Effect Of Antibody Concentration Research Articles

A SERS-based immunoassay for porcine circovirus type 2 using multi-branched gold nanoparticles

We report on a facile immunoassay for porcine circovirus type 2 (PCV2) based on surface enhanced Raman scattering (SERS) using multi-branched gold nanoparticles (mb-AuNPs) as substrates. The mb-AuNPs in the immunosensor act as Raman reporters and were prepared via Tris base-induced reduction and subsequent reaction with p-mercaptobenzoic acid (pMBA). They possess good stability and high SERS activity. Subsequently, the modified mb-AuNPs were covalently conjugated to the monoclonal antibody (McAb) against the PCV2 cap protein to form SERS immuno nanoprobes. These were captured in a microtiterplate via a immunoreaction in the presence of target antigens. The effects of antibody concentration, reaction time and temperature on the sensitivity of the immunoassay were investigated. Under optimized assay conditions, the Raman signal intensity at 1,076 cm−1 increases logarithmically with the concentrations of PCV2 in the concentration ranging from 8 × 102 to 8 × 106 copies per mL. The limit of detection is 8 × 102 copies per mL. Compared to conventional detecting methods such as those based on PCR, the method presented here is rapid, facile and very sensitive.

Tyramide signal amplification for analysis of kinase activity by intracellular flow cytometry

Intracellular flow cytometry permits quantitation of diverse molecular targets at the single-cell level. However, limitations in detection sensitivity inherently restrict the method, sometimes resulting in the inability to measure proteins of very low abundance or to differentiate cells expressing subtly different protein concentrations. To improve these measurements, an enzymatic amplification approach called tyramide signal amplification (TSA) was optimized for assessment of intracellular kinase cascades. First, Pacific Blue, Pacific Orange, and Alexa Fluor 488 tyramide reporters were shown to exhibit low nonspecific binding in permeabilized cells. Next, the effects of antibody concentration, tyramide concentration, and reaction time on assay resolution were characterized. Use of optimized TSA resulted in a 10-fold or greater improvement in measurement resolution of endogenous Erk and Stat cell signaling pathways relative to standard, nonamplified detection. TSA also enhanced assay sensitivity and, in conjunction with fluorescent cell barcoding, improved assay performance according to a metric used to evaluate high-throughput drug screens. TSA was used to profile Stat1 phosphorylation in primary immune system cells, which revealed heterogeneity in various populations, including CD4+ FoxP3+ regulatory T cells. We anticipate the approach will be broadly applicable to intracellular flow cytometry assays with low signal-to-noise ratios.

Hepatitis B surface antibody purification with hepatitis B surface antibody imprinted poly(hydroxyethyl methacrylate-N-methacryloyl- l-tyrosine methyl ester) particles

Hepatitis B surface antibody imprinted poly(hydroxyethyl methacrylate-N-methacryloyl- l-tyrosine methyl ester) particles were prepared for the purification of hepatitis B surface antibody from human plasma. N-methacryloyl- l-tyrosine methyl ester was chosen as a complexing agent for hepatitis B surface antibodies. Hepatitis B surface antibody imprinted poly(hydroxyethyl methacrylate-N-methacryloyl- l-tyrosine methyl ester) particles were characterized by surface area measurements, swelling test, scanning electron microscopy, elemental analysis, and Fourier transform infrared spectroscopy. Ethylene glycol (1.0 M) was used as desorption agent. Adsorption studies were performed from hepatitis B surface antibody and anti-hepatitis A antibody positive human plasma. Effects of antibody concentration, contact time, N-methacryloyl- l-tyrosine methyl ester content and temperature on the adsorption capacity were investigated. The amount of hepatitis B surface antibody adsorbed per unit mass increased with increasing hepatitis B surface antibody concentration, then reached saturation. Maximum hepatitis B surface antibody adsorption amount was 21.4 mIU/mg. Adsorption process reached the equilibrium in 60 min. Competitive adsorption of hepatitis B surface antibody, total anti-hepatitis A antibody and total immunoglobulin E was investigated for showing the selectivity. Hepatitis B surface antibody-imprinted particles could adsorb hepatitis B surface antibody 18.3 times more than anti-hepatitis A antibody and 2.2 times more than immunoglobulin E. It can be concluded that hepatitis B surface antibody-imprinted particles have significant selectivity for hepatitis B surface antibody.

Effects of antibody concentration on the separation of human natural killer cells in a commercial immunomagnetic separation system.

The magnetic separation of a cell population based on cell surface markers is a critical step in many biological and clinical laboratories. In this study, the effect of antibody concentration on the separation of human natural killer cells in a commercial, immunomagnetic cell separation system was investigated. Specifically, the degree of saturation of antibody binding sites using a two-step antibody sandwich was quantified. The quantification of the first step, a primary anti-CD56-PE antibody, was achieved through fluorescence intensity measurements using a flow cytometer. The quantification of the second step, an anti-PE-microbeads antibody reagent, was achieved through magnetophoretic mobility measurements using cell tracking velocimetry. From the results of these studies, two different labeling protocols were used to separate CD56+ cells from human, peripheral blood by a Miltenyi Biotech MiniMACS cell separation system. The first of these two labeling protocols was based on company recommendations, whereas the second was based on the results of the saturation studies. The results from these studies demonstrate that the magnetophoretic mobility is a function of both primary and secondary antibody concentrations and that mobility does have an effect on the performance of the separation system. As the mobility increased due to an increase in bound antibodies, the positive cells were almost completely eliminated from the negative eluent. However, with an increase in bound antibodies, and thus mobility, the total amount of positive cells recovered decreases. It is speculated that these cells are irreversibly retained in the column. These results demonstrate the complexity of immunomagnetic cell separation and the need to further optimize the cell separation process.

Evaluation of Monoclonal Antifibrin Antibodies by Their Binding to Human Blood Clots

The primary goal of this study was to develop a method for evaluating fibrin-specific antibodies as thrombus detecting agents. The apparatus and assay conditions were chosen by testing antibody 64C5, which binds to the amino terminus of the fibrin beta chain, for its ability to bind to human blood clots. Using 125I-labeled antibody 64C5, the effects of antibody concentration, clot shape, clot mass, temperature, and flow rate were tested. Increased antibody binding was observed when antibody concentration, clot mass and temperature were increased. Under one set of conditions, six 125I-labeled monoclonal antifibrin antibodies (four specific for the beta chain, two specific for the alpha chain) were tested for their binding to retracted clots of human blood. Two radioiodinated antidigoxin antibodies were used as a control. Beta chain-specific antibody 59D8, which provided the highest level of binding to clot, bound 14-fold better than the control antidigoxin antibody. Neither alpha chain-specific antibody bound to clotted blood. To examine the in vivo relevance of the in vitro binding to clots, the uptake of antibody 64C5 was assessed for its binding to human fibrin clotted within the jugular vein of a rabbit. The correlation coefficient between in vitro and in vivo uptake as a function of clot weight was calculated to be 0.91. Thus, in vitro binding of monoclonal antifibrin antibodies to human blood clots was judged to be a realistic method for the comparison and selection of an ideal antifibrin antibody for detailed in vivo testing.

A paradoxical effect of antibody concentration on vWF-dependent platelet agglutination distinguishes between botrocetin and ristocetin-induced agglutination.

Several heterologous antisera directed against either human or porcine von Willebrand factor (vWF), inhibited botrocetin-induced vWF-dependent agglutination at high concentrations but were found to enhance this reaction at low concentrations. Purified IgG from these immune sera also potentiated botrocetin-induced agglutination as did its F(ab)'2 fragments. However, monovalent Fab fragments of the purified IgG did not. The requirement for a divalent antigen combining region suggests that one possible mechanism of enhanced agglutination may involve intra or inter-molecular cross-linking of vWF multimers by the antibody. Another possible mechanism could be a conformational change in the vWF molecule induced by antibody binding. Such a conformational change may provide additional active sites on the vWF molecule that, in the presence of botrocetin, lead to enhancement of the agglutination reaction. Antisera to human vWF that showed this paradoxical inhibitory/enhancing effect on botrocetin-induced agglutination also inhibited ristocetin-induced platelet agglutination at high concentrations, but failed to enhance agglutination at any concentration. The different effects of these antisera on botrocetin and ristocetin-induced platelet agglutination suggest that no single mechanism can explain the action of both of these mediators.

Effect of antibody concentration on opsonic requirements for phagocytosis in vitro of Streptococcus pneumoniae types 7 and 19.

We investigated the effect of the concentration of type-specific antibody to pneumococcal polysaccharide (PPS) on opsonic requirements for phagocytosis in vitro of Streptococcus pneumonia types 7 and 19. We measured the uptake by human neutrophils of radiolabeled S. pneumoniae opsonized with either complement-intact, complement-depleted (by heat inactivation), or alternative complement pathway-activated (by magnesium dichloride-ethylene glycol tetraacetic acid (MgEGTA) chelation) immune sera with varying concentrations of antibody from individuals immunized with polyvalent PPS vaccine. Increased opsonization was found with increasing concentrations of type-specific antibody in the sera. Higher concentrations of antibody were required to opsonize type 7 than type 19 bacteria, both in the presence and absence of complement activity. Type 19 bacteria were more efficiently opsonized via the alternative complement pathway than type 7. For both types, antibody and the alternative complement pathway provided most of the opsonic activity in sera with lower concentrations of type-specific antibody. At high antibody concentrations, effective opsonization occurred in heat-activated sera, indicating the requirement for complement could be overcome with sufficient amounts of antibody alone.

Passive sensitization in vitro: effect of antibody concentration on the lag period and velocity.

Strips of guinea pig ileum were sensitized in vitro in various concen trations of rabbit antibody prepared against ovalbumin. The dependence of the first-order velocity constants upon antibody concentration was hyperbolic. The limiting velocity constant was 0.0097 per minute, and the antibody concentration giving half the limiting velocity was 0.0035 milligrams per milliliter. The lag period varied inversely with the logarithm of the antibody concentration.

Effect of Antibody Concentration and Temperature upon Physical Adsorption and Histamine Release

Summary An investigation was made into the nature of passive in vitro sensitization of guinea pig ileum by parallel studies of the effect of temperature on the physical adsorption of antiovalbumin and the magnitude of histamine release following challenge with antigen. The uptake of radioiodinated rabbit γ-globulin by strips of guinea pig ileum was studied for several concentrations of protein at temperatures ranging from 20° to 37°C. There was no essential difference in the rates of physical adsorption among the several categories tested, the average first order velocity constants being 0.020 and 0.024 min-1 for 20° and 37°C, respectively. Tissues immersed for 60 min in concentrations of antiovalbumin ranging from 0.046 to 0.273 mg/ml at 20°C and 27°C, and from 0.009 to 0.228 mg/ml at 37°C, took up labeled antibody according to a simple function of the bulk phase concentration. The relationship between the bulk phase concentration and the amount physically adsorbed was not affected by temperature. Antibody that had been adsorbed to tissue during the first incubation phase could be eluted by washing with Tyrode's solutions, but the amount eluted was not influenced by temperature. When these tissues were subsequently challenged in the presence of 0.1 mg/ml ovalbumin a constant proportion of label was removed but, as control experiments showed, this was no greater than that which could be achieved by washing with Tyrode's. The release of histamine from tissues of actively immunized animals was increased by elevating the temperature during the challenge step. Variations of temperature during the adsorption step that preceded challenge had no effect upon histamine release if the subsequent challenge temperature was constant. Although the amount of antibody physically adsorbed was influenced only by bulk phase concentration and not by temperature, the quantity of histamine released for a given degree of physical adsorption was greatly increased as the temperature of the adsorption step was elevated, the activation energy of this process being 18 kcal. Since both physical adsorption and histamine release depend upon the concentration of antibody in the bulk phase, the relationship between these two processes is not a specious one. It is our view that the effect of temperature observed in these studies is to activate receptor sites in such a way as to increase the proportion of histamine-bearing cells which are in a critical state preceding challenge with antigen.