Degree Of Non-specific Binding Research Articles

Affinity Purification of Anti-Parkin Antibodies from Rabbit Serum for their Application in Immunohistochemistry

Background:Parkin is a mitochondrial autophagy protein that is associated with Parkinson’s Disease (PD), as well as auditory function. Parkin dysfunction in the context of PD allows accumulation of protein aggregates, leading to dopaminergic neurotoxicity. Previous work has demonstrated that Parkin plays an essential role in normal auditory function, as Prkn-/- mice exhibit significantly reduced hearing sensitivity. However, Prkn-/- mice also experience a protective effect from cochlear hair cell death and hearing loss caused by aminoglycosides, which are commonly used antibiotics that can cause permanent hearing loss. Multiple antibodies marketed as suitable for immunohistochemistry (IHC) have been validated using Prkn-/- tissues; however, they exhibit non-specific activity. This project aims to generate a highly specific antibody for accurate and reliable detection of Parkin expression.
 Project Methods:Rabbits were immunized using two Parkin peptides, notated P1 and P2, and the resulting serum was collected. A two-step purification process was utilized in this project, first isolating the IgGs from serum via protein A/G columns, and secondly using affinity purification to obtain specific antibodies against antigens P1 and P2. Additionally, two different protocols for affinity purification were tested and compared. Purity of serum, IgG, and specific antibodies was assessed by Western blot and immunofluorescence.
 Results:Utilizing the first purification protocol, IHC antibodies were applied in Western blot, and all demonstrated a prominent ~50kD band specific to Parkin, with some degree of nonspecific binding. Immunostaining confirmed functionality of the IHC antibodies and revealed those against P2 exhibited higher binding specificity. The second purification protocol generated IHC antibodies of similar, if not slightly superior specific interaction.
 Conclusion/Implications:Further investigation of Parkin expression would facilitate a better understanding of how it may be associated with the development of PD, as well as auditory function, with the potential of utilizing Parkin as a therapeutic target in PD treatment and preventing aminoglycoside ototoxicity.

A high molar activity 18F-labeled TAK-875 derivative for PET imaging of pancreatic \u03b2-cells

BackgroundThe free-fatty acid receptor-1 (FFA-1) is expressed by β-cells and is a promising target for molecular imaging of functional β-cell mass. Recently, the ((3-[18F]fluoropropyl)sulfonyl)propoxy-derivative of the high-affinity FFA-1 agonist TAK-875 ([18F]7) was reported. Here we describe the preparation of this tracer in high molar activity using a purification method permitting separation of [18F]7 from a structurally-related by-product and evaluation of the tracer in rats as a potential FFA-1 PET imaging agent.ResultsThe radiotracer was produced by nucleophilic radio-fluorination of the tosylate precursor and deprotection of the methyl ester. Semi-preparative HPLC with a C18 column revealed that [18F]7 co-eluted with a non-radioactive impurity. Mass spectrometry identified the impurity as the alkene-containing elimination by-product. A pentafluorophenyl-functionalized HPLC column was found to separate the two compounds and allowed for purification of [18F]7 in high molar activity. A strong anion-exchange resin was used to reformulate [18F]7 in high concentration. Starting from 96 to 311 GBq of [18F]fluoride, 3.8–15.4 GBq of pure [18F]7 (end of synthesis (EOS)) was prepared (RCY 8.3% ± 1.1% decay-corrected, n = 4) in high molar activity (166–767 GBq/μmol at EOS). This PET agent was evaluated in rats using dynamic microPET/CT imaging, ex vivo biodistribution, and radio-metabolite studies. MicroPET/CT exhibited high uptake of the tracer in the abdominal area. There was no measurable decrease of the PET signal in the pancreatic area in rats pre-treated with saturating doses (30 mg/kg) of TAK-875. Biodistribution studies corroborated the microPET/CT results. Radiometabolism analyses revealed high compound stability with only the parent molecule detected in the pancreas.ConclusionsAnalysis of the crude reaction mixture and identification of the elimination by-product allowed for the development of a fully automated process to prepare the TAK-875-derived PET agent [18F]7 in high purity and high molar activity. Even though the radiotracer exhibited high in vivo stability, microPET/CT and biodistribution results confirmed recent reports demonstrating that lipophilic analogs of TAK-875 display a high degree of non-specific binding, masking any specific binding to FFA-1 in pancreatic β-cells. Future development of TAK-875-derived PET tracers should focus on reducing non-specific binding in the pancreatic tissue.

Directed Assembly of End-Functionalized Single Wall Carbon Nanotube Segments.

A key impediment to the implementation of a nanoelectronics technology based on single wall carbon nanotubes (SWCNTs) is the inability to arrange them in a manner suitable for integration into complex circuits. As a step toward addressing this problem, we explore the binding of fixed-length, end-functionalized SWCNT segments to lithographically defined nanoscale anchors, such that individual SWCNTs can be placed with control over position and orientation. Both monovalent and bivalent bindings are explored using covalent and noncovalent binding chemistries. Placement efficiency is assessed in terms of overall yield of SWCNT binding, as well as binding specificity and the degree of nonspecific binding. Placement yields as high as 93% and 79% are achieved, respectively, for covalent binding and for binding through DNA hybridization. Orientational control of the SWCNT segments is achieved with 95% and 51% efficiency for monovalent and bivalent bindings, respectively. This represents a new approach that could pave the way toward complex SWCNT devices and circuits.

Label free detection of specific protein binding using a microwave sensor.

The specific binding of streptavidin to biotinylated protein A was demonstrated using a microwave detection system. In control experiments, the degree of non-specific binding was negligible. The method of detection was used to monitor the adsorption of two other proteins, cytochrome c and glucose oxidase, on to the IDE microwave sensor surface. The response of the sensor was also examined on different substrate materials, with detection of protein binding observed obtained on both smooth, conductive (gold) and on rough, insulating (hydroxyapatite) surfaces.

Can [125I]-Iodocyanopindolol Label β3-Adrenoceptors in Rat Urinary Bladder?

β3-Adrenoceptors have been demonstrated to mediate urinary bladder smooth muscle relaxation but proof of their expression at the protein level has been missing because of lack of suitable antibodies or radioligands. As among various available radioligands [125I]-iodocyanopindolol ([125I]-ICYP) exhibited the smallest problems in labeling cloned human β3-adrenoceptors in previous studies, we have explored its suitability to label β3-adrenoceptors in rat urinary bladder in saturation and competition radioligand binding experiments. Rat lung was used as an internal control and exhibited all characteristics expected from this tissue with regard to β1/β2-adrenoceptor labeling. Saturation and competition binding studies with [125I]-ICYP in rat bladder yielded saturable binding sites with an affinity compatible with β3-adrenoceptors. In competition experiments various agonists and antagonists largely exhibited a profile compatible with a population consisting largely of β3-adrenoceptors. However, the binding competition properties of ICI 118,551 and SR 59,230A were not easily explained by the idea of labeling a homogeneous β3-adrenoceptor population but interpretation of the data was limited by a high degree of non-specific binding in [125I]-ICYP concentrations required to label the receptors. We conclude that [125I]-ICYP can be used to label tissue β3-adrenoceptors but results obtained with this ligand have to be interpreted with caution.

Cross species applicability of abundant protein depletion columns for ribulose-1,5-bisphosphate carboxylase/oxygenase

In plants, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is an important enzyme in the Calvin cycle, catalyzing the first step of carbon fixation. Because of its critical role in photosynthesis, RuBisCO comprises 30–60% of the total protein content in green leaf tissue and represents a major protein which can interfere with determination of lower abundance proteins in plant proteomics. A potential solution to aid in the determination of low level proteins in plant proteomics are RuBisCO immunodepletion columns. Two formats, spin and LC, of Seppro™ IgY RuBisCO depletion columns were evaluated for cross species applicability. The spin and LC columns were found to deplete arabidopsis RuBisCO by greater than 90 and 98%, respectively, and automation could be achieved with the LC format. Canola RuBisCO was depleted to a similar extent, and there was evidence suggesting that corn and tobacco RuBisCO were also highly depleted in flow through fractions. Model proteins were spiked into samples to provide insight into the degree of non-specific binding. Finally, improved detection and identification of lower abundance proteins was demonstrated after depletion.

Characterizing the Glycocalyx of Poultry Spermatozoa: I. Identification and Distribution of Carbohydrate Residues Using Flow Cytometry and Epifluorescence Microscopy

The aim of the present work was to use a battery of lectins to 1) delineate the carbohydrate content of sperm glycocalyx in the turkey and chicken using flow cytometry analysis, and 2) evaluate the distribution of existing sugars over the sperm plasma membrane surface with epifluorescent microscopy. Carbohydrate groups (corresponding lectins) that were investigated included galactose (GS-I, Jacalin, RCA-I, PNA), glucose and/or mannose (Con A, PSA, GNA), N-acetyl-glucosamine (GS-II, s-WGA, STA), N-acetyl-galactosamine (SBA, WFA), fucose (Lotus, UEA-I), sialic acid (LFA, LPA), and N-acetyl-lactosamine (ECA). Spermatozoa were assessed before and after treatment with neuraminidase to remove sialic acid. Mean fluorescence intensity (MnFI) was used as indicator of lectin binding for flow cytometry analysis. Nontreated spermatozoa from both species showed high MnFI when incubated with RCA-I, Con A, LFA, and LPA, as did chicken spermatozoa incubated with s-WGA. Neuraminidase treatment increased the MnFI for most lectins except LFA and LPA, as expected. Differences in MnFI between species included higher values for s-WGA and ECA in chicken spermatozoa and for WFA in turkey spermatozoa. Microscopy revealed segregation of some sugar residues into membrane-specific domains; however, the 2 staining techniques (cell suspension vs fixed preparation) differed in identifying lectin binding patterns, with fixed preparations yielding a high degree of nonspecific binding. We conclude that 1) the glycocalyx of turkey and chicken spermatozoa contains a diversity of carbohydrate groups, 2) these residues are extensively masked by sialic acid, 3) the glycocalyx composition is species-specific, and 4) some glycoconjugates appear to be segregated into membrane-specific domains. Characterization of the poultry sperm glycocalyx is the first step in identifying the physiological impact of semen storage on sperm function.

High non-specific binding of the β1-selective radioligand 2-125I-ICI-H

As results of cardiac biopsies suggest, myocardial beta(1) -adrenoceptor density is reduced in patients with chronic heart failure. However, changes in cardiac beta(2)-adrenoceptors vary. With suitable radiopharmaceuticals single photon emission computed tomography (SPECT) and positron emission tomography (PET) offer the opportunity to assess beta-adrenoceptors non-invasively. Among the novel racemic analogues of the established beta(1)-selective adrenoceptor antagonist ICI 89.406 the iodinated 2-I-ICI-H showed high affinity and selectivity to beta(1)-adrenoceptors in murine ventricular membranes. The aim of this study was its evaluation as a putative sub-type selective beta(1)-adrenergic radioligand in cardiac imaging. Competition studies in vitro and in vivo were used to investigate the kinetics of 2-I-ICI-H binding to cardiac beta-adrenoceptors in mice and rats. In addition, the radiosynthesis of 2-(125)I-ICI-H from the silylated precursor 2-SiMe(3)-ICI-H was established. The specific activity was 80 GBq/ micro mol, the radiochemical yield ranged from 70 to 80%. The unlabelled compound 2-I-ICI-H showed high beta(1)-selectivity and -affinity in the in vitro competition studies. In vivo biodistribution studies apparently showed low affinity to cardiac beta-adrenoceptors. The radiolabelled counterpart 2-(125)I-ICI-H showed a high degree of non-specific binding in vitro and no specific binding to cardiac beta(1)-adrenoceptors in vivo. Because of its high non-specific binding 2-(125)I-ICI-H is no suitable radiotracer for imaging in vivo.

Non-specific binding of anti-human IgE peroxidase-linked conjugates to legume proteins in immunoblots

Non-specific binding was observed in immunoblotting of legume proteins with commercial anti-human IgE and IgG antibodies conjugated with peroxidase when incubations were conducted without prior incubation with human sera of legume-allergic individuals. The non-specific binding was encountered with ten legume species. It was not associated with endogenous peroxidase or the peanut or soybean lectins. The degree of non-specific binding diminished with roasted peanut extracts as compared to raw peanut extracts. The cause of this non-specific binding remains unknown, although it may be related to the animal source of the antiserum or the method of conjugation of the antibody with peroxidase. This interference in immunoblotting can be easily detected with controls lacking incubation with human sera.

The Effects of Radioisotopes Used in Nuclear Medicine on Diagnostic Radioimmunoassay Testing: Is There Any Significant Interference?

The administration of radioisotopes for diagnostic nuclear medicine scans and therapeutic procedures is quite prevalent today. A period of interference with the counting of a radioimmunoassay [RIA] test may occur with the serum of a patient receiving an in vivo radionuclide that decays by gamma emission. Because the logistics of precounting all specimens may be cumbersome and prohibitive, it is important to determine the degree of this interference. In this study, the authors evaluate the potential interference of the most commonly used radioisotopes with RIA studies. For two months (March and August 1988) 10,650 patient serum specimens were counted for significant background gamma radiation before RIA testing. Forty-three patients, on whom 105 RIA tests were performed, were identified as having preassay gamma radiation in their serum. With the use of selective energy windows for each different interfering radionuclide, proportional determinations were made as to the amount of interfering gamma radiation spilling into the iodine 125 test marker window. It was shown that initial whole serum pretest gamma counts as high as 111,000 counts/minute did not significantly affect the results of the RIA. Because of the meticulous washing and decanting procedures required in modern RIA and the monoclonal nature of most antibodies used currently, it appears the degree of nonspecific binding of this potentially interfering radiation is minuscule. The energy level of cobalt 57, however, and many of the other commonly used radioisotopes, overlaps so closely that it is difficult to window for this interference. It is possible, therefore, that this distinction cannot be made and folate and vitamin B12 test systems using cobalt 57 markers may have to be routinely prescreened. The authors conclude that the requirement for prescreening of all RIA test samples for interfering gamma radiation is unnecessary (1987 CAP Commission on Laboratory Accreditation Inspection Checklist, Section VII, Question 07.0290).

The mechanism of decreased Na +-dependent d-glucose transport in brush-border membrane vesicles from rabbit kidneys with experimental Fanconi syndrome

In our previous paper (Yanase, M. et al. (1983) Biochim. Biophys. Acta 733, 95–101) we reported that the Na +-dependent d-glucose uptake into brush-border membrane vesicles is decreased in rabbits with experimental Fanconi syndrome (induced by anhydro-4-epitetracycline). In the present paper we investigate the mechanism underlying this decrease. d-Glucose is taken up into the osmotically active space in anhydro-4-epitetracycline-treated brush-border membrane vesicles and exhibits the same distribution volume and the same degree of nonspecific binding and trapping as in control brush-border membrane vesicles. The passive permeability properties of control and anhydro-4-epitetracycline-treated brush-border membrane vesicles are shown to be the same as measured by the time-dependence of l-glucose efflux from brush-border membrane vesicles. d-Glucose flux was measured by the equilibrium exchange procedure at constant external and internal Na + concentrations and zero potential. Kinetic analyses of Na +-dependent d-glucose flux indicate that V max in anhydro-4-epitetracycline-treated brush-border membrane vesicles (79.3 ± 7.6 nmol/min per mg protein) is significantly smaller than in control brush-border membrane vesicles (141.3 ± 9.9 nmol/min per mg protein), while the K m values in the two cases are not different from each other (22.3 ± 0.9 and 27.4 ± 1.8 nM, respectively). These results suggest that Na +-dependent d-glucose carriers per se are affected by anhydro-4-epitetracycline, and that this disorder is an important underlying mechanism in the decreased Na +-dependent d-glucose uptake into anhydro-4-epitetracycline-treated brush-border membrane vesicles.

Sodium-dependent succinate transport in renal outer cortical brush border membrane vesicles.

The transport of succinate into outer cortical brush border membrane vesicles (early proximal tubule) was studied. Succinate is taken up into an osmotically active space and exhibits the same distribution volume and the same degree of nonspecific binding and trapping as D-glucose. Succinate uptake is markedly enhanced by sodium and slightly enhanced by lithium but shows no stimulation by other monovalent cations tested. Kinetic analysis of the sodium-dependent component of succinate flux indicates a single transport site obeying Michaelis-Menten kinetics (Km = 1 mM and Vmax = 50 nmol X min -1 X mg protein -1 as measured under zero trans conditions at 100 mM NaCl and 28 degrees C with delta psi = 0). Direct evidence is given that succinate transport is coupled to sodium and is rheogenic, involving the net transfer of positive charge. The sodium:succinate coupling stoichiometry is found to be 2:1 by two independent methods.

Characterization of estrogen binding in uterine cytosol from the fetal rhesus monkey.

Cytosol receptor binding of 17 beta-estradiol was demonstrated in the uterus of the late-gestation, fetal rhesus monkey. Sucrose density gradient analysis performed in low-ionic strength buffer indicated a binding component with a sedimentation coefficient of 6-7 S. Under high-ionic strength conditions, the component shifted to a sedimentation coefficient of approximately 4 S. The specificity of the receptor for estrogens was indicated by inhibition of [3H]estradiol binding by both natural and synthetic estrogen competitors, but not by progesterone. Saturation analysis indicated a high degree of nonspecific binding with saturation of specific binding occurring at 2-3 nM. Computer-assisted Scatchard analysis of the data resolved a one-receptor model having a limited number of binding sites and an apparent dissociation constant of 10(-10) M. The interaction of estrogens with the fetal uterus and the cellular mechanisms which permit this interaction are discussed in relation to the development of models for extrapolation to the human.

Improved preparation of poly(A) Sepharose and the isolation of oligo(U) containing heterogeneous nuclear RNA from Friend erythroleukemic cells.

When poly(A) sepharose (prepared according to previously published procedures) was stored in aqueous buffer at 4 degrees C for 5 days or longer, it bound nonspecifically a high percentage of the input RNA which could not be eluted with formamide. We have found that treatment with ethanolamine, followed by dehydration with ethanol yielded poly(A) sepharose which was stable for many months and possessed a low degree of nonspecific binding. Chromatography on poly(A) sepharose permitted the specific isolation of that fraction of Friend erythroleukemic cell heterogeneous nuclear RNA (hnRNA) which contained oligo(U) sequences. Approximately 10% of the hnRNA which contained a poly(A) sequence [poly(A+)] also contained an oligo(U) sequence. Interestingly, prior HCHO denaturation of the hnRNA enhanced binding of the poly(A+) oligo(U+) hnRNA to poly(A) sepharose by tenfold. This suggested that the oligo(U) sequence may be in a region with secondary structure, possibly an intramolecular duplex with the 3' poly(A). Friend cell oligo(U) sequences ranged from 20 to 50 nucleotides in length and, thus, were similar to the oligo(U) sequences which heretofore had only been shown to be present in HeLa cell hnRNA. These results established that rodent cell hnRNA contain oligo(U) sequences and demonstrate, for the first time, that hnRNA containing both a poly(A) and an oligo(U) sequence can be separated from other classes of hnRNA. In addition, conditions are presented for the removal of HCHO from nucleic acid.

Immunological and biochemical characteristics of acid citrate eluates from tumour cells: a major non-immunoglobulin component.

Using competitive double-antibody radioimmunoassays we have shown that immunoglobulin (especially IgA) can be recovered in pH 3.5, 0.12M acid citrate eluates of freshly excised CCH1 tumour-cell suspensions. Studies with 125I-labelled eluates indicate that such preparations exhibit a variable, but appreciable, degree of non-specific binding to unrelated syngeneic tumour and normal tissues. PAGE/SDS gel electrophoresis of the labelled eluates revealed the presence of a major non-immunoglobulin component of 33-36K dalton which could account in part for the non-specific binding observed. This component was also detected in similar eluates from cultured CCH1 tumour and in all other tumour-cell eluates examined to date. In contrast, preliminary data suggest that it is less prevalent in acid citrate eluates from normal tissue, with the exception of peritoneal-exudate cells. The possible origins, nature and significance of this non-immunoglobulin component are discussed.