Use of a biomimetic peptide in the design of a competitive binding assay for biotin and biotin analogues

A competitive binding assay for biotin, biocytin, and desthiobiotin utilizing a genetically engineered enzyme-ligand conjugate is described herein. This assay is unique in that the enzyme-ligand conjugate consists of the streptavidin binding peptide Strep-tag II, which mimics the binding of biotin to streptavidin, rather than biotin itself. This allows for the construction of a well-defined, oligosubstituted enzyme-ligand conjugate for which the site of attachment of the ligand on the enzyme is known precisely. The assay has detection limits of 5 x 10(-8) M for biotin, 1 x 10(-7) M for biocytin, and 2 x 10(-6) M for desthiobiotin, and it serves as a model system in that it demonstrates the feasibility of using enzyme-ligand conjugates in which a peptide mimic of the analyte ligand is genetically fused to the enzyme. This avoids the problems associated with covalent attachment of the ligand to the enzyme, such as multiple substitution of the ligand and variability of the site of attachment. To our knowledge, this is the first example of using an enzyme-peptide mimic conjugate to detect a nonpeptide analyte.     

Neoglycoconjugates of mannan with bovine serum albumin and their interaction with lectin concanavalin A.

Neoglycoconjugates were prepared from mannan isolated from yeast Saccharomyces cerevisiae and activated by periodate oxidation to create aldehyde groups. Various degrees of oxidation introduced 11-28 aldehyde groups per mannan molecule and simultaneously resulted in a molar mass decrease from 46 to 44.5-31 kDa. The activated mannans were subsequently conjugated with bovine serum albumin forming neoglycoconjugates. Some parameters of these mannan-bovine serum albumin conjugates were characterized: saccharide content 25-30% w/w, molar mass within the range 169-246 kDa, and polydispersion (M(w)/M(n)) from 2.8 to 3.6. The interaction of these conjugates with lectin concanavalin A was studied using three different methods: (i) quantitative precipitation in solution; (ii) sorption to concanavalin A immobilized on bead cellulose; and (iii) kinetic measurement of the interaction by surface plasmon resonance. Quantitative precipitation assay showed only negligible differences in the precipitation course of original mannan and the corresponding mannan-bovine serum albumin conjugates. Both the sorption method (equilibrium method) and the surface plasmon resonance measurement (kinetic method) demonstrates that the values of dissociation constant K(D) of all synthetic neoglycoconjugates were within the range 10(-7) - 10(-8) mol x L(-1) (close to K(D) = 10(-8) mol x L(-1) determined by the sorption method for the original mannan). In conclusion, characterization of synthetic neoglycoconjugates confirmed that the method used for their preparation retained the ability of mannan moiety to interact with concanavalin A.     

Enzymatic solid-phase assay for biotin and a biotin-benzodiazepine conjugate

A novel enzymatic ligand binding assay for biotin and its benzodiazepine conjugate is based on their binding to horseradish peroxidase-avidin conjugate (A-P) followed by the uptake of biotin-unsaturated A-P onto polystyrene beads coated with biotin-BSA. The detection limit is 1.3 x 10(-16) mol per tube (300 microL) with a 3.3 x 10(-12) M A-P solution and varies with the conjugate concentration employed. The coefficient of variation for 10 repetitive assays of 10(-15) mol of biotin is 6.22%.     

Characteristics of aldosterone antisera related to the duration of immunization.

Three antisera specific to aldosterone and elicited with different aldosterone protein conjugates (aldosterone-3-oxine rabbit serum albumin and aldosterone-3-oxime bovine gamma-globulin) were studied by radioimmunological methods at various times subsequent to first-immunization. A considerable variability of the parameters important in radioimmunoassay was observed over the whole experimental period. Titer, sensitivity and specificity of two antisera tended to increase as long as the animals were boosted. In the third they did not change in a uniform way.     

Production and characterization of a monoclonal antibody to biotin.

Monoclonal antibodies to biotin have been prepared by using biotin linked to keyhole limpet haemocyanin (KLH) as the antigen. Spleen cells obtained from mice immunized with biotin-KLH were fused with the myeloma cell line NS-1. The resulting hybridomas were screened for the production of antibodies to biotin using an enzyme-linked immunosorbent assay. Clones producing antibodies to biotin were isolated by limiting dilution methods. Four cell lines, each derived originally from a different fusion, were chosen for the production of monoclonal antibodies. The monoclonal antibodies obtained have been characterized with respect to their ability to interact with biotin, biotin-bovine serum albumin, biotin-KLH and biocytin as well as to inhibit biotin-dependent enzymes. They have been used to produce cellular biotin deficiency in vitro for studies of biotin function.     

Coupling of peptides to protein carriers by mixed anhydride procedure

Carboxyl groups of succinylated bovine serum albumin were activated by isobutylchloroformate in dimethylformamide solution. Subsequent reaction of the mixed anhydride with amino groups of the added peptide provided rapid and efficient coupling of peptide to protein. For different peptides the yield of coupling was equal to 40-100%. These values corresponded to 20-50 mol peptide bound/mol protein. Immunization of rabbits with these conjugates produced antisera to peptides with titers of 1:1000-1:3000 (estimated by enzyme-linked immunosorbent assay).     

Preparation of specific antisera to 15alpha-hydroxyestrogens.

The synthesis of haptens of 15alpha-hydroxyestrone, 15alpha-hydroxyestradiol, and 15alpha-hydroxyestriol (estetrol) was undertaken, to obtain specific antisera required for enzyme immunoassay. 3-(1-Carboxypropyl) ethers of these 15alpha-hydroxyestrogens were prepared and conjugated with bovine serum albumin and horseradish peroxidase. The specificity of antisera elicited against bovine serum albumin conjugates was checked by the enzyme immunoassay by using horseradish peroxidase-labeled antigen, and proved to be satisfactory in terms of cross-reactivities to related compounds.     

Water proton relaxation rate enhancements and association constants for Mn(II) to serum proteins determined by NMR T1 measurements

The water proton relaxation rate enhancement of Mn(II) bound to bovine serum albumin (BSA) and the association constant for manganese to BSA have already been determined, but such determinations have not been done for human serum albumin (HSA) and other human serum proteins and also for human serum. In this work, NMR T1 values in aqueous solutions of serum proteins and serum were measured versus increasing concentration of Mn(II). Proton relaxation rate enhancements (epsilon*) caused by different manganese concentrations were determined for each solution and 1/epsilon* was fitted against concentrations of Mn(II). Proton relaxation rate enhancements (epsilonb) of Mn(II) bound to albumin, gamma-globulin, (alpha+beta)-globulins and serum were found to be 13.69, 3.09, 8.62, and 10.87, respectively. Free and bound manganese fractions, resulted from each addition of Mn(II) to the sample, were determined by using corresponding (epsilon*) and the epsilonb values. Association constants for Mn(II) to HSA and gamma-globulin were calculated as 1.84 x 10(4) M(-1) and 2.35 x 10(4) M(-1), respectively. Present data suggest that the proton relaxation rate enhancement of Mn(II) in serum is caused by Mn(II) bound to various serum constituents. Data also suggest that association constants for Mn(II) to gamma-globulin are nearly the same as that to HSA.     

Isolation of conjugates of albumin and ribonuclease

A method for separation of albumin-ribonuclease (RNase) conjugates has been proposed, based on the use of macroporous silicates. It was established that about 76% of ligand-free human serum albumin (LFHSA) formed complexes with enzymes. It was shown that most of the conjugates of albumin and pancreatic RNase contained up to 2 mol enzyme per 1 mol LFHSA. The conjugates of albumin and bacterial RNase, isolated from the cells of the strain Bacillus intermedius 7P, displayed higher specific activities, containing, on average, 2.3 mol RNase per 1 mol LFHSA (for the conjugates with molecular weights below 92 kDa) or 3.3 mol RNase per 1 mol protein carrier (for the conjugates with higher molecular weight).     

Separation of Albumin–Ribonuclease Conjugates

A method for separation of albumin–ribonuclease (RNase) conjugates has been proposed based on the use of macroporous silicates. It was established that about 76% of ligand-free human serum albumin (LFHSA) formed complexes with enzymes. It was shown that most of the conjugates of albumin and pancreatic RNase contained up to 2 mol enzyme per 1 mol LFHSA. The conjugates of albumin and bacterial RNase isolated from cells of the strain Bacillus intermedius 7P displayed higher specific activities, containing, on average, 2.3 mol RNase per 1 mol LFHSA (for the conjugates with molecular weights below 92 kDa) or 3.3 mol RNase per 1 mol protein carrier (for the conjugates with higher molecular weight).     

Intermolecular interaction of avidin and PEGylated biotin

The equilibrium binding constants and stoichiometries between PEGylated biotins and avidin have been studied for a range of PEGylated biotin molecular weights. These studies show that as the molecular weight of PEG (polyethylene glycol) increases over the range 588, 3400, and 5000 g/mol, the equilibrium dissociation constants of PEGylated biotins with avidin increase to approximately 10 (-8) M compared with 10 (-15) M for the biotin-avidin complex. The stoichiometries of PEGylated biotins with avidin are 4:1 for 588 and 3400 g/mol PEG and 1:1 for 5000 g/mol PEG. The data demonstrate that the equilibrium binding constant and the stoichiometry of the avidin-biotin-PEG complex system can be adjusted by the length of PEG chains. This approach may be used with PEGylated biotin analogues for pretargeting in drug delivery, such as a biotin-PEGylated enzyme for converting an inactive prodrug into a cytotoxin. When a PEG chain is chosen as an appropriate spacer, the length of the PEG chain must be considered because PEG can block the binding sites on avidin.     

Dynamics of fluorescence dequenching of ostrich-quenched fluorescein biotin: a multifunctional quantitative assay for biotin

We describe a simple and rapid quantitative assay for biotin and biotin conjugates. The assay is based on the kinetic analysis of the enhancement of fluorescence of streptavidin/fluorescein biotin complexes in the presence of biotin. The kinetic response of fluorescence enhancement is proportional to the concentration of biotin. Standard calibration curves based on the kinetic response are obtained and detection limits of approximately 10(-9)M are established. Because the assay is amenable for use in small volumes of 5-50 microL or bead-based assays, the detection limits can be extended to the femtomole range. Since the assay depends on kinetic analysis, routine quantitation can be achieved without reference to standard curves. The dynamic aspects allow the assay to be extended to a broader range of applications including its use as an indicator of reagent mixing in laminar-flow assays carried out in microfluidic devices.     

Comparison of biotin binding protein of pregnant rat serum with rat serum albumin

The purified biotin binding protein of pregnant rat serum was shown to be immunologically similar to rat serum albumin as assessed by a sensitive radioimmunoassay. In radioimmunoassay for rat biotin binding protein, the binding of [¹²⁵I] rat biotin binding protein to anti-chicken egg yolk biotin binding protein antibodies was displaced by both rat serum (10–100 nl) and purified rat serum albumin (0.1–10 ng). Similarly, in radioimmunoassay for rat serum albumin the binding of [¹²⁵I] rat serum albumin to either anti-rat serum albumin antibodies or anti-chicken egg yolk biotin binding protein antibodies was displaced by unlabelled rat biotin binding protein at comparable concentration range (0·5–10 ng). Significant fractions of radioiodinated rat biotin binding protein and rat serum albumin bound to antibodies to chicken egg yolk biotin binding protein. In immature rats, the circulating half-lives of rat biotin binding protein and rat serum albumin were determined to be 12 and 17 h respectively. The rat biotin binding protein and rat serum albumin were analysed by techniques that exploit their physicochemical properties. They displayed similar electrophoretic mobilities in alkaline as well as denaturing sodium dodecyl sulphate-polyacrylamide gels. However, in nonequilibrium pH gradient polyacrylamide gel electrophoresis, they resolved clearly. In two-dimensional tryptic peptide map analysis, the two proteins showed similarities as well as significant differences in the relative distribution patterns of their iodopeptides. These results showed that the primary structure of rat biotin binding protein and rat serum albumin were different in finer details despite the fact that they shared significant immunological cross-reactivity.     

Fluorescence studies of the interactions of serum albumin and rat alpha1-fetoprotein with aflatoxin B1. Specificity and binding parameters.

The interaction of bovine serum albumin and rat alpha1-fetoprotein with aflatoxin B1 has been followed by the fluorescence quenching of the protein in presence of the ligand. The binding parameters (n, number of sites and Kd, dissociation constant) have been determined for the bovine serum albumin-alflatoxin B1 system: n = 3.5 and Kd = 3.1 +/- 0.5 . 10(-5) M and for the alpha-fetoprotein-aflatoxin system: n = 4 and Kd = 3.7 +/-0.5 . 10(-5) M. The competition of anilino-naphthalene-sulfonate and aflatoxin B1 for the same hydrophobic sites on bovine serum albumin has been demonstrated. The fluorescence quenching of various proteins (lysozymes, egg-albumin, gamma-globulin) by aflatoxin B1 have shown that there is not a strict specificity of aflatoxin towards proteins.     

A streptavidin-biotin binding system that minimizes blocking by endogenous biotin

Pretargeted radioimmunotherapy specifically targets radiation to tumors using antibody-streptavidin conjugates followed by radiolabeled biotin. A potential barrier to this cancer therapy is the presence of endogenous biotin in serum, which can block the biotin-binding sites of the antibody-streptavidin conjugate before the administration of radiolabeled biotin. Serum-derived biotin can also be problematic in clinical diagnostic applications. Due to the extremely slow dissociation of the biotin-streptavidin complex, this endogenous biotin can irreversibly block the biotin-binding sites of streptavidin and reduce therapeutic efficacy, as well as reduce sensitivity in diagnostic assays. We tested a streptavidin mutant (SAv-Y43A), which has a 67-fold lower affinity for biotin than wild type streptavidin, and three bivalent bis-biotin constructs as replacements for wild-type streptavidin and biotin used in pretargeting and clinical diagnostics. Biotin dimers were engineered with certain parameters including water solubility, biotinidase resistance, and linker lengths long enough to span the distance between two biotin-binding sites of streptavidin. The bivalent biotins were compared to biotin in exchange, retention, and off-rate assays. The faster off-rate of SAv-Y43A allowed efficient exchange of prebound biotin by the biotin dimers. In fluorescent competition experiments, the biotin dimer ligands displayed high avidity binding and essentially irreversible retention with SAv-Y43A. The off-rate of a biotinidase-stabilized biotin dimer from SAv-Y43A was 4.36 x 10(-)(6) s(-)(1), over 640 times slower compared to biotin. These findings strongly suggest that employing a mutant streptavidin in concert with a bivalent biotin can mitigate the deleterious impact of endogenous biotin, by allowing exchange of bound biotin and retention of the biotin dimer carriers.     

Active immunization to luteinizing hormone releasing hormone to inhibit the induction of mammary tumors in the rat

Immunization of female rats with a bovine serum albumin-luteinizing hormone releasing hormone conjugate results in suppression of dimethylbenzanthracene mammary tumor incidence. Tumor incidence was 1.3, and 1.29 tumors per rat in bovine serum albumin alone (n = 10) and unimmunized (n = 18) control groups, but no tumors were found in the bovine serum albumin-luteinizing hormone releasing hormone conjugate immunized animals (n = 10). In a second experiment immunization with bovine serum albumin-luteinizing hormone releasing hormone conjugates reduced tumor incidence to 0.3 tumors per rat (n = 10) from the 1.2 tumors per animal seen in the control animals (n = 10) immunized with bovine serum albumin alone. Bovine serum albumin-luteinizing hormone immunization caused the production of anti-LHRH antibodies, an interruption of estrous cycles, lowered serum estradiol and progesterone levels, and atrophy of the ovaries and uteri. Immunization BSA-hormone conjugates is a novel anti-tumor strategy.     

Molecular characteristics of bovine serum albumin-dextran conjugates

Bovine serum albumin (BSA)-dextran conjugates were prepared by using the Maillard reaction; depending on the ratio of dextran to BSA used, about 0.5-1 mol of dextran could be bound to 1 mol of native BSA. SDS-PAGE patterns revealed that BSA and dextran had been covalently bonded. Structural analyses by fluorescence spectroscopy and circular dichroism indicated that the BSA surface in each conjugate was covered with dextran without any great disruption of the native conformation. The conjugates could be grouped into two fractions on the basis of the weight-average molecular mass measured: the main fraction at 1.95-2.35 x 10(5) g/mol and a less-abundant fraction with aggregates greater than 1.50 x 10(6) g/mol. High-performance size-exclusion chromatography in conjunction with multi-angle laser light scattering detection revealed that the BSA-dextran conjugates prepared by using the Maillard reaction had various molar masses and radii.     

Determination of biotin (vitamin H) by the high-performance affinity chromatography with a trypsin-treated avidin-bound column

A method for measuring biotin by affinity-chromatography was developed using a trypsin-treated avidin silica gel column. Elution was by a linear gradient of propan-2-ol in an acidic phosphate buffer system containing 0.7 M NaCl (pH 2.4). Biotin was derivatized with 9-anthryldiazomethane (ADAM) to the fluorescent biotin-ADAM ester and a linear calibration line was obtained from 0 to 1.39 pmol with a detection limit of 69.5 fmol. Biotin was measured after hydrolysis in 2.25 M sulphuric acid for 1h at 120 degrees C and the recovery for biocytin was 65.7+/-2.53%, and hence a correction factor of 1.52 was used for the total biotin analysis. The recovery of added biotin from the serum was more than 98% using this correction factor of 1.52. Biotin was also measured in nutritional supplemental foods and foodstuffs, and we found that chicken egg yolk, "natto", rice bran, royal jelly, and dried yeast contained highest levels of biotin. Biotin was also found in ferments by Bacillus natto, yeast, and some acetic acid bacterium. Storage foods such as beans, nuts and eggs also contained abundant biotin. Biotin was also determined and replacement monitored in the serum of suspected biotinidase deficiency patients. This affinity-chromatographic method for biotin determination was shown to be a robust and reliable and is well suited for biochemical and nutritional research.     

Antibodies to oxidative DNA damage: Characterization of antibodies to 8-oxopurines

The 8-oxo-7,8-dihydropurines (8-oxopurines) are important cellular premutagenic lesions produced in DNA by free radicals. Specific antibodies were prepared to detect these lesions. For antigens, 8-oxo-7,8-dihydroadenosine (8-oxoAdo) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) were synthesized from the bromonucleosides, and the immunogens were produced by conjugating these to either bovine serum albumin or rabbit serum albumin by the periodate method. Polyclonal antibodies specific for the haptens were elicited from rabbits immunized with the BSA conjugates. The antibodies to 8-oxoAdo (anti-8-oxoAdo) and 8-oxoGuo (anti-8-oxoGuo) precipitated the homologous antigens in an Ouchterlony gel diffusion assay and no cross-reactivity was observed toward the normal nucleosides or to the heterologous 8-oxopurine. Specificity was also examined by hapten inhibition of antibody reactivity with the homologous conjugates using ELISA. For anti-8-oxoAdo, the IC50 for 8-oxoAdo was 8 µmol/L and 8-bromoadenosine, guanosine, and inosine did not inhibit, even at concentrations of 1.25 mmol/L. Similarly, the IC50 for anti-8-oxoGuo for 8-oxoGuo was 0.1 µmol/L. 8-Methoxyguanosine also inhibited the reaction but was about 500-fold less effective than the eliciting hapten. Other nucleosides tested did not inhibit at concentrations up to 100 µmol/L. Both antibodies could easily detect the corresponding damage in x-irradiated fl DNA at a dose of 7.5 Gy and both antibodies recognized the corresponding lesion in duplex DNA; however, with anti-8-oxoGuo the signal was reduced about 50% compared to single-stranded DNA. In order to determine the exact amount of each lesion produced in irradiated DNA, and to standardize the ELISA signal, both products were measured after alkaline phosphatase digestion of x-irradiated calf thymus DNA using high-pressure liquid chromatography (HPLC) coupled to an electrochemical detector. Anti-8-oxoGuo could detect ten 8-oxoG residues and anti-8-oxoAdo could detect two 8-oxoA residues per 10 000 nucleotides. Thus, these antibodies should be useful for the detection and measurement of 8-oxopurines in cellular DNA.