developed as therapeutic candidates. This is particularly important where direct pathogen binding assays are complicated by requirements for biosafety level III or IV for pathogen handling. In this study, we describe the development of a recombinant, anti-idiotype monoclonal antibody termed E1 for the detection of a fully human, serotype-specific, therapeutic antibody candidate for the BSLIII pathogen Dengue virus termed 14c10 hG1. E1 was generated by naïve human Fab phage library panning technology and subsequently engineered as a monoclonal antibody. We show that E1 is highly specific for the fully-folded form of 14c10 hG1 and can be employed for the detection of this antibody in healthy human subjects' serum by enzyme linked immunosorbent assay. In addition, we show that E1 is capable of blocking the binding of 14c10 hG1 to dengue virus serotype 1. Finally, we show that E1 can detect 14c10 hG1 in mouse serum after the administration of the therapeutic antibody in vivo. E1 represents an important new form of ancillary reagent that can be utilized in the clinical development of a therapeutic human antibody candidate.

Effects of anti-phospholipase A(2) antibody supplementation on dry matter intake feed efficiency, acute phase response, and blood differentials of steers fed forage- and grain-based diets.

To determine whether supplementation of anti-phospholipase A antibody (aPLA) would alter voluntary DMI, feed efficiency (FE), acute-phase protein concentration, and blood differentials (BD) due to a change in diet from a forage-based to a grain-based diet, individual daily DMI was measured on 80 cross-bred steers during a 141-d period. On d 0, steers were blocked by BW and randomly assigned to receive a growing forage diet containing 1) no additive (CON; = 20), 2) inclusion of 30 mg of monensin and 8.8 mg of tylosin per kg of diet DM (MT; = 20), 3) inclusion of an aPLA supplement at 0.4% of the diet DM (0.4% aPLA; = 20), and 4) inclusion of an aPLA supplement at 0.2% of the diet DM (0.2% aPLA; = 20). On d 60, steers were transitioned into a grain-based diet (90% concentrate) over a 21-d "step-up" period while continuing to receive their supplement treatments and were maintained on the high-grain diet until the end of the trial on d 141. On d 0, 60, 81, and 141, individual shrunk BW was recorded. Blood samples were collected on d 60, 63, 65, 67, 70, 72, 74, 77, 79, 81, and 84 for determination of concentration of plasma ceruloplasmin, haptoglobin, and BD. During the growing forage-diet period, steers from the 0.2% aPLA and 0.4% aPLA treatments had lower ( < 0.05) residual feed intake (RFI; -0.12 ± 0.13 and -0.22 ± 0.13 kg/d, respectively) than steers from the CON treatment (0.31 ± 0.13 kg/d). During the grain-based diet period, the 0.2% aPLA (-0.12 ± 0.10 kg/d), 0.4% aPLA (0.36 ± 0.10 kg/d), and MT (0.10 ± 0.10 kg/d) steers had greater ( = 0.04) RFI than CON steers (-0.37 ± 0.10 kg/d). During the transition phase, white blood cell counts were greater ( = 0.04) for the 0.2% aPLA treatment (13.61 × 10 ± 0.42 × 10 cells/µL) than the 0.4% aPLA and MT treatments (12.16 × 10 ± 0.42 × 10 and 12.37 × 10 ± 0.42 × 10 cells/µL, respectively) and concentrations of lymphocytes also were greater ( = 0.01) for the 0.2% aPLA treatment (7.66 × 10 ± 0.28 × 10 cells/µL) than the 0.4% aPLA and MT treatments (6.71 × 10 ± 0.28 × 10 and 6.70 × 10 ± 0.28 × 10 cells/µL, respectively). Concentrations of plasma ceruloplasmin and haptoglobin were reduced ( < 0.05) for CON compared to aPLA steers (22.2 ± 0.83 vs. 24.4 ± 0.83 mg/dL and 0.18 ± 0.05 vs. 0.26 ± 0.05 mg/mL, respectively). Supplementation of aPLA improved FE of steers fed a forage-based growing diet but not when feeding grain-based diets. The 0.4% aPLA and MT treatments had decreased white blood cell counts and concentration of lymphocytes during the transition period compared to the 0.2% aPLA treatment, and CON steers had reduced concentrations of plasma ceruloplasmin and haptoglobin during the diet transition phase.

State of the art and perspectives in food allergy (part II): therapy.

Currently management of food allergy is mainly based on absolute avoidance of the offending food(s) and the use of rescue medication. However, the risk of severe or life-threatening reactions due to inadvertent exposure, nutritional imbalance and social isolation raises the demand of disease-modifying treatments. The aim of the different treatments is to allow patients to safely ingest the offending food(s). However this unresponsiveness can be transient and requires continued treatment (desensitization) and has to be permanent and sustained also after stopping the treatment (tolerance). This review focuses on non-allergen specific (anti-IgE, Chinese herbal formula, etc..) and allergen specific treatments for food allergy. The anti-IgE treatment is at the moment the only non-allergen-specific therapy, for which some data on a temporarily clinical efficacy have been provided. Regarding allergen-specific treatments, different protocols (oral, sublingual, subcutaneous and epicutaneous) with natural, heat treated or recombinant food allergens have been investigated. Although promising, results of the different clinical trials are heterogeneous. In particular data on long-term effects are lacking. At the moment food specific immunotherapy can be considered an experimental interventional strategy, limited to research, and not yet ready for routine use.

Intussusception secondary to anaphylactic reaction to salmon roe (ikura).

Anaphylactic food reaction often involves gastrointestinal symptoms, such as vomiting and abdominal pain, but to date, there have been no publications documenting the association between food hypersensitivity and intussusception. Herein is reported the case of a 2-year-old boy with intussusception accompanied by anaphylactic food reaction. The patient without known allergies complained of severe abdominal pain following ingestion of salmon roe for the first time. Dyspnea, wheezing and generalized urticaria also developed. Subsequently, he had stools containing jelly-like blood with mucus. At hospital arrival, physical examination identified an abdominal mass in the right lower quadrant; imaging confirmed the diagnosis of colo-colic intussusception. This patient was successfully treated with enema and no pathological findings were identified via radiology. Laboratory results supported the presence of IgE-mediated allergy to salmon roe in the present patient. To our knowledge, this is the first report to describe the possible association between intussusception and a hypersensitive food reaction.

Rapid polyclonal desensitization with antibodies to IgE and FcεRIα.

BACKGROUND: Rapid desensitization, a procedure in which persons allergic to an antigen are treated at short intervals with increasing doses of that antigen until they tolerate a large dose, is an effective, but risky, way to induce temporary tolerance. OBJECTIVE: We wanted to determine whether this approach can be adapted to suppress all IgE-mediated allergies in mice by injecting serially increasing doses of monoclonal antibodies (mAbs) to IgE or FcεRIα. METHODS: Active and passive models of antigen- and anti-IgE mAb-induced IgE-mediated anaphylaxis were used. Mice were desensitized with serially increasing doses of anti-IgE mAb, anti-FcεRIα mAb, or antigen. Development of shock (hypothermia), histamine and mast cell protease release, cytokine secretion, calcium flux, and changes in cell number and FcεRI and IgE expression were evaluated. RESULTS: Rapid desensitization with anti-IgE mAb suppressed IgE-mediated immediate hypersensitivity; however, some mice developed mild anaphylaxis during desensitization. Rapid desensitization with anti-FcεRIα mAb that only binds FcεRI that is not occupied by IgE suppressed both active and passive IgE-mediated anaphylaxis without inducing disease. It quickly, but temporarily, suppressed IgE-mediated anaphylaxis by decreasing mast cell signaling through FcεRI, then slowly induced longer lasting mast cell unresponsiveness by removing membrane FcεRI. Rapid desensitization with anti-FcεRIα mAb was safer and longer lasting than rapid desensitization with antigen. CONCLUSION: A rapid desensitization approach with anti-FcεRIα mAb safely desensitizes mice to IgE-mediated anaphylaxis by inducing mast cell anergy and later removing all mast cell IgE. Rapid desensitization with an anti-human FcεRIα mAb may be able to prevent human IgE-mediated anaphylaxis.

Crystal structure of two anti-porphyrin antibodies with peroxidase activity.

We report the crystal structures at 2.05 and 2.45 Å resolution of two antibodies, 13G10 and 14H7, directed against an iron(III)-αααß-carboxyphenylporphyrin, which display some peroxidase activity. Although these two antibodies differ by only one amino acid in their variable λ-light chain and display 86% sequence identity in their variable heavy chain, their complementary determining regions (CDR) CDRH1 and CDRH3 adopt very different conformations. The presence of Met or Leu residues at positions preceding residue H101 in CDRH3 in 13G10 and 14H7, respectively, yields to shallow combining sites pockets with different shapes that are mainly hydrophobic. The hapten and other carboxyphenyl-derivatized iron(III)-porphyrins have been modeled in the active sites of both antibodies using protein ligand docking with the program GOLD. The hapten is maintained in the antibody pockets of 13G10 and 14H7 by a strong network of hydrogen bonds with two or three carboxylates of the carboxyphenyl substituents of the porphyrin, respectively, as well as numerous stacking and van der Waals interactions with the very hydrophobic CDRH3. However, no amino acid residue was found to chelate the iron. Modeling also allows us to rationalize the recognition of alternative porphyrinic cofactors by the 13G10 and 14H7 antibodies and the effect of imidazole binding on the peroxidase activity of the 13G10/porphyrin complexes.

Induction of anti-anti-idiotype antibodies against sulfated glycosaminoglycans reduces atherosclerosis in apolipoprotein E-deficient mice.

OBJECTIVE: The pathogenesis of atherosclerosis is associated with the early retention of low-density lipoproteins that are trapped in the extracellular matrix of the arterial intima by interaction with glycosaminoglycan side chains of proteoglycans. Mutant mouse/human chimeric antibodies of the murine monoclonal antibody P3, which react with N-glycolyl-containing gangliosides and sulfated glycosaminoglycans, were tested for their potentially antiatherogenic properties through the induction of an idiotypic antibody network that may specifically interfere with the binding of low-density lipoproteins to proteoglycan side chains, low-density lipoprotein modification, and foam cell formation. METHODS AND RESULTS: Apolipoprotein E-deficient mice fed a high-fat, high-cholesterol diet received 5 to 6 doses of chP3R99 or chP3S98 mutant antibodies, showing high and low reactivity, respectively, against their respective antigens. Both chimeric antibodies elicited an immunodominant anti-idiotypic response in the absence of adjuvant. A striking (40%-43%) reduction (P<0.01) in total lesion areas was observed in 18-week-old mice immunized with chP3R99, but not chP3S98, compared with PBS-treated mice. The antiatherosclerotic effect was associated with increased mice sera reactivity against heparin and sulfated glycosaminoglycans, including chondroitin and dermatan sulfate. In addition, purified IgG from chP3R99-immunized mice blocked the retention of apolipoprotein B-containing lipoproteins within the arterial wall of apolipoprotein E(-/-) mice. CONCLUSIONS: The present study supports use of active immunization and the mounting of an idiotypic antibody network response against glycosaminoglycans as a novel approach to target atherosclerosis.

Cotinine-conjugated aptamer/anti-cotinine antibody complexes as a novel affinity unit for use in biological assays.

Aptamers are synthetic, relatively short (e.g., 20-80 bases) RNA or ssDNA oligonucleotides that can bind targets with high affinity and specificity, similar to antibodies, because they can fold into unique, three-dimensional shapes. For use in various assays and experiments, aptamers have been conjugated with biotin or digoxigenin to form complexes with avidin or anti-digoxigenin antibodies, respectively. In this study, we developed a method to label the 5' ends of aptamers with cotinine, which allows formation of a stable complex with anti-cotinine antibodies for the purpose of providing another affinity unit for the application in biological assays using aptamers. To demonstrate the functionality of this affinity unit in biological assays, we utilized two well-known aptamers: AS1411, which binds nucleolin, and pegaptanib, which binds vascular endothelial growth factor. Cotinine-conjugated AS1411/ anti-cotinine antibody complexes were successfully applied to immunoblot, immunoprecipitation, and flow cytometric analyses, and cotinine-conjugated pegaptanib/ anti-cotinine antibody complexes were used successfully in enzyme immunoassays. Our results show that cotinine-conjugated aptamer/anti-cotinine antibody complexes are an effective alternative and complementary technique for aptamer use in multiple assays and experiments.

Novel gold-capped nanopillars imprinted on a polymer film for highly sensitive plasmonic biosensing.

Herein, a nanoporous alumina was fabricated to use as a mold in transforming nanopillar structures onto a thin film polymer by thermal nanoimprint lithography (NIL). The size of the pores was successfully controlled by varying the applied voltages and etching time. These nanoporous structures were transferred to the Cyclo-olefin polymer (COP) film surface from the porous mold by a thermal nanoimprinting process. A plasmonic substrate was fabricated by sputtering a thin layer of gold onto this nanopillar polymer structure, and the refractive index response in a variety of media was evaluated. Finally, the biosensing capacity of this novel plasmonic substrate was verified by analysis of Human immunoglobulin and achieved a minimum detection limit of 1.0 ng/mL. With the advantages of mass production with consistent reproducibility stemming from the nanoimprint fabrication process, our gold-capped polymeric pillars are ready for the transition from academic interest into commercialization systems for practical use in diagnostic applications.

Time-resolved fluorescence resonance energy transfer as a versatile tool in the development of homogeneous cellular kinase assays.

Homogeneous cellular assays can streamline product detection in the drug discovery process. One commercially available assay employing time-resolved fluorescence resonance energy transfer (TR-FRET) that detects phosphorylated products was used to evaluate inhibitors of the receptor tyrosine kinase AXL in a cell line expressing an AXL-green fluorescent protein fusion protein. This TR-FRET assay was modified to evaluate the phosphorylation state of the AXL family member MER in a cell line expressing MER with a V5 tag by adding a fluorescein-labeled anti-V5 antibody. This homogeneous cellular assay was further modified to evaluate the nonreceptor tyrosine kinase focal adhesion kinase (FAK) in cell lines that expressed an untagged kinase by the inclusion of a commercially available anti-FAK antibody conjugated with an acceptor dye. The methods described here can be further adapted for TR-FRET detection of other cellular kinase activities.

Cotinine-conjugated aptamer/anti-cotinine antibody complexes as a novel affinity unit for use in biological assays

Aptamers are synthetic, relatively short (e.g., 20-80 bases) RNA or ssDNA oligonucleotides that can bind targets with high affinity and specificity, similar to antibodies, because they can fold into unique, three-dimensional shapes. For use in various assays and experiments, aptamers have been conjugated with biotin or digoxigenin to form complexes with avidin or anti-digoxigenin antibodies, respectively. In this study, we developed a method to label the 5' ends of aptamers with cotinine, which allows formation of a stable complex with anti-cotinine antibodies for the purpose of providing another affinity unit for the application in biological assays using aptamers. To demonstrate the functionality of this affinity unit in biological assays, we utilized two well-known aptamers: AS1411, which binds nucleolin, and pegaptanib, which binds vascular endothelial growth factor. Cotinine-conjugated AS1411/anti-cotinine antibody complexes were successfully applied to immunoblot, immunoprecipitation, and flow cytometric analyses, and cotinine-conjugated pegaptanib/anti-cotinine antibody complexes were used successfully in enzyme immunoassays. Our results show that cotinine-conjugated aptamer/anti-cotinine antibody complexes are an effective alternative and complementary technique for aptamer use in multiple assays and experiments.

Extracelluar matrix metalloproteinase as a novel target for pancreatic cancer therapy.

The objective of this study was to evaluate extracellular matrix metalloproteinase (EMMPRIN) as a novel target in orthotopic pancreatic cancer murine models. MIA PaCa-2 human pancreatic tumor cells were implanted in groups 1 and 3-7, whereas MIA PaCa-2 EMMPRIN knockdown cells were implanted in group 2. Dosing with anti-EMMPRIN antibody started immediately after implantation for groups 1-3 (residual tumor model) and at 21 days after cell implantation for groups 4-7 (established tumor model). Groups 3, 5, and 7 were treated with anti-EMMRPIN antibody (0.2-1.0 mg) twice weekly for 2-3 weeks, whereas the other groups served as the control. In the residual tumor model, tumor growth of anti-EMMPRIN-treated group was successfully arrested for 21 days (15 ± 4 mm(3)), which was significantly lower than that of the EMMPRIN knockdown group (80 ± 15 mm(3); P=0.001) or the control group (240 ± 41 mm(3); P<0.001). In the established tumor model, anti-EMMPRIN therapy lowered tumor volume increase by approximately 40% compared with the control, regardless of the dose amount. Ki67-expressed cell density of group 5 was 939 ± 150 mm(-2), which was significantly lower than that of group 4 (1709 ± 145 mm(-2); P=0.006). Microvessel density of group 5 (30 ± 6 mm(-2)) was also significantly lower than that of group 4 (53 ± 5 mm(-2); P=0.014), whereas the microvessel size of group 5 (191 ± 22 µm(2)) was significantly larger than that of group 4 (113 ± 26 µm(2); P=0.049). These data show the high potential of anti-EMMPRIN therapy for pancreatic cancer and support its clinical translation.

Vaccine approaches for food allergy.

The prevalence of food allergy appears to be increasing. Hypersensitivity reactions to foods account for significant morbidity and mortality. The current standard of care for treatment of food allergies is limited to diligent dietary avoidance and prompt pharmacotherapy should an unexpected ingestion result in a reaction. Complex interactions between dietary antigens, the gastrointestinal flora, and the gut associated mucosal system drive host immune responses towards oral tolerance or hypersensitivity. Oral tolerance is achieved by regulatory T cell suppression of immune responses and by clonal anergy. Many novel therapies to treat food allergies are currently under investigation. Most utilize antigen-specific strategies in an attempt to induce oral tolerance. Oral immunotherapy (OIT) has been the focus of much attention. Early studies had established the safety and efficacy of OIT, but its ability to induce long-term tolerance versus a state of desensitization remains to be firmly established. Nevertheless, recent advances in our understanding of oral tolerance induction has increased optimism that disease-modifying therapies for food allergies will soon be the standard of care.

Effects of dietary soy intake on maternal thyroid functions and serum anti-thyroperoxidase antibody level during early pregnancy.

Soy and its isoflavones have been suggested to suppress thyroperoxidase (TPO), induce goiter, inhibit deiodinase, and modulate immune functions. This study initially investigated the effects of dietary soy consumption on maternal thyroid functions and anti-TPO antibody (TPOAb) production during early pregnancy. Data were collected through questionnaire from 505 women enrolled during early pregnancy by random sampling in Shenyang, China. Based on soy intake frequency, the subjects were divided into three groups (frequent [three or more times per week], conventional [more than twice per month but less than three times per week], and occasional [two or fewer times per month]). Serum thyrotropin (TSH), free thyroxine (FT(4)), and TPOAb were measured by chemiluminescence immunoassay. Additionally, the concentrations of two primary isoflavones (daidzein and genistein) and creatinine were assessed in the spot urine samples from representative subjects (about 20%) randomly selected from the three groups. The percentages of frequent, conventional, and occasional consumers were 18.6%, 62.6%, and 18.8%, respectively. No difference was found in age, medical records, family history of thyroid diseases, serum FT(4), TSH, and TPOAb levels, TPOAb-positive percentages, or prevalence of thyroid dysfunctions among the groups. Both urinary daidzein and genistein levels were significantly higher in the frequent consumers compared with the other two groups. No correlations were found between urinary isoflavone levels and serum FT(4) or TSH. Urinary isoflavone levels were not significantly different between TPOAb-positive and -negative women among the randomly selected representative subjects. On the whole, our findings suggest dietary soy consumption during early pregnancy is not associated with the development of thyroid dysfunction or autoimmunity.

Detection of low-affinity anti-drug antibodies and improved drug tolerance in immunogenicity testing by Octet(®) biolayer interferometry.

We assessed the utility of the FortéBio Octet(®) system for detection of anti-drug antibodies (ADAs) against an investigational therapeutic human IgG1 monoclonal antibody (mAb), CNTO X. To understand the relative merits of this technology, key performance requirements were compared with two popularly accepted ADA detection methods, a step-wise bridging ELISA and a Meso Scale Discovery (MSD) homogeneous (single step binding) bridging ECLIA. When used to detect 13 monoclonal ADAs of varying affinities and one polyclonal ADA, all three methods demonstrated their greatest apparent sensitivity to the polyclonal sample (1, 6, and 130 ng/mL, respectively for ECLIA, ELISA, and Octet). Sensitivity to monoclonal ADAs tended to vary in accordance with their affinities, however, the sensitivity of the Octet method varied much less between ADAs. As a result, the above ranking became reversed such that Octet was the most and ELISA least sensitive for detection of low-affinity ADAs. With regard to drug tolerance, the presence of CNTO X could lead to false-negative assay results, although each method was affected to a different degree, with the Octet method tolerating up to 10 times more drug than the ECLIA method, which in turn tolerated up to 10 times more than the ELISA. Finally, the ECLIA and Octet methods were applied to the bioanalysis of cynomolgus monkey sera from a pre-clinical multiple dose study of CNTO X. Octet indicated 3 positive animals developed ADA as early as day 15 of the dosing phase while drug was present at nearly 1mg/mL. ECLIA detected only one of these, and only in a day 57 recovery sample after drug had cleared from circulation. We conclude that the Octet is a promising platform for detection of lower affinity ADAs and is particularly suitable for ADA detection when drug persists at levels that negatively impact bridging immunoassays.