Biotin as a masking agent in chorionic gonadotropin assays utilizing biotinylated antibodies.

Biotin interference in streptavidin/biotin-based immunoassays has been recently recognized as a confounding factor in clinical settings. Depending on the nature of the assay, the presence of excess biotin in patient samples can cause falsely high or low results. One of the platforms known to be affected, Roche Cobas, is widely used in anti-doping laboratories to test for intact chorionic gonadotropin (hCG) in urine. While biotin levels in blood have been well studied, less is known about urinary biotin due to its limited clinical significance. Having analyzed over 4,000 urine samples, we have established a reference range for urinary biotin with a median concentration of approximately 12 ng/ml. However, a significant number of samples contain much higher amounts, with a maximum approaching 10 µg/ml, suggesting biotin supplementation. Consequently, the tolerance of hCG STAT assay towards biotin was investigated over a wide concentration range. The apparent hCG concentration was found to decrease almost linearly as biotin increased from 100 to 1,000 ng/ml, with only 10% of the expected value reported by the assay as biotin reached 1,000 ng/ml. Further increase of biotin resulted in a progressive, albeit more moderate, decline in measured hCG concentration. To avoid a false negative result in the context of anti-doping analysis, it is highly recommended to monitor biotin in urine and perform diafiltration before hCG measurement in samples with elevated biotin to remove the interference.

Fluorescence correlation spectroscopy study of the complexation of DNA hybrids, IgG antibody, and a chimeric protein of IgG-binding ZZ domains fused with a carbohydrate binding module.

Fluorescence correlation spectroscopy (FCS) was used to characterize the molecular interactions between the four components of a DNA recognition system. A fluorescent DNA probe was used to assess: (i) the hybridization with a complementary biotin-labeled target, (ii) the complexation of the resulting hybrid and an anti-biotin antibody, and (iii) the binding of the latter complex to a ZZ-CBM fusion protein that combines small synthetic IgG Fc-binding Z domains with a carbohydrate binding module (CBM). These binding interactions were monitored by exposing the fluorescent DNA probe to different amounts and combinations of the other molecules in solution. Through the analysis of FCS autocorrelation curves, an association constant (Ka) of 2.9 × 107 M-1 was estimated for DNA·DNA hybridization, and the presence of (non-) complementary target DNA in solution could be discriminated. The specific capture of biotinylated DNA hybrids by anti-biotin IgG was verified, with an apparent Ka of 2.5 × 106 M-1. The increment in the diffusion time measured when the DNA·DNA:antibody complexes were in contact with the ZZ-CBM fusion protein suggested that the binding occurs at a stoichiometric ratio of DNA/antibody complex to fusion larger than 1 : 1. The FCS-derived information obtained is useful to gain insight into molecular interactions involved in diagnostic assays.

Factitious Graves' Disease Due to Biotin Immunoassay Interference-A Case and Review of the Literature.

CONTEXT: Biotin (vitamin B7) is an essential co-factor for four carboxylases involved in fatty acid metabolism, leucine degradation, and gluconeogenesis. The recommended daily intake (RDI) of biotin is approximately 30 µg per day. Low-moderate dose biotin is a common component of multivitamin preparations, and high-dose biotin (10 000 times RDI) has been reported to improve clinical outcomes and quality of life in patients with progressive multiple sclerosis. Biotin is also a component of immunoassays, and supplementation may cause interference in both thyroid and non-thyroid immunoassays. OBJECTIVE: To assess whether biotin ingestion caused abnormal thyroid function tests (TFTs) in a patient through assay interference. DESIGN: We report a patient with biotin-associated abnormal TFTs and a systematic review of the literature. SETTING: A tertiary endocrine service in Hamilton, New Zealand. RESULTS: The patient had markedly abnormal TFTs that did not match the clinical context. After biotin cessation, TFTs normalized far more rapidly than possible given the half-life of T4, consistent with assay interference by biotin. Multiple other analytes also tested abnormal in the presence of biotin. CONCLUSION: Biotin ingested in moderate to high doses can cause immunoassay interference. Depending on the assay format, biotin interference can result in either falsely high or low values. Interference is not limited to thyroid tests and has the potential to affect a wide range of analytes. It is important for clinicians to be aware of this interaction to prevent misdiagnosis and inappropriate treatment.

Visualising single molecules of HIV-1 and miRNA nucleic acids.

BACKGROUND: The scarcity of certain nucleic acid species and the small size of target sequences such as miRNA, impose a significant barrier to subcellular visualization and present a major challenge to cell biologists. Here, we offer a generic and highly sensitive visualization approach (oligo fluorescent in situ hybridization, O-FISH) that can be used to detect such nucleic acids using a single-oligonucleotide probe of 19-26 nucleotides in length. RESULTS: We used O-FISH to visualize miR146a in human and avian cells. Furthermore, we reveal the sensitivity of O-FISH detection by using a HIV-1 model system to show that as little as 1-2 copies of nucleic acids can be detected in a single cell. We were able to discern newly synthesized viral cDNA and, moreover, observed that certain HIV RNA sequences are only transiently available for O-FISH detection. CONCLUSIONS: Taken together, these results suggest that the O-FISH method can potentially be used for in situ probing of, as few as, 1-2 copies of nucleic acid and, additionally, to visualize small RNA such as miRNA. We further propose that the O-FISH method could be extended to understand viral function by probing newly transcribed viral intermediates; and discern the localisation of nucleic acids of interest. Additionally, interrogating the conformation and structure of a particular nucleic acid in situ might also be possible, based on the accessibility of a target sequence.

A nanoparticle-based sensor for visual detection of multiple mutations.

Disposable dipstick-type DNA biosensors in the form of lateral flow strips are particularly useful for genotyping in a small laboratory or for field testing due to their simplicity, low cost and portability. Their unique advantage is that they enable visual detection in minutes without the use of instruments. In addition, the dry-reagent format minimizes the pipetting, incubation and washing steps. In this work, we significantly enhance the multiplexing capabilities of lateral flow strip biosensors without compromising their simplicity. Multiplex genotyping is carried out by polymerase chain reaction (PCR) followed by a single primer extension reaction for all target alleles, in which a primer is extended and biotin is incorporated only if it is perfectly complementary to the target. Multiallele detection is achieved by multiple test spots on the membrane of the sensor, each comprising a suspension of polystyrene microspheres functionalized with capture probes. The products of the primer extension reaction hybridize, through specific sequence tags, to the capture probes and are visualized by using antibiotin-conjugated gold nanoparticles. This design enables accommodation of multiple spots in a small area because the microspheres are trapped in the fibres of the membrane and remain fixed in site without any diffusion. Furthermore, the detectability is improved because the hybrids are exposed on the surface of the trapped microspheres rather than inside the pores of the membrane. We demonstrate the specificity and performance of the biosensor for multiallele genotyping.

Macromolecular scaffolds for immobilizing small molecule microarrays in label-free detection of protein-ligand interactions on solid support.

We explored two macromolecular scaffolds, bovine serum albumin (BSA) and polyvinyl alcohol (PVA), as chemically complementary platforms for immobilizing small molecule compounds on functionalized glass slides. We conjugated biotin molecules to BSA and amine-derivatized PVA and subsequently immobilized the conjugates on epoxy-functionalized glass slides through reaction of free amine residues on BSA and PVA with surface-bound epoxy groups. We studied binding reactions of such immobilized small molecule targets with solution-phase protein probes using an oblique-incidence reflectivity difference scanning optical microscope. The results showed that both BSA and amine-derivatized PVA were effective and efficient as carriers of small molecules with NHS residues and fluoric residues and for immobilization on epoxy-coated solid surfaces. A significant fraction of the conjugated small molecules retain their innate chemical activity.

Comparison of neutralizing antibody assays for receptor binding and enzyme activity of the enzyme replacement therapeutic Naglazyme (galsulfase).

Most patients receiving Naglazyme (galsulfase, rhASB) enzyme replacement therapy for mucopolysaccharidosis type VI develop an antibody response. To evaluate the impact of this response, two in vitro neutralizing antibody (NAb) assays were developed based on the two steps of the mechanism of action. Neutralization of enzyme activity was detected by inhibition of rhASB cleavage of a fluorogenic substrate. Neutralization of receptor binding was detected by decreased binding of labeled rhASB to immobilized soluble receptor. For the enzyme activity NAb assay, serum pretreatment was required to isolate antibodies from interfering phosphate ions, with sensitivity of < or =5 microg/mL. The receptor binding NAb assay used a five-fold dilution, with sensitivity of < or =40 microg/mL. Cutpoints for percent inhibition were based on 95% confidence intervals from naïve sera. Clinical samples were similarly likely to be positive in both assays than positive for neutralization of only one step in the mechanism of action. The two NAb assays yielded complementary information about potential neutralization of rhASB. Relative estimated sensitivity between neutralization assays did not correlate with the number of positive clinical samples or patients. In vitro NAb assays based on a well-understood mechanism of action provide specific information about the NAb mechanism.

Antigen targeting to dendritic cells with bispecific antibodies.

We have developed a universal DC targeting vehicle that could be a convenient method to deliver any type of antigen to DC. P125, a quadroma (hybrid-hybridoma) secreting bispecific monoclonal antibodies (bsmAb), with one paratope specific for mouse DC DEC-205 and another paratope specific for biotin, was developed by PEG-fusion of the two parental hybridomas and selected by a fluorescence activated cell sorter. The bsmAb were purified using a biotin-Agarose column and the bsmAb activity was demonstrated using ELISA method employing mouse bone marrow DC and biotinylated BSA. Both confocal microscopy and ELISA studies have shown enhanced binding and internalization of biotinylated and FITC-labelled M13 to DC cell in the presence of bsmAb. In vivo studies in mice with biotinylated OVA has shown that in the presence of bsmAb and anti-CD40 mAb, both humoral and cell-mediated responses can be augmented. In addition, only a low concentration of antigen (500 fold less) is required using bsmAb to achieve a similar immune response in mice that were immunized using complete Freund's adjuvant. In the absence of traditional adjuvants, bsmAb targeting of biotinylated antigens to DC could be an alternative, convenient method to deliver antigens to DC. Moreover, this method could be an alternative method to ex vivo stimulation of DC to overcome DC defects and for treatment of cancer.

Enhanced immune response to T-independent antigen by using CpG oligodeoxynucleotides encapsulated in liposomes.

Immunostimulatory CpG oligodeoxynucleotides (ODN) have been tested as immunoadjuvants for various vaccines including T-cell independent (TI) antigens. Findings from previous reports suggest that close physical association of CpG ODN to the antigen could enhance its adjuvant effect. As an alternative to chemical conjugation of CpG ODN to the antigen, the current study is aimed at determining the benefit of using liposomes as a carrier for CpG ODN to improve the immune response to biotinylated liposomes (Bx-liposomes), a model of a TI antigen. Liposomes with suboptimal concentration of hapten (1% biotin) were not immunogenic. However, when CpG ODN encapsulated in Bx-liposomes were used to immunize mice, a hapten-specific response was obtained as indicated by antibody-mediated elimination of re-administered Bx-liposomes. CpG ODN co-administered with empty Bx-liposomes could not achieve the same effect, indicating the requirement for encapsulation of the adjuvant. Using both intravenous (i.v.) and subcutaneous (s.c.) immunization methods, it was found that IgM levels, but not IgG levels were elevated. Immunization in nude mice confirmed that the immune response obtained was TI. The use of non-CpG ODN and an ODN with alternatively flanked CpG motifs showed no adjuvant effect. Incorporation of poly(ethylene)glycol (PEG)-modified lipid in liposomes enhanced the immune response even further. In conclusion, our data shows that liposomes are a useful delivery vehicle for CpG ODN as an immune adjuvant for TI antigens.

A novel anionic modification of N-glycans on mammalian endothelial cells is recognized by activated neutrophils and modulates acute inflammatory responses.

We previously reported an unusual carboxylated modification on N:-glycans isolated from whole bovine lung. We have now raised IgG mAbs against the modification by immunization with biotinylated aminopyridine-derivatized glycans enriched for the anionic species and screening for Abs whose reactivities were abrogated by carboxylate neutralization of bovine lung glycopeptides. One such Ab (mAb GB3.1) was inhibited by carboxylated bovine lung glycopeptides and other multicarboxylated molecules, but not by glycopeptides in which the carboxylate groups were modified. The Ab recognized an epitope constitutively expressed on bovine, human, and other mammalian endothelial cells. Stimulated, but not resting, neutrophils bound to immobilized bovine lung glycopeptides in a carboxylate-dependent manner. The binding of activated neutrophils to immobilized bovine lung glycopeptides was inhibited both by mAb GB3.1 and by soluble glycopeptides in a carboxylate-dependent manner. The Ab also inhibited extravasation of neutrophils and monocytes in a murine model of peritoneal inflammation. This inhibition of cell trafficking correlated with the increased sequestration but reduced transmigration of leukocytes that were found to be adherent to the endothelium of the mesenteric microvasculature. Taken together, these results indicate that these novel carboxylated N:-glycans are constitutively expressed on vascular endothelium and participate in acute inflammatory responses by interaction with activated neutrophils.

Biotin carboxyl carrier protein and carboxyltransferase subunits of the multi-subunit form of acetyl-CoA carboxylase from Brassica napus: cloning and analysis of expression during oilseed rape embryogenesis.

In the oilseed rape Brassica napus there are two forms of acetyl-CoA carboxylase (ACCase). As in other dicotyledonous plants there is a type I ACCase, the single polypeptide 220 kDa form, and a type II multi-subunit complex analogous to that of Escherichia coli and Anabaena. This paper describes the cloning and characterization of a plant biotin carboxyl carrier protein (BCCP) from the type II ACCase complex that shows 61% identity/79% similarity with Anabaena BCCP at the amino acid level. Six classes of nuclear encoded oilseed rape BCCP cDNA were clones, two of which contained the entire coding region. The BCCP sequences allowed the assignment of function to two previously unassigned Arabidopsis expressed sequence tag (EST) sequences. We also report the cloning of a second type II ACCase component from oilseed rape, the beta-carboxyltransferase subunit (betaCT), which is chloroplast-encoded. Northern analysis showed that although the relative levels of BCCP and betaCT mRNA differed between different oilseed rape tissues, their temporal patterns of expression were identical during embryo development. At the protein level, expression of BCCP during embryo development was studied by Western blotting, using affinity-purified anti-biotin polyclonal sera. With this technique a 35 kDa protein thought to be BCCP was shown to reside within the chloroplast. This analysis also permitted us to view the differential expression of several unidentified biotinylated proteins during embryogenesis.

Immunogenicity of immunoliposomes: reactivity against species-specific IgG and liposomal phospholipids.

Immunoliposomes with surface-linked avidin-biotinylated mouse IgG2a were prepared from dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylglycerol (DMPG) and biotinylated dipalmitoylphosphatidylethanolamine (DPPE-biotin) in the molar ratio 10:1:0.1 with or without 5 mol% poly(ethylene glycol) dipalmitate (PEG-(C18)2). The ability of IgG2a immunoliposomes to elicit anti-IgG2a antibodies in mice was compared with alum and N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP). IgG2a 5 microgram/mouse) did not elicit an IgG1 antibody response after 4 s.c. injections. Alum-adsorbed IgG2a elicited 2.1 +/- microgram IgG1 antibody/ml serum, whereas MDP elicited 24.3 +/- microgram/ml serum. IgG2a immunoliposomes elicited 12.4 +/- 3.7 microgram IgG1 antibody/ml serum, while immunoliposomes containing lipophilic PEG-(C18)2 elicited 21.4 +/- 5.1 microgram IgG1 antibody/ml serum. Elicited antibodies were specific for IgG2a, with no cross-reactivity with IgG2b. Anti-DPPC or anti-DMPG IgG antibody levels did not change during immunization. Anti-DPPE IgG antibody levels were slightly but significantly elevated during immunization, and there was a significant increase in the level of anti-DPPE-biotin antibodies. These results demonstrate that immunoliposomes prepared with species-specific antibody are immunogenic and induce significant levels of isotypespecific antibody upon repeated injection.

Photoaffinity labeling of antibodies for applications in homogeneous fluoroimmunoassays.

A homogeneous noncompetitive immunoassay based on photoaffinity labeling techniques is described. Using this method, a fluorophore (reporter) can be specifically attached to an antibody in the vicinity of its antigen-combining sites. Upon antigen binding, changes in the fluorescence spectrum of the reporter molecule are often observed. Two fluorophores, pyrene and dansyl, were evaluated for this purpose. Also, this technology is ideal for fluorescence energy-transfer immunoassays that require labeling of the antibody with either a donor or acceptor fluorophore. In such cases, a fluorescent dye can be specifically attached near the antigen-combining site, where it can participate in high-efficiency energy transfer with its complementary fluorophore attached to the antigen.

Monoclonal anti-biotin antibodies simulate avidin in the recognition of biotin.

The sequence of the VH gene of a monoclonal anti-biotin antibody was determined. Biotin-binding motifs, similar to those in avidin and streptavidin, were identified in complementary determining regions 2 and 3, suggesting that natural selection of functional motifs may occur in unrelated protein types.

Characterization of the adjuvant-free serological response to protein antigens coupled to antibodies specific for class II MHC determinants.

Having previously demonstrated that coupling protein antigens to anti-class II major histocompatibility complex (MHC) monoclonal antibodies provides a means of inducing adjuvant-independent IgG responses to the 'targeted' antigen, we have now explored the parameters of this response in more detail. Using as the model system egg avidin coupled to biotinylated anti-class II MHC mAbs, the kinetics of the avidin-specific 1 degrees IgG response, the IgG subclass distribution of the 2 degrees anti-avidin response, and the immunogenicity of anti-class II-avidin conjugates in H-2-recombinant mice have been investigated. In addition, the use of avidin as a bridging molecule in the creation of immunoconjugates involving third-party proteins has been further characterized, and the immunogenicity of defined soluble conjugates in mice bearing the appropriate class II MHC gene products has been demonstrated. These findings extend our understanding of the immunoconjugate approach to adjuvant-independent immunization, and support its exploration as an alternative means of subunit vaccine design.

Adjuvant-free IgG responses induced with antigen coupled to antibodies against class II MHC.

The generation of strong serological responses to protein antigens in experimental animals usually requires the use of potent adjuvants, most of which cannot be used in human or veterinary vaccines because of deleterious side effects. Attempting to circumvent this problem, we have assessed an adjuvant-free antigen-delivery system based on the hypothesis that antigen coupled to monoclonal antibodies (mAbs) specific for class II major histocompatibility complex (MHC) determinants should be 'targeted' onto antigen-presenting cells, thus facilitating recognition by helper T cells. We found that the biotin-binding protein avidin could generate a serological response in mice, without adjuvant, when injected coupled to a biotinylated anti-class II MHC mAb. Equivalent amounts of avidin mixed with the non-biotinylated form of the same mAb failed to elicit a response. A targeting effect was demonstrated at low levels of injected conjugate because only mice bearing the appropriate class II antigens responded. Responses were also seen with a protein antigen other than avidin, offering a new, adjuvant-free approach to subunit vaccine construction.

Antigenic presentation of small molecules and peptides conjugated to a preformed iscom as carrier.

The aim of the present study was to elaborate a carrier system for haptens and synthetic peptides, making them immunogenic without addition of Freund's adjuvants. As carriers, preformed iscoms and micelles as well as BSA have been compared. The iscoms and micelles were prepared with envelope proteins of an influenza virus. As a model hapten, the small molecules of biotin were coupled to iscoms to determine the optimum epitope density for induction of an enhanced antibody response to the hapten. The most efficient carrier tested was the preformed iscom at an epitope density of ten biotin molecules per viral protein in the iscom. This carrier system exceeded the efficacy of both the preformed micelles and BSA, the latter with or without addition of Freund's adjuvant. A favourable epitope density could not be achieved when each of two different synthetic peptides was conjugated to iscoms. Epitope densities higher than one to three peptide molecules per protein lead to polymerization of either the peptide or the carrier. The coupling agent was glutardialdehyde.