Amount Of Nonspecific Binding Research Articles

Complement C1q in plasma induces nonspecific binding of poly(acrylic acid)-coated upconverting nanoparticle antibody conjugates

Upconverting nanoparticles are attractive reporters for immunoassays, because their high specific activity and lack of autofluorescence background enable their detection at extremely low concentrations. However, the sensitivity achieved with heterogeneous sandwich immunoassays using nanoparticle reporters is generally limited by the nonspecific binding of nanoparticle antibody conjugates to solid supports. In this study, we characterized plasma components associated with elevated nonspecific binding of poly(acrylic acid)-coated upconverting nanoparticles in heterogeneous two-step sandwich immunoassays. Plasma was consecutively fractionated using various chromatographic methods by selecting after each step the fractions producing the highest nonspecific binding of upconverting nanoparticle conjugates in an immunoassay for cardiac troponin I. Finally, the proteins in the fractions associated with highest amount of nonspecific binding were separated by gel electrophoresis and identified with mass spectrometry. The results indicated that complement component C1q was present in the fractions associated with the highest signal from nonspecific binding. The interference was not limited to only poly(acrylic acid)-coated nanoparticles or certain antibody combination, but occurred more generally. The interference was removed by increasing the ionic strength of the assay buffer in the sample incubation step or by adding a negatively charged blocker to bind on positively charged C1q, suggesting that the interaction is mostly electrostatic. Hence, we assume that the interference is likely to affect various negatively charged nanoparticles. The identification of complement component C1q as the major interfering protein allows for more rational design of countermeasures in future immunoassay development utilizing nanoparticle reporters.Graphical abstract

Affinity membrane development from PBT nonwoven by photo-induced graft polymerization, hydrophilization and ligand attachment

Nonwoven fabrics are of great interest as potential materials for bioseparations due to their interconnected porous structure, relatively high surface area and low cost. In this paper we focus on the development of a potentially disposable affinity membrane for pathogen removal from biological systems such as human plasma. Poly glycidyl methacrylate (polyGMA) was grafted on the fiber surface of a polybutylene terephthalate (PBT) nonwoven using photo-induced graft polymerization. SEM and FTIR were used to characterize the pore structure and surface chemistry of the resulting material. To minimize nonspecific protein binding and hydrophilize the material, diethylene glycol (DEG) and diol groups were attached covalently to the grafted layer of polyGMA. The amount of nonspecific binding was quantified by the adsorption of bovine serum albumin (BSA) and an E. coli extract. The results showed that the grafted matrix containing DEG or diol groups bound significantly less total protein, compared with unmodified material. The DEG modified membrane was further developed by attachment of a specific proprietary ligand that binds to the prion protein, the agent responsible for transmissible spongiform encephalopathies. The affinity membrane showed good selectivity for the capture of prion protein from hamster brain homogenate.

Next‐generation Immunoprecipitation Kits Validated for Functional Applications

The study of protein function and interaction is essential for understanding many biological processes. We have developed three new immunoprecipitation (IP) kits with improved protocols and buffers that provide excellent antigen recovery with the least amount of nonspecific binding using 2 to 10 µg of antibody. The Pierce IP Direct kit contains our AminoLink Plus Coupling resin for directly crosslinking purified antibodies to the matrix for specific elution of the antigen. The Pierce IP Classic kit contains Protein A/G Plus Agarose that binds immunoglobulins from a variety of species. The Protein A/G Plus resin is also used in the Pierce IP Crosslinked kit which is designed to purify and crosslink the antibody to the resin before performing the immunoprecipitation procedure. With the new kits, we have demonstrated immunoprecipitations of a diverse selection of proteins including membrane proteins, protein phosphatases and kinases, transcription factors, and cell cycle regulation proteins. We show that the new Thermo Scientific Pierce IP Kits immunoprecipitate protein complexes and are compatible with mass spectrometry and enzymatic assays.

In vivo [123I]CNS‐1261 binding to D‐serine‐activated and MK801‐blocked NMDA receptors: A storage phosphor imaging study in rats

Disturbances of activity of the glutamatergic neurotransmitter system in the brain are present in many neuropsychiatric disorders. The N-methyl-D-aspartate (NMDA) receptor is the most abundant receptor of the glutamatergic system. In the neurodegenerative events of Alzheimer's disease, excessive activation of NMDA receptors may contribute to neuronal death. Inhibition of NMDA receptor activation may have neuroprotective effects and (semi)quantitative imaging of the activated system may help in the selection of patients for such inhibition therapies. In this study we evaluated [(123)I]CNS-1261 binding in the rat brain. This radiotracer binds in vivo to the MK801 binding site of activated NMDA receptors. To determine the optimal time point for ex vivo assessments after bolus injection [(123)I]CNS-1261 binding in rats, we performed a time course biodistribution study using dissection techniques. [(123)I]CNS-1261 binding was also studied in the rat brain using autoradiography by means of storage phosphor imaging, with prior facilitation of NMDA receptor activation by injection of the potent coagonist D-serine and after blocking of the NMDA receptor binding site by MK801 injection in D-serine pretreated rats. Measurements of [(123)I]CNS-1261 uptake matched the distribution of similar tracers for the MK801 binding site of the NMDA receptor and revealed an optimal time point of 2 h post injection for the assessment of tracer distribution in the rat brain. The blocking experiments indicated specific binding of [(123)I]CNS-1261 to NMDA receptors but also a considerable amount of nonspecific binding. Facilitation of NMDA receptor activation by D-serine did not result in an enhancement of binding of the radiotracer in the NMDA receptor-rich rat hippocampus compared to the untreated group, as measured by autoradiography. In conclusion, our study has shown that [(123)I]CNS-1261 binding is influenced by NMDA receptor availability. However, high nonspecific binding limits quantification and small changes in receptor availability are unlikely to be detected.

Immunosensing Based on Site-Directed Immobilization of Antibody Fragments and Polymers that Reduce Nonspecific Binding

Antibody Fab'-fragments can be directly coupled onto gold, and the space between the fragments can be filled with protein repellent disulfide bearing polymers. Coupling of the antibody Fab'-fragments, and thus both the amount of nonspecific binding and antigen binding but also the ability to regenerate the layer, is dependent on the immobilization procedure. First, the immobilization has taken place by coupling the Fab'-fragments to the surface and thereafter attaching the polymer in the remaining space between the antibodies. Second, the Fab'-fragments have been added after the surface has been coated by polymer. Third, the Fab'-fragments and polymer have been added onto the surface from the same solution. Up to 80% of the antigen could be removed during regeneration, if proper concentrations of polymer and Fab'-fragments were immobilized onto the gold surface. Only about 60% of the antigen could be removed, when the fragments were coupled directly onto a clean Au surface before the polymer or if low concentrations of polymer were attached onto gold before the Fab'-fragments. The first immobilization method, however, showed the highest response to antigen.

Limit of Resolution of Flow Cytometry for the Detection of Selected Bacterial Species

The enumeration of bacteria in dental plaque samples is a vital but time-consuming procedure that uses standard cultural methods. Flow cytometry has proven to be a useful tool for the analysis of eukaryotic cells. In the present investigation, the utility of this technology for the enumeration of bacteria in mixtures was explored. Rabbit antisera were produced against the putative periodontal pathogens A. actinomycetemcomitans, B. intermedius, B. gingivalis, E. corrodens, W. recta, B. forsythus, as well as the frequently isolated supragingival species S. sanguis. Cross-reactive antibodies were removed by absorption, and the specificity of each antiserum was confirmed by being tested against a panel of 235 oral microbial strains (79 genera; 94 species) by means of ELISA. Conditions were established for the indirect immunofluorescent labeling of cells without agglutination with use of a goat anti-rabbit Ig-FITC second antibody. When an internal bead standard was used, it was found that unstained bacteria were enumerated by light-scattering parameters with poor efficiency (less than 3%). However, cells exposed to FITC either in the presence of specific or non-specific first antibody were enumerated with high efficiency (102.6 +/- 29.3%), indicating that a small amount of non-specific binding of fluorochrome facilitates bacterial detection. Clear discrimination between specifically- and non-specifically-stained bacteria was achieved with all six rabbit antisera. Mixtures of known composition were made (1) with pure cultures or (2) with a known species and supragingival plaque devoid of that species by culture. The results from both approaches with various species combinations revealed that the limit of resolution for accurate quantitation of a selected species was approximately 5%, although specific organisms could be detected qualitatively when present at approximately 1%.