Repellant effect of neem formulation and aqeuous extract of Melia azedarach on greenhouse whitefly (Trialeurodes vaporariorum Westwood, Hemiptera: Aleyrodidae)

The present study assessed the repellence activities of two biopesticides viz. a formulation of neem, neem baan and aqueous extract of Melia azedarach (Dharek) kernels against crawlers of greenhouse whitefly Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae). The maximum repellency (22.07%) was recorded at 10 % concentration of dharek extract followed by Neem Baan at 0.0025 % concentration (18.33%). The minimum repellency (4.71%) was observed at 0.0005 % concentration of Neem baan. These results indicate a potential use of neem baan and aqueous dharek kernel extracts in management of greenhouse whitefly

Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire.

Staphylococcus aureus is both an important pathogen and a human commensal. To explore this ambivalent relationship between host and microbe, we analysed the memory humoral response against IsdB, a protein involved in iron acquisition, in four healthy donors. Here we show that in all donors a heavily biased use of two immunoglobulin heavy chain germlines generated high affinity (pM) antibodies that neutralize the two IsdB NEAT domains, IGHV4-39 for NEAT1 and IGHV1-69 for NEAT2. In contrast to the typical antibody/antigen interactions, the binding is primarily driven by the germline-encoded hydrophobic CDRH-2 motifs of IGHV1-69 and IGHV4-39, with a binding mechanism nearly identical for each antibody derived from different donors. Our results suggest that IGHV1-69 and IGHV4-39, while part of the adaptive immune system, may have evolved under selection pressure to encode a binding motif innately capable of recognizing and neutralizing a structurally conserved protein domain involved in pathogen iron acquisition

Exploring the Dynamic Range of the Kinetic Exclusion Assay in Characterizing Antigen-Antibody Interactions

Therapeutic antibodies are often engineered or selected to have high on-target binding affinities that can be challenging to determine precisely by most biophysical methods. Here, we explore the dynamic range of the kinetic exclusion assay (KinExA) by exploiting the interactions of an anti-DKK antibody with a panel of DKK antigens as a model system. By tailoring the KinExA to each studied antigen, we obtained apparent equilibrium dissociation constants (KD values) spanning six orders of magnitude, from approximately 100 fM to 100 nM. Using a previously calibrated antibody concentration and working in a suitable concentration range, we show that a single experiment can yield accurate and precise values for both the apparent KD and the apparent active concentration of the antigen, thereby increasing the information content of an assay and decreasing sample consumption. Orthogonal measurements obtained on Biacore and Octet label-free biosensor platforms further validated our KinExA-derived affinity and active concentration determinations. We obtained excellent agreement in the apparent affinities obtained across platforms and within the KinExA method irrespective of the assay orientation employed or the purity of the recombinant or native antigens

Determining the Binding Affinity of Therapeutic Monoclonal Antibodies towards Their Native Unpurified Antigens in Human Serum

<div><p>Monoclonal antibodies (mAbs) are a growing segment of therapeutics, yet their <i>in vitro</i> characterization remains challenging. While it is essential that a therapeutic mAb recognizes the native, physiologically occurring epitope, the generation and selection of mAbs often rely on the use of purified recombinant versions of the antigen that may display non-native epitopes. Here, we present a method to measure both, the binding affinity of a therapeutic mAb towards its native unpurified antigen in human serum, and the antigen’s endogenous concentration, by combining the kinetic exclusion assay and Biacore’s calibration free concentration analysis. To illustrate the broad utility of our method, we studied a panel of mAbs raised against three disparate soluble antigens that are abundant in the serum of healthy donors: proprotein convertase subtilisin/kexin type 9 (PCSK9), progranulin (PGRN), and fatty acid binding protein (FABP4). We also determined the affinity of each mAb towards its purified recombinant antigen and assessed whether the interactions were pH-dependent. Of the six mAbs studied, three did not appear to discriminate between the serum and recombinant forms of the antigen; one mAb bound serum antigen with a higher affinity than recombinant antigen; and two mAbs displayed a different affinity for serum antigen that could be explained by a pH-dependent interaction. Our results highlight the importance of taking pH into account when measuring the affinities of mAbs towards their serum antigens, since the pH of serum samples becomes increasingly alkaline upon aerobic handling. </p> </div

KinExA-determined affinities of three mAbs (J17, 2B2, and 33B12) towards their purified recombinant antigens (left) and native antigens in human serum (right).

<p>The data are presented in the same way as in Figure 2.</p

KinExA-determined apparent affinities for four mAbs (J16, J17, 19F7, and 21B8) towards their purified recombinant antigens (left) and their serum antigens (right) at stable, non-neutral pH values.

<p>Titration curves are presented as described in Figure 2.</p

Influence of pH on the apparent affinity (top) and apparent activity (bottom) of different mAbs towards their purified recombinant antigens.

<p>The K_D values and mAb activities for each interaction were obtained from a single curve KinExA analysis performed at different pH values that spanned the pH range encountered during serum experiments. The bars represent the best fit values and the error bars represent the 95% confidence interval. The arrows indicate the trend observed with increasing pH and the range of best fit values for K_D and activity. Only sweet spot experiments enabled a determination of both the K_D and the mAb activity (no mAb activity is reported for 19F7 and 33B12 because those curves were mostly K_D-controlled). The antigen concentrations used were 128 pM rhPCSK9, 42 pM rhPGRN, 21 pM rhPGRN, 100 pM rhFABP4, and 1nM rhFABP4 (from left to right).</p

Human serum titrated with anti-PCSK9 mAb J16.

<p>(A) Raw data trace of fluorescence (in Volts) as a function of time recorded by the KinExA instrument for a typical experiment: (I) packing of mAb-coated beads inside the flow cell; (II) baseline signal; (III) auto-fluorescence signal obtained from serum components (presumably porphyrins); (IV) buffer wash; (V) detection of bead-captured PCSK9 with a Dylight-labeled mAb; and (VI) buffer wash, after which the final fluorescence signal for bead-captured PCSK9 is recorded (relative to the baseline signal). (B) Global fit of normalized data obtained from titrating J16 into different dilutions of serum prepared in PBS. (C) Error plots for K_D and PCSK9 concentration for the global analysis in panel B with best fit values (solid line) and 95% confidence interval (dotted lines). (D) Comparison of the fits obtained for single-curve and multi-curve (global) analysis of the data in panel B. The PCSK9 concentration is back-calculated for undiluted serum. Open and closed symbols indicate independent experiments performed with the same dilution factor. </p