Competitive ELISA Method Research Articles

ELISA for Detection of Soya Proteins in Meat Products

Indirect competitive ELISA method for the detection of soya proteins in meat products was developed. The detection limit of the method is 0.5% of the weight of added soya protein. A total of 131 meat product samples such as salamis or sausages from the Czech Republic market were investigated for the presence of soya proteins. Soya proteins were detected in 84% of the investigated samples without any declaration on the package of the product. The use of vegetable additives, namely soya in meat products in the market of the Czech Republic is very frequent and the restriction of its usage by legislation relates only to some kinds of durable products and ham (Act 264/2003 Coll.). The need for sensitive inspecting methods for soya protein detection is not only associated with the economic aspect (adulteration), but mainly with consumer health protection in case of allergy to soya proteins.

Toxin-screening and identification of bacteria isolated from highly toxic marine gastropod Nassarius semiplicatus

Bacteria isolated from a highly toxic sample of gastropod Nassarius semiplicatus in Lianyungang, Jiangsu Province in July 2007, were studied to probe into the relationship between bacteria and toxicity of nassariid gastropod. The toxicity of the gastropod sample was 2 × 10 2 mouse unit (MU) per gram of tissue (wet weight). High concentration of tetrodotoxin (TTX) and its analogues (TTXs) were found in the digestive gland and muscle of the gastropod, using high performance liquid chromatography coupled with mass chromatography (LC-MS). Bacterial strains isolated from the digestive gland were cultured and screened for TTX with a competitive ELISA method. Tetrodotoxin was detected in a proportion of bacterial strains, but the toxin content was low. Partial 16S ribosomal DNA (rDNA) of the TTX-producing strains was then sequenced and compared with those published in the GenBank to tentatively identify the toxic strains. It was found that most of the toxic strains were closely affiliated with genus Vibrio, and the others were related to genus Shewanella, Marinomonas, Tenacibaculum and Aeromonas. These findings suggest that tetrodotoxin-producing bacteria might play an important role in tetrodotoxin accumulation/production in N. semiplicatus.

A sensitive and reliable quantification method for Bisphenol A based on modified competitive ELISA method

Bisphenol A (BPA), generally known as bisphenols, has been identified as a potential estrogenic substance. BPA must be conjugated to carrier protein and BSA was commonly used. 4,4-Bis(4-hydroxyphenyl) valeric acid (BHPVA) has a bisphenolic structure and a long carbon chain with a reactive carboxyl group on the end. In this study, BHPVA-BSA was used to produce polyclonal antibody against bisphenolic structure, and a modified competitive ELISA method for quantification of BPA was developed. This system was based on BHPVA-BSA for polyclonal antibody production against bisphenolic structure, and BHPVA-HRP for determination of BPA substituting detection antibody in competitive reaction. Recovery was assessed at 10 different concentrations (2-1000 ng/ml) of BHPVA, and the recovery range was from 96.3% to 107.2%. The variation was from 6.2% to 9.8% for intra assay and from 10.1% to 12.6% for inter assay. The quadratic was used to establish the curve regression. The range was found to be between 2 and 1000 ng/ml. This modified competitive ELISA method has proven to be a very useful tool for quantification of BPA without the unexpected interaction of BSA and anti-BSA polyclonal antibody.

Seasonal occurrence and toxicity of Microcystis in impoundments of the Huron River, Michigan, USA

Occurrence and toxicity of Microcystis spp. were measured by ELISA in three impoundments of the Huron River in southeast Michigan, USA. Assays were conducted weekly from June through October 2005. Additional samples were collected to assess microcystin concentrations throughout the drinking water treatment process at the Ann Arbor Water Treatment Facility. Water column stability, nutrient concentrations, and N:P ratios were examined as potential predictors of phycocyanin and microcystin. Microcystin was found in two of the impoundments at seasonally varying concentrations. Microcystis presence was associated with N:P ratios between 40 and 80 by moles, and toxin levels typically peaked one to two weeks after a peak in phycocyanin. The toxin was also detected at low levels at all stages of the drinking water treatment process. Freezing and thawing water samples prior to analysis yielded maximum microcystin assay concentrations. Experiments indicated that the competitive ELISA method is susceptible to false negative reporting. This is the first report of algal toxins in this catchment, and results demonstrate that sensitive and rapid analytical methods offer the chance to link the dynamics of toxin production with environmental conditions.

Uptake of 2S Albumin Allergens, Ber e 1 and Ses i 1, across Human Intestinal Epithelial Caco-2 Cell Monolayers

We have investigated the absorption rates of two purified major allergen 2S albumins, Ber e 1 from Brazil nuts (Bertholletia excelsa Humb. & Bonpl.) and Ses i 1 from white sesame seeds (Sesamum indicum L.), across human intestinal epithelial Caco-2 cell monolayers following gastrointestinal digestion in vitro. The transport from apical to basolateral side in cell monolayers was evaluated by RP-HPLC-UV and indirect competitive ELISA methods, being confirmed by western-blotting analysis. Significant amounts (approximately 15-25 nmol micromol(-1) initial amount/h) of intact Ber e 1 and Ses i 1 were found in the basolateral side. The absorption rates of both plant allergens through the cell monolayer were shown to be constant during the whole incubation period (4 h at 37 degrees C), verifying that the permeability of the membrane was not altered by the allergen digests. Our findings revealed that both purified 2S albumin allergens may be able to survive in immunologically reactive forms to the simulated harsh conditions of the gastrointestinal tract to be transported across the Caco-2 cell monolayers, so that they would be able to sensitize the mucosal immune system and/or elicit an allergic response.

P113 A BIOCHEMICAL APPROACH FOR ANALYZING CHONDROITIN SULPHATE IN BLOOD CIRCULATION AND PURITY TESTING IN SUPPLEMENTS AND DRUGS

Methods: Urine samples were obtained in 354 consecutive patients, referred to the department of rheumatology for one of the following diseases [Osteoarthritis (OA), rheumatoid arthritis (RA), ankylosing spondylitis (AS), undetermined inflammatory arthritis (UIA), osteoporosis (OP)] and in 66 controls (CTRL). CTX I and II were assayed using a competitive ELISA method. CTX I and II correlations were studied by linear regression. Differences in CTX levels between groups were studied with analysis of variance, taking into account age, sex and BMI. Results: Cross sectional study. 144 males and 276 females; mean (SD) age 59(17); mean CTX I 228.9 (204) ng/mL; mean CTX II 375.7(408.6) ng/mL. CTX I was significantly higher in females than in males (p=0.006). There was a correlation between CTX I and CTX II concentrations (R=0.34, p 0.2). Conclusions: Both CTX I and II were increased in muscoskeletal diseases. Compared to CTRL, CTX I levels were signifcantly increased only in patients with OP. By contrast CTX II were significantly increased in OA, inflammatory rheumatisms but also OP. Surprisingly CTX II was as high in OP as in inflammatory diseases and in OA. The influence of the treatments on CTX levels remains to be investigated.

Development of Competitive Direct Enzyme-linked Immunosorbent Assay for the Detection of Gentamicin Residues in the Plasma of Live Animals

Competitive direct ELISA was developed to detect gentamicin residues. Mice immunized with gentamicin-keyhole limpet hemocyanin (KLH) conjugate developed good antiserum titers, which gradually increased with booster injections, indicating immunization was successfully processed. Monoclonal antibody against gentamicin was prepared using hybridoma cells cloned by limit dilution of fused cells. IgG was purified from ascites fluid of hybridoma cell-injected mice through ammonium sulfate precipitation and Sephadex G-25 gel filtration. After the gel filtration, fractions of high antibody titer were further purified through affinity chromatography on protein A/G column. Monoclonal antibody against gentamicin was confirmed as IgG1, which has kappa light chain. Cross-reactivities (CR50) of gentamicin monoclonal antibody to other aminoglycosides (kanamycin, neomycin, and streptomycin) were less than 0.005%, indicating the monoclonal antibody was highly specific for gentamicin. Standard curve constructed through competitive direct ELISA showed measurement range (from 80 to 20% of B/B0 ratio) of gentamicin was between 1 and 40 ng/ml, and 50% of B/B0 ratio was about 4 ng/ml. The gentamicin concentration rapidly increased to 1,300 ng/ml after the intramuscular administration up to 2 h, then sharply decreased to less than 300 ng/ml after 4 h of withdrawal, during which the elimination half-life (t1/2) of gentamicin in the rabbit plasma was estimated to be 1.8 h. Competitive direct ELISA method developed in this study using the prepared monoclonal antibody is highly sensitive for gentamicin, and could be useful for detecting gentamicin residues in plasma of live animals. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 10 : 1498-1504)

A non-radioactive method for angiotensin II receptor binding studies using the rat liver

Introduction: A new non-radioactive method based on competitive ELISA has been developed for binding studies on angiotensin II (Ang II) receptors. Method: Rat liver membrane was used as the source of angiotensin receptors and FITC-angiotensin II (FITC-Ang II) was used as the labeled ligand with an affinity similar to unlabeled Ang II. The effects of different concentrations of Ang II, losartan, CGP-42112A and saralasin were studied on FITC-Ang II binding. Results: The Ki values for Ang II, losartan and CGP-42112A were calculated as 0.52 ± 0.22 nM, 6 ± 3 nM and 0.15 ± 0.07 nM, respectively. Saralasin inhibited the binding of labeled ligand biphasically, revealing two different populations of Ang receptor with different affinities for saralasin. About 74% of the binding sites were more sensitive to saralasin with a Ki value of 0.32 ± 0.04 nM while saralasin showed a Ki value of 2.7 ± 0.8 nM for the remaining binding sites. Discussion: The competitive ELISA method developed in this work yields Ki values for angiotensin antagonists similar to those obtained by others using radiolabeled ligands. The simplicity of this method makes it a suitable alternative to radioligand studies for routine analysis of interaction of drugs with angiotensin receptors.

Development of ELISA and immunochromatographic assay for the detection of neomycin

Background Reliable analytical methods are required to monitor neomycin residue levels in the livestock products. In particular, a more simple and rapid detection method is required in the veterinary fields. Methods Competitive direct ELISA and immunochromatographic assay were developed using monoclonal antibody to detect neomycin in the animal plasma and milk. Results No cross-reactivity of the antibody was observed with other aminoglycosides based on competitive direct ELISA methods, indicating that the antibody is highly specific for neomycin. Based on the standard curves, the detection limits were determined to be 6.85 ng/ml in PBS, 3.61 ng/ml in plasma, and 2.73 ng/ml in milk, respectively. Recoveries of neomycin from spiked plasma and milk at levels of 50–200 ng/ml ranged from 87% to 108%. Concentration of intramuscularly injected neomycin was successfully monitored in the rabbit plasma through competitive direct ELISA. Immunochromatographic method was also developed using colloidal gold-conjugated monoclonal antibody. Through this method, the detection limits were estimated to be about 10 ng/ml of neomycin in PBS, plasma, and milk. Conclusions Immunochromatographic assay developed in this study is suitable for the simple screening of neomycin residues in the veterinary field. Observed positives can be confirmed using a more sensitive laboratory method such as competitive direct ELISA.

Detection of methyl parathion using immuno-chemiluminescence based image analysis using charge coupled device

A novel method based on immuno-chemiluminescence and image analysis using charge coupled device (CCD) for the qualitative detection of methyl parathion (MP) with high sensitivity (up to 10 ppt) is described. MP antibodies raised in poultry were used as a biological sensing element for the recognition of MP present in the sample. The immuno-reactor column was prepared by packing in a glass capillary column (150 microl capacity) MP antibodies immobilized on Sepharose CL-4B through periodate oxidation method. Chemiluminescence principle was used for the detection of the pesticide. Light images generated during the chemiluminescence reaction were captured by a CCD camera and further processed for image intensity, which was correlated with pesticide concentrations. K(3)Fe(CN)(6) was used as a light enhancer to obtain detectable light images. Different parameters including concentrations of K(3)Fe(CN)(6), luminol, urea H(2)O(2), antibody, addition sequence of reactants and incubation time to obtain best images were optimized. The results obtained by image analysis method showed very good correlation with that of competitive ELISA for methyl parathion detection. Competitive ELISA method was used as a reference to compare the results obtained by CCD imaging.

Comparison of fluorescence polarization assay, indirect ELISA and competitive ELISA methods for diagnosis of Brucella melitensis-infection in small ruminants

Three serological tests, the fluorescence-polarization assay (FPA), the indirect ELISA (iELISA) and the competitive ELISA (cELISA), were compared in detecting Brucella melitensis antibodies in 454 serum samples from sheep (71 from animals which had aborted, 228 from animals without a reported abortion for the last 2 years, 155 samples from animals vaccinated with the REV-1 vaccine) and 251 serum samples from goats (animals with sporadic abortions and of unknown vaccination-status). The sensitivity of the three tests was 85, 93.5 and 88.5% and their specificity 96, 94.5 and 98.5%, for the FPA, iELISA and cELISA, respectively. The agreement between the three methods was 63.5% when testing samples from abortion cases, 92% when testing samples from apparently healthy sheep and 84% when testing samples from vaccinated sheep. Agreement between the three methods increased, when the possibility of true infection decreased; it was higher when the samples were likely to be from brucella-free animals. It appears that iELISA would be valuable for use at the early stage of a control program, whilst cELISA at its final stage. The FPA did not appear to be as accurate as the other two tests, but it was the easier, cheaper and quicker to perform.

Molecular evolution inferred from immunological cross-reactivity of immunoglobulin G among Chiroptera and closely related species.

We examined the relationships between Megachiroptera and Microchiroptera, and between Chiroptera and other closely related species by the cross-reactivity of immunoglobulin epitopes. Rabbit polyclonal antibody to bat IgG was used for determining the cross-reactivity by a competitive ELISA method. Megachiroptera and Microchiroptera showed high cross-reactivity, over 95.3%, with each other. However, primates and insectivores showed very low cross-reactivity, 8.6 to 20.2% and 5.3 to 12.7%, respectively. These results suggest that suborders of Chiroptera are monophyletic and Chiroptera have a relatively closer relationship to primates than to insectivores.

Prevalence of hepatitis A antibody in children and adolescents with chronic liver disease

To evaluate the prevalence of Hepatitis A virus (HAV) in children and adolescents with chronic liver disease in a unit of pediatric hepatology. Between May 1999 and February 2001, we studied the prevalence of anti-HAV in 60 children and adolescents with chronic liver disease, aged between 1 and 16 years, from the Unit of Pediatric Hepatology of the Hospital de Clínicas de Porto Alegre. The total anti-HAV was determined by a commercially available competitive ELISA method (Abbott), and compared with age, sex, race, etiologic diagnosis and family income of each patient. A one-year old child was not included in the study because she presented twice with undetermined anti-HAV results. Among the other 59 patients, 14 (24%) presented a positive result of total anti-HAV. The ages of test-positive subjects varied between 1 and 16 years old (mean=7.7 years, median=8.5). The differences between positive and negative groups in relation to age, sex and race were not statistically significant. Family income was lower in anti-HAV positive patients, but this difference was not significant. The differences between the etiologies of liver diseases were probably more related to the age than to the etiologies of the diseases. In the studied population, the majority (76%) of children and adolescents with chronic liver disease are susceptible to hepatitis A virus infection and, consequently, they could present a more severe disease or even fulminant hepatitis A. We strongly suggest that these subjects receive Hepatitis A inactivated vaccine.

Effect of Conjugation of Cow Milk Whey Protein with Polyethylene Glycol on Changes in their Immunoreactive and Allergic Properties

Polyethylene glycol (PEG) was conjugated with milk proteins to determine the changes of the immunoreactive properties of α-lactalbumin (α-la) and β-lactoglobulin (β-lg). The competitive ELISA method was applied to determine the antigenicity of modified protein, using rabbit polyclonal antibodies. The immunoreactivity decreased to 0.90 μg ml-1 α-la (which corresponded with 0.05%) and 0.14 μg ml-1 β-lg (which corresponded with 0.08% of the immunoreactivity observed in raw milk). The determination of allergenicity of modified whey proteins was carried out on a group of 10 allergic patients and it was insignificantly weaker than that of a control sample of milk allergens.

Immunological detection of syringopeptins produced by Pseudomonas syringae pv. lachrymans

Several strains of plant pathogenic Pseudomonas are known to produce phytotoxic lipodepsipeptides (syringomycin, syringopeptins and related compounds) in vitro. However, detection of these compounds in organic extracts from diseased plant tissues has been attempted by chromatographic methods for syringomycin only. A macromolecular derivative of syringopeptins (KLH-SP25A+B) was used to raise polyclonal antibodies in rabbit. The antiserum was able to recognize free syringopeptins (SP22and SP25) with an estimated detection limit of 0.05 μg per well in the indirect ELISA, and 0.01 μg per well in the competitive ELISA. Cross-reaction with other structurally related lipodepsipeptides, e.g. syringomycins and pseudomycin A, was less than 20%. Using the competitive ELISA method developed in this study, relatively abundant quantities of the toxins, i.e. approximately 0.1 mg of syringopeptins per gram fresh tissue, were detected when an aqueous extract from cotyledons of zucchini infected with a strain of P. syringae pv. lachrymans was tested.

The Gene Expression of Coagulation Factor VIII in Mammalian Cell Lines

Both the full-length and B domain-deleted cDNA of factor VIII were constructed in plasmid pcDNA3, respectively, and successfully expressed in Cos-7 cells. The yield of recombinant factor VIII-ΔB (0.4 U/mL/10 6cells/day) was approximately four times higher than that of the recombinant factor VIII. In addition, it was indicated that the gene expression of factor VIII is specific for cells from different tissues. The highest expression level was found in the hepatocellular carcinoma line SMMC-7721, followed by kidney, ovary, and lung cell lines. To compare the efficiency of gene expression of recombinant factor VIII, the factor VIII-ΔB gene was further reconstructed in different forms in the expression plasmid pCMV-dhfr for transient gene expression in Chinese hamster ovary cells. The redundant 5′- and 3′-untranslated sequences of factor VIII-ΔB were deleted. The cDNA encoding the heavy and light chains of factor VIII were constructed, respectively. Among them the high yield of the recombinant factor VIII was found in the coexpression of the heavy and light chain cDNA fragments of factor VIII. The deletion of the redundant 5′-untranslated sequence of factor VIII-ΔB was also beneficial for gene expression. As expected, the gene coexpression of factor VIII-ΔB and von Willibrand Factor cloned by the long-polymerase chain reaction method was also helpful for enhancing the expression level of recombinant factor VIII. A monoclonal antibody raised against factor VIII was prepared and used for the specific assay of recombinant factor VIII by the competitive ELISA method, the assay results were consistent with those determined by the one-stage bioassay.

Detection of native and recombinant Bacillus macerans cyclodextrin glycosyltransferase using microtiter elisa techniques

Direct, sandwich, and competitive ELISA methods were developed to quantify Bacillus macerans cyclodextrin glycosyltransferase (CGTase) and its thioredoxin fusion molecule using rabbit polyclonal antibodies. The direct ELISA method was used to demonstrate equivalent molar response of both native CGTase and the thioredoxin-CGTase fusion protein to antibody binding. Sandwich ELISA showed the most sensitive detection range (0.2 ∼ 50 μg ml −1) against CGTase. We improved a competitive ELISA method by coating the microtiter plate with denatured CGTase in 6 m guanidine-HCI solution, resulting in enhanced, rapid response. This competitive ELISA method was specific, precise, and rapid when the method was used to quantify CGTase and monitor production of CGTase and its thioredoxin fusion protein in the recombinant E. coli. These methods are specifically useful when detecting and quantifying recombinant CGTase proteins in which mutations may have reduced or eliminated enzymatic activity.

Alternative G1m, G2m and G3m Allotypes of IGHG Genes Correlate with Atopic and Nonatopic Pathways of Immune Regulation in Children with Bronchial Asthma

Most genetic studies of bronchial asthma deal with IgE responsiveness. The manner by which allergens trigger IgE production and activate mast cells suggests that several genetic loci may be involved. Several reports of candidate genes include chromosome 6 and HLA antigens, chromosome 14q11 and the α chain of the T cell receptor, chromosome 11q32 and the β chain of the high-affinity IgE receptor and chromosome 5 and the gene cluster for IL-4, respectively. In addition, the immunoglobulin heavy chain G (IGHG) genes on chromosome 14q32 have been associated with both atopic and non atopic bronchial asthma in children. In order to further investigate the role of IGHG genes in asthmatic children, the phenotypes of patients with homozygous but alternative IGHG genes were investigated. IGHG gene expression of patients with childhood asthma was determined by serum Gm allotypes with a quantitative competitive indirect ELISA method. The groups consisted of 24 children with the homozygous G3m(b/b)-G1m(f/f )-G2m(n/n) and 16 with the alternative G3m(g/g)-G1m(a/a)-G2m(–n/–n) genes. The two different genotypes were investigated for serum IgE (PRIST), serum IgG subclass levels (radial immunodiffusion), Gm allotype levels (competitive ELISA), IgA and IgM levels (radial immunodiffusion), peripheral blood eosinophils, specific IgE antibodies (skin prick test, SPT, or radioallergosorbent test, RAST), number of peripheral blood CD lymphocyte markers (flow cytometry) and serum IL-4 and IFN-γ levels (ELISA). Comparison of the two genotypes in children with bronchial asthma revealed significantly increased IgE (p < 0.001), increased specific IgE (p < 0.001), as investigated by SPT or RAST (n = 10 allergens tested), increased number of peripheral blood eosinophils (p < 0.01), increased serum IgG1(f/f )(p < 0.001), IgG2(n/n)(p < 0.001) and IgG3(b/b)(p < 0.01) levels, and decreased CD8 given in percent of the total number of peripheral lymphocytes, (p < 0.02) in the G3m(b/b)-G1m(f/f )-G2m(n/n) genotype. The asthmatic children with the G3m(g/g)-G1m(a/a)-G2m(–n/–n) genes instead showed low IgE levels, practically no specific IgE antibodies, a lower number of peripheral blood eosinophils, lower IgG1(a/a), IgG2(–n/–n) and IgG3(g/g) serum levels and higher CD8 lymphocyte numbers. The results show that the IGHG3(b/b)-IGHG1(f/f )-IGHG2(n/n) genes are in linkage disequilibrium with allergen-specific high-responding IGHE genes and present the atopic phenotype of bronchial asthma, while the IGHG3(g/g)-IGHG1(a/a)-IGHG2(–n/–n) genes present the nonatopic phenotype of childhood asthma. The two genotypes with different amino acid epitopes of their constant heavy γ1, γ2 and γ3 chains presented qualitatively different IgG1, IgG2 and IgG3 molecules, respectively, and also different serum IgG1, IgG2 and IgG3 levels, together with different numbers of peripheral blood eosinophils and CD8 lymphocytes. The two IGHG genotypes represent different pathways of human immune regulation. An association of atopic IGHG genotype with other candidate genes for atopy could be suggested.

ELISA for the determination of saikosaponin a, an active component of Bupleuri Radix.

In order to quantify saikosaponin a (SSA), one of the major active components of Bupleuri Radix, a competitive and indirect ELISA method was developed. High titer rabbit polyclonal antibodies (pAbs) were raised against a conjugate of SSA and bovine serum albumin, coupled with a periodate oxidation method. SSA competitively inhibited the binding of rabbit anti-SSA pAbs to SSA-ovalbumin on the solid phase, a coated antigen on the well. The quantity of pAbs bound to the well was monitored using a peroxidase-conjugated anti-rabbit IgG as a secondary antibody, and tetramethylbenzidine solution as a substrate. The measuring range extended from 50 pg/ml to 20 ng/ml of SSA, with a detection limit of 40 pg/ml (5.13 pM). Antibodies showed some cross-reactivity with saikosaponin c (12.74%). However, the antibodies showed only slight cross-reactivities with saikosaponin d (0.3%), which differs from SSA only in the stereochemistry of the 16-hydroxyl group, and the artificial saikosaponins, saikosaponin b1 (2.1%) and saikosaponin g (0.53%). The specific and sensitive ELISA is especially suited for determination of SSA in samples when only small quantities of materials can be extracted for analysis.

Regulation of the flavin redox potential by flavin-binding antibodies.

Single-chain Fv antibody fragments binding different flavin forms [10-(5'-carboxybutyl-)flavin (Fl[ox]) and 10-(5'-carboxybutyl)-1,5-dihydroflavin (Fl[red])] have been generated from an antibody phage-display library to study how a protein environment regulates the redox potential, starting from a protein other than a natural flavoprotein. These 'flavobodies' are characterized by time-resolved and steady-state fluorescence spectroscopy, by competitive ELISA methods (mapping of the antigen-binding site), and by molecular modelling. The three-dimensional models of the antigen-binding sites are consistent with the experimental results. Binding of anti-Fl(red) 5 to flavin increases the redox potential, mainly due to an Arg residue interacting with the flavin N1. Thus anti-Fl(red) 5 shows an 'oxidase-like' redox-potential behaviour, confirming the idea that positively charged residues in the vicinity of N1 increase the redox potential. The results obtained with anti-Fl(ox), which do not resemble a natural flavoprotein, show that when the pyrimidine-like nucleus of the flavin is not involved in binding, the redox potential is not significantly affected. These results are in contrast to those obtained with chicken riboflavin-binding protein.