Avidin-biotin Reaction Research Articles

Intra-articular kinetics of a cartilage targeting cationic PEGylated protein for applications in drug delivery

Cartilage targeting cationic glycoprotein Avidin was PEGylated to synthesize a multi-arm Avidin (mAv) nano-construct with high drug loading content. Here we investigate mAv biodistribution and kinetics over a 7-day period following intra-articular (IA) administration in rat knee joints. Labeled mAv was injected into healthy rat knees, and joint tissues (articular cartilage, menisci, ligaments, tendons, fat pad) were harvested following sacrifice at 6h, 1, 4 and 7 days. Its IA biodistribution and retention were measured using fluorescence microscopy. Tissue localization was compared in young vs old rats by immunohistochemistry. mAv chondrotoxicity and immune response were evaluated to determine safe carrier dose limits. mAv penetrated through the full thickness of rat cartilage and other joint tissues within 6h, remaining detectable within most joint tissues over 7 days. Intra-tissue uptake correlated strongly with tissue GAG concentration, confirming the dominant role of electrostatic interactions between positively charged mAv and the negatively charged aggrecan proteoglycans. mAv was uptaken by chondrocytes and also penetrated the osteocyte lacuno-canalicular system of peri-articular bone in both young and old rats. mAv did not cause cytotoxicity at concentrations up to 300μM but elicited a dose dependent immunogenic response. mAv's ability to target a variety of joint tissues, chondrocytes, and peri-articular osteocytes without sequestration in synovial fluid makes it a versatile carrier for delivering a wide range of drugs for treating a broad class of musculoskeletal diseases. Drugs can be conjugated using simple aqueous based avidin-biotin reaction, supporting its clinical prospects.

Charge-Based Multiarm Avidin Nanoconstruct as a Platform Technology for Applications in Drug Delivery.

Here we describe methods for synthesizing cationic multiarm Avidin (mAv) nanoconstruct that has a wide range of applications in drug delivery and imaging for a variety of negatively charged tissues. The multiarm structure provides multiple sites for covalent conjugation of drugs. We use avidin-biotin reaction that gives the flexibility for conjugating any desired biotinylated drug to mAv by simple mixing at room temperature. We also describe methods to control hydrolysis rates of ester linkers to enable sustained (and tunable) drug release rates in therapeutic doses.

Detection of Lung Cancer Cells in Solutions Using a Terahertz Chemical Microscope.

Cancer genome analysis has recently attracted attention for personalized cancer treatment. In this treatment, evaluation of the ratio of cancer cells in a specimen tissue is essential for the precise analysis of the genome. Conventionally, the evaluation takes at least two days and depends on the skill of the pathologist. In our group, a terahertz chemical microscope (TCM) was developed to easily and quickly measure the number of cancer cells in a solution. In this study, an antibody was immobilized on a sensing plate using an avidin-biotin reaction to immobilize it for high density and to improve antibody alignment. In addition, as the detected terahertz signals vary depending on the sensitivity of the sensing plate, the sensitivity was evaluated using pH measurement. The result of the cancer cell detection was corrected using the result of pH measurement. These results indicate that a TCM is expected to be an excellent candidate for liquid biopsies in cancer diagnosis.

Avidin-biotin technology to synthesize multi-arm nano-construct for drug delivery

Here we describe methods for synthesizing a cationic, multi-arm Avidin (mAv) nano-construct that has a wide range of applications in drug delivery and imaging of negatively charged tissues. We use Avidin-biotin technology that gives the flexibility for conjugating biotinylated Dexamethasone to mAv by simple mixing at room temperature. We also describe methods to control hydrolysis rates of ester linkers to enable sustained (and tunable) drug release rates in therapeutic doses.•Multi-arm structure provides multiple sites for covalent conjugation of drugs•Use of Avidin-biotin reaction gives multi-arm nano-construct a modular design enabling conjugation and delivery of similar sized biotinylated drugs.

Acoustic Sensing Based on Density Shift of Microspheres by Surface Binding of Gold Nanoparticles.

Herein, we propose a concept for sensing based on density changes of microparticles (MPs) caused by a biochemical reaction. The MPs are levitated by a combined acoustic-gravitational force at a position determined by the density and compressibility. Importantly, the levitation is independent of the MPs sizes. When gold nanoparticles (AuNPs) are bound on the surface of polymer MPs through a reaction, the density of the MPs dramatically increases, and their levitation position in the acoustic-gravitational field is lowered. Because the shift of the levitation position is proportional to the number of AuNPs bound on one MP, we can determine the number of molecules involved in the reaction. The avidin-biotin reaction is used to demonstrate the effectiveness of this concept. The number of molecules involved in the reaction is very small because the reaction space is small for an MP; thus, the method has potential for highly sensitive detection.

Ultrasensitive sensing platform for platelet-derived growth factor BB detection based on layered molybdenum selenide-graphene composites and Exonuclease III assisted signal amplification.

A highly sensitive and ultrasensitive electrochemical aptasensor for platelet-derived growth factor BB (PDGF-BB) detection is fabricated based on layered molybdenum selenide-graphene (MoSe2-Gr) composites and Exonuclease III (Exo III)-aided signal amplification. MoSe2-Gr is prepared by a simple hydrothermal method and used as a promising sensing platform. Exo III has a specifical exo-deoxyribonuclease activity for duplex DNAs in the direction from 3' to 5' terminus, however its activity is limited on the duplex DNAs with more than 4 mismatched terminal bases at 3' ends. Herein, aptamer and complementary DNA (cDNA) sequences are designed with four thymine bases on 3' ends. In the presence of target protein, the aptamer associates with it and facilitates the formation of duplex DNA between cDNA and signal DNA. The duplex DNA then is digested by Exo III and releases cDNA, which hybridizes with signal DNA to perform a new cleavage process. Nevertheless, in the absence of target protein, the aptamer hybridizes with cDNA will inhibit the Exo III-assisted nucleotides cleavage. The signal DNA then hybridizes with capture DNA on the electrode. Subsequently, horse radish peroxidase is fixed on electrode by avidin-biotin reaction and then catalyzes hydrogen peroxide and hydroquinone to produce electrochemical response. Therefore, a bridge can be established between the concentration of target protein and the degree of the attenuation of the obtained signal, providing a quantitative measure of target protein with a broad detection range of 0.0001-1 nM and a detection limit of 20 fM.

Sensitive quantitation of Ochratoxin A in cocoa beans using differential pulse voltammetry based aptasensor

In this work, we propose for the first time a sensitive Ochratoxin A (OTA) detection in cocoa beans using competitive aptasensor by differential pulse voltammetry (DPV). In the proposed method, biotin labeled and free OTA competed to bind with immobilized aptamer onto the surface of a screen printed carbon electrode (SPCE), and percentage binding was calculated. The detection was performed after adding avidin-ALP to perform avidin–biotin reaction; the signal was generated through a suitable substrate 1-naphthyl phosphate (1-NP), for alkaline phosphatase (ALP). The cocoa samples were extracted and purified using molecular imprinted polymer (MIP) columns specifically designed for OTA. The developed aptasensor showed a good linearity in the range 0.15–5ng/mL with the limit of detection (LOD) 0.07ng/mL and 3.7% relative standard deviation (RSD). The aptasensor displayed good recovery values in the range 82.1–85% with 3.87% RSD, thus, demonstrated the efficiency of proposed aptasensor for such matrices.

Expression of Ephrin in Retinal Neovascularization and Iris Rubeosis

We investigated expression of ephrin-B2 and Eph-B4 in the retinal tissues of six primate eyes with neovascularization and iris rubeosis secondary to laser-induced central retinal vein occlusion and in tissue from 10 human eyes with proliferative diabetic retinopathy. Two primate eyes with rubeosis and retinal neovascularization were enucleated 1, 2 and 4 weeks after the creation of central retinal vein occlusion. Antibodies were localized using the avidin-biotin reaction. In the primate eyes, ephrin-B2 was negative at I week and positive at 2 and 4 weeks in the rubeotic tissue, but was positive only at 2 weeks in the retinal neovascular membrane. Eph-B4 was negative in all the primate eye specimens. In the human tissue, ephrin-B2 was detected in two of the five eyes with rubeosis and three of the five eyes with retinal neovascularization. These data suggest that ephrin-B2 is a key regulator of neovascularization.

Electrochemical biosensing based on universal affinity biocomposite platforms

Rigid conducting biocomposites are versatile and effective transducing materials for the construction of a wide range of amperometric biosensors such as immunosensors, genosensors and enzymosensors, particularly if the transducer is bulk-modified with universal affinity biomolecules. The strept(avidin)-graphite-epoxy biocomposite could be considered as an universal immobilization platform whereon biotinylated DNAs, oligonucleotides, enzymes or antibodies can be captured by means of the highly affinity (strept)avidin-biotin reaction. Universal affinity biocomposite-based biosensors offer many potential advantages compared to more traditional electrochemical biosensors commonly based on a biologically surface-modified transducer. The integration of many materials into one matrix is their main advantage. As biological bulk-modified materials, the conducting biocomposites act not only as transducers, but also as reservoir for the biomaterial. After its use, the electrode surface can be renewed by a simple polishing procedure, establishing a clear advantage of these approaches relative to classical biosensors and other common biological assays. Moreover, the same material is useful for the analysis of many molecules whose determinations are based on genetic, enzymatic or immunological reactions. The different strategies for electrochemical genosensing, immunosensing and enzymosensing, all of them being dependent on the presence of a redox enzyme marker for the generation of the electrochemical signal, based on this universal affinity biocomposite platform are all presented and discussed.

Development of an ELISA for Chum Salmon ( Oncorhynchus keta) Growth Hormone

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of low levels of serum growth hormone (GH) in chum salmon ( Oncorhynchus keta). The antiserum to GH (a-rsGH) was obtained from a rabbit immunized with recombinant chum salmon GH. The noncompetitive ELISA was performed by a sandwich method using a-rsGH rabbit IgG as the first antibody, its biotinylated Fab′ fragment as the second antibody, and the avidin-biotin reaction for signal amplification. This assay could be run in 3 days and routinely detected GH at concentrations as low as 0.5 ng/ml. The development of an ELISA for GH made possible quantification of serum GH levels. In this assay system, parallel dilution curves were obtained using purified chum salmon GH and GH's from several species of the genus Oncorhynchus.

Immunoelectron-microscopic localization of synaptophysin in the organ of Corti of the guinea pig.

Synaptophysin has been localized previously in the mammalian cochlea at the light-microscopic level and in few reports by electron microscopy using either a preincubation procedure or the avidin-biotin reaction. Here we present results of the electron-microscopic analysis for postembedding immunoreactivity of synaptophysin in the cochlea of the guinea pig of LR-White-embedded samples. Strong synaptophysin immunoreactivity is located in the cytoplasm of the efferent nerve endings at the base of inner and outer hair cells. Besides this, some antibodies to synaptophysin were also identified in the cytoplasm of outer hair cells. To get more information about the cellular content of synaptophysin, the density of the immunoreaction per square micrometer was determined for the outer hair cells of the second turn of the cochlea counting the gold particles in numerous ultrathin sections. The role of synaptophysin identified in the cytoplasm of outer hair cells is discussed.

Lectins in ocular drug delivery: An investigation of lectin binding sites on the corneal and conjunctival surfaces

Ocular drug delivery is limited both by patient acceptability and by the limited time that the dosage form is retained within the precorneal region. The aim of this investigation was to identify lectin receptors within the precorneal region as potential targets for a lectin containing ocular dosage form, thus facilitating prolonged drug delivery. Initial ex-vivo studies evaluated the binding, after 15 min contact with intact (unfixed) rat corneal and conjunctival epithelia, of a panel of 36 lectins of varying sugar specificities. Tissues were sectioned and examined using light microscopy. Lectin binding was identified using a standard histological procedure involving the avidin-biotin reaction, and the formation of a brown precipitate from a 3,3'-diaminobenzidine tetrahydrochloride (DAB) solution. All lectins were observed to bind to both the cornea and conjunctiva to varying degrees. There was little evidence of a specific receptor or structural type of lectin that particularly favoured binding. The lectins from Solanum tuberosum (potato) and Helix pomatia (edible snail) which are specific for the terminal sugar residues N-acetyl- D-glucosamine and N-acetyl- D-galactosamine respectively, appeared to be the most promising and displayed clear visual evidence of binding to ocular tissues after only 10 s contact. The stability of binding of these two lectins was assessed using a video microscopy method of evaluation, and binding in a second species investigated using unfixed porcine cornea. Both lectins were found to show rapid, substantial and stable binding ex-vivo, and these will be taken forward in future work for quantitative in vivo studies and toxicological evaluation.

Streptavidin-reactive protein in Fasciola hepatica

Streptavidin-binding protein was found in an extract of Fasciola hepatica, and the protein was analyzed by the inhibition assay using avidin or D-biotin, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using peroxidase-conjugated streptavidin. Inhibition assays revealed that the protein could bind to streptavidin by a mechanism similar to the avidin-biotin reaction, and the molecular size of the protein was estimated by SDS-PAGE to be 71.7 kDa.

Use of drug-specific antibodies to identify ethidium adducts produced in Trypanosoma brucei by photoaffinity labeling

A photoreactive azido analog of the trypanocide ethidium bromide, 3-amino-8-azido-5-ethyl-6-phenylphenanthridinium chloride, attached covalently to calf thymus DNA (CT DNA) by photoaffinity labeling, was used to generate antibodies for the drug analog. The specificity of the antiserum was tested using enzyme-linked immunoadsorbant assays (ELISA) against immobilized antigen (photoaffinity labeled DNA) and by both the avidin-biotin peroxidase reaction and indirect immunofluorescence performed on smears of drug treated trypanosomes. The reaction of the antiserum with the covalently bound drug adduct was diminished effectively by prior incubation with an excess of ethidium monoazide, ethidium diazide, and ethidium bromide, and to a lesser extent by the DNA-ethidium complex, the diazide-DNA or RNA adduct, and the monoazide-RNA adduct. DNA which had been photoaffinity labeled with either the propidium or the acridine moiety did not react. The antiserum recognition of DNA photoaffinity labeled with ethidium monoazide was based on the substituted phenanthridinium ring system of the parent ethidium, as evidenced by competition binding studies involving the free monoazido analog (EA1), the diazido analog (EA2), and the parent compound, ethidium bromide (EB). This approach and the sensitivity it provides should prove useful for identifying the distribution and fate of covalently bound drugs resulting from antiparasitic drug treatment, and for studying their roles in antiparasitic action.

The localization of synaptophysin in the organ of Corti of the human as shown by immunoelectron microscopy.

Synaptophysin, or p38, a polypeptide of molecular weight 38 kD, is a calcium-binding membrane protein found in synaptic vesicles of neurons and smooth surfaced vesicles of neuroendocrine cells. Six human neonatal and infant temporal bones were fixed in paraformaldehyde and glutaraldehyde, decalcified in EDTA and were than immunoreacted for synaptophysin (ICN Biomedicals) using the avidin-biotin reaction (ABC kit, Vector Labs). The tissue was then prepared for light microscopic surface preparation, radial sections of 5 microns, and serial section electron microscopy. At a light microscopic level, the inner spiral bundle, tunnel spiral bundle, upper tunnel crossing fibers and the base of outer hair cells were stained. At the base of outer hair cells, the immunoreactivity was seen to decrease from the base to the apex and from the first to third outer hair cells. At an electron microscopic level, immunoreactivity at the base of outer hair cells was limited to vesiculated efferent fibers. The degree of immunoreactivity between adjacent efferent fibers varied significantly. Immunoreactive vesiculated endings were also found in the supranuclear region of outer hair cells.

An ELISA-like assay for hyaluronidase and hyaluronidase inhibitors

Hyaluronic acid (HA) is a prominent molecule in the extracellular matrix and is enriched whenever there is rapid tissue proliferation, regeneration and repair. HA is degraded in part by hyaluronidases (HA'ases) that are not well characterized. We have developed a novel ELISA-like rapid assay for HA'ases and their inhibitors. The assay is based on a high affinity biotinylated HA-binding peptide derived from tryptic digests of proteoglycan core protein of bovine nasal cartilage and the avidin-biotin reaction. HA-coated plates were incubated with serial dilutions of Streptomyces HA'ase, and the undegraded HA was measured. This established a standard curve for HA'ase activity against which all unknown enzyme samples were compared. The assay is easily modified to also serve a measure of HA'ase inhibitors. For detection of inhibitors, aliquots of sample were preincubated with a known activity of HA'ase and inhibition of HA degradation by the mixture was measured. We have used this assay to document the presence of potent HA'ase inhibitors in fetal calf sera. These techniques will aid in the purification and characterization of Ha'ases and their inhibitors.

Biotin-dextran: a sensitive anterograde tracer for neuroanatomic studies in rat and monkey

The properties of a new anterograde tracer, biotin-dextran (BD), are reported. Iontophoretic or pressure injections of BD in the spinal cord of rats and monkeys revealed terminal-like BD label in many regions of the brain. The BD label was visualized by an avidin-biotin reaction combined with diaminobenzidine, with and without nickel enhancement. This reaction resulted in permanent label and revealed the fine morphology of terminal boutons and en-passant endings. Biotin-dextran is an excellent anterograde tracer that can also be visualized at the electron microscopic level, used in combination with other retrograde and anterograde tracers and with immunohistochemical labeling of neurotransmitters.

Chemiluminescent and Bioluminescent Assays for Polyadenylic Acid and Applications to DNA Probe Assay and Immunoassay

We developed a chemiluminescent (CL) assay for polyadenylic acid (poly A) based on the enzymatic cycling reaction following the conversion to ATP by pyruvate kinase/polynucleotide phosphoryrase enzyme reaction. Poly A was also measured with the bioluminescent (BL) assay using luciferase from firefly Photinuspyralis. The detection limits of poly A by CL and BL assay were 10 amol and 2 amol, respectively. We prepared the biotinylated poly A using biotin hydrazide and glutaraldehyde as a bifunctional coupling reagent to apply the poly A assay to a general avidin-biotin reaction system. When the CL assay and the BL assay of biotinylated poly A are applied to the DNA probe assay of λ-phage DNA and the immunoassay of thyroid-stimulating hormone (TSH) using avidin-biotin reaction as detection system, 500 pg assay−1 well of λ-phage DNA and 3 μU ml−1 of TSH were determined.

Enzyme immunoassay for placental protein 4 (PP4) and its possible diagnostic significance in patients with genital tract cancer

We have established an enzyme immunoassay for placental protein 4 (PP4), by using avidin-biotin binding reaction, and set its normal range below 10.9 ng/ml (mean + 2 sigma). Throughout the menstrual cycle, the serum PP4 profile was similar to that of serum progesterone. In the follicular and ovulatory phase, PP4 remained relatively low, with the mean levels of 1.5 ng/ml and 1.8 ng/ml, respectively. In the luteal phase, the mean level was 3.2 ng/ml. In normal pregnancy, serum PP4 levels were low irrespective of gestational age, with a mean level of 3.0 ng/ml. There was only one case in which the serum PP4 level over 10.9 ng/ml. Mean serum PP4 levels and the frequencies of elevated serum PP4 levels were respectively 6.3 ng/ml and 11% in patients with benign ovarian neoplasms, 4.7 ng/ml and 6% in patients with endometriosis, and 5.5 ng/ml and 18% in patients with uterine myomata. The frequency of raised PP4 levels was 48% and the mean value was 13.3 ng/ml in patients with endometrial carcinoma, and the values were 44% and 13.4 ng/ml respectively in patients with cervical carcinoma. In patients with ovarian malignancy, the respective values were 15% and 7.0 ng/ml. The results did not relate to clinical stages of disease (FIGO), while the frequencies of elevated serum PP4 in patients with uterine carcinoma was over 40% in stage I diseases. Compared with other tumor markers such as carcino-embryonic antigen (CEA), tissue polypeptide antigen (TPA) and cancer antigen 125 (CA125), PP4 seems to be more promising as a marker of endometrial carcinoma.(ABSTRACT TRUNCATED AT 250 WORDS)

Competitive enzyme immunoassay for human chorionic somatomammotropin using the avidin-biotin system

Human chorionic somatomammotropin (HCS) is determined by an enzyme immunoassay where HCS competes with biotin-labeled HCS for insolubilized anti-HCS antibodies. Enzymelabeled avidin is then used to reveal the amount of bound HCS. The system proves to be sensitive (1 ng/ml of HCS can be detected) and results agree with radioimmunoassay determinations (correlation coefficient = 0.979). Kinetics of the avidin-biotin reaction and coating of polystyrene wells are also investigated.