Enhancement of Binding Affinity of Anti-Hapten Polyclonal IgG Recognizing Mitragynine using Affinity Purification

Antibodies are glycoproteins found in peritoneal fluid, serum, and blood. The antibody-based assay has been used for broad applications such as immunodiagnostic and other biomedical applications. Depending on the intended application, a highly purified polyclonal antibody could be used as an alternative. Purification of antibodies from anti-sera has been proven as one of the methods to enhance the binding affinity of antibodies towards its antigen. We report herein the enhancement of the binding affinity of anti-hapten polyclonal IgG recognizing mitragynine using affinity purification. Serum from the terminal bleed of New Zealand White (NZW) rabbits immunized with mitragynine conjugated with cationized– bovine serum albumin at methyl ester (C22-MG-cBSA), or aromatic ether modification (C9-MG-cBSA) were subjected to HiTrap Protein G affinity purification using fast protein liquid chromatography (FPLC). The elution peak from chromatography fractions was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Here, we report the binding of polyclonal antibodies produced from inoculation of either C22-MG-cBSA or C9-MG-cBSA immunogens of which mitragynine-ovalbumin (MG-OVA) was used as coating antigen in the ELISA assay. Non purified anti-sera from C22-MG-cBSA-inoculated rabbits showed higher titer than C9-MG-cBSA at 1/128 000 and 1/32 000 dilutions, respectively. The affinity of purified poly-IgGs from rabbits immunized with C22-MG-cBSA showed a mean Kd value of 7.965 × 10-6 μM, which was lower than those immunized with C9-MG-cBSA at mean Kd of 1.390 × 10-4 μM. In addition, the purified poly- IgGs showed higher binding towards MG-OVA than non-purified anti-sera at comparable protein concentrations. These results indicated that the higher binding affinity of purified polyclonal IgG is due to the reduced competition among polyclonal antibodies with non- IgG proteins that co-existed in the non-purified anti-sera after the affinity purification.

Generalized calibration across LC-setups for generic prediction of small molecule retention times

MotivationAccurate prediction of liquid chromatographic retention times from small molecule structures is useful for reducing experimental measurements and for improved identification in targeted and untargeted MS. However, different experimental setups (e.g. differences in columns, gradients, solvents, or stationary phase) have given rise to a multitude of prediction models that only predict accurate retention times for a specific experimental setup. In practice this typically results in the fitting of a new predictive model for each specific type of setup, which is not only inefficient but also requires substantial prior data to be accumulated on each such setup.ResultsHere we introduce the concept of generalized calibration, which is capable of the straightforward mapping of retention time models between different experimental setups. This concept builds on the database-controlled calibration approach implemented in PredRet, and fits calibration curves on predicted retention times instead of only on observed retention times. We show that this approach results in significantly higher accuracy of elution peak prediction than is achieved by setup-specific models.

Semi-automation of process analytics reduces operator effect

The aim of this study was to semi-automate process analytics for the quantification of common impurities in downstream processing such as host cell DNA, host cell proteins and endotoxins using a commercial liquid handling station. By semi-automation, the work load to fully analyze the elution peak of a purification run was reduced by at least 2.41 h. The relative standard deviation of results among different operators over a time span of up to 6 months was at the best reduced by half, e.g. from 13.7 to 7.1% in dsDNA analysis. Automation did not improve the reproducibility of results produced by one operator but released time for data evaluation and interpretation or planning of experiments. Overall, semi-automation of process analytics reduced operator-specific influence on test results. Such robust and reproducible analytics is fundamental to establish process analytical technology and get downstream processing ready for Quality by Design approaches.

DO-MS: Data-Driven Optimization of Mass Spectrometry Methods

The performance of ultrasensitive LC-MS/MS methods, such as Single-Cell Proteomics by Mass Spectrometry (SCoPE-MS), depends on multiple interdependent parameters. This interdependence makes it challenging to specifically pinpoint bottlenecks in the LC-MS/MS methods and approaches for resolving them. For example, low signal at MS2 level can be due to poor LC separation, ionization, apex targeting, ion transfer, or ion detection. We sought to specifically diagnose such bottlenecks by interactively visualizing data from all levels of bottom-up LC-MS/MS analysis. Many search engines, such as MaxQuant, already provide such data, and we developed an open source platform for their interactive visualization and analysis: Data-driven Optimization of MS (DO-MS). We found that in many cases DO-MS not only specifically diagnosed bottlenecks but also enabled us to rationally optimize them. For example, we used DO-MS to diagnose poor sampling of the elution peak apex and to optimize it, which increased the efficiency of delivering ions for MS2 analysis by 370%. DO-MS is easy to install and use, and its GUI allows for interactive data subsetting and high-quality figure generation. The modular design of DO-MS facilitates customization and expansion. DO-MS is available for download from GitHub: github.com/SlavovLab/DO-MS

Extraction and Separation of Angiotensin-Converting Enzyme Inhibitor from Enzymatic Hydrolysate of Oyster

The oyster shell was a common sort of marine shellfish, and its meat and shell serve as traditional medicinal materials. To invest the method in which ACEI (Angiotensin-Converting Enzyme Inhibitor) can be prepared, this experiment took oyster as study objective, employed inhibitory coefficient as standard and utilized tryptase as an enzyme. Moreover, this study carried out the chromatogram separation of the hydrolysate by using Sephadex G-25, and measured inhibition coefficient as well as relative molecular weight distribution of ACEI from hydrolysate. The result showed that after the chromatogram separation, the highest activity was determined at the second elution peak at which the active coefficient of ACEI was 89.51%, and that the relative molecular weight distributed between 1842Da and 1396Da.

CROMATOGRAFIA DE AFINIDADE COM ÍON CU2+: ELIMINAÇÃO DE FALSO-POSITIVOS NA DETECÇÃO DE ENTEROTOXINA ESTAFILOCÓCICA

Avaliou-se a aplicação da cromatografia de afinidade com íon Cu2+ para minimizar a interferência de componentes alimentares na detecção de enterotoxinas estafilocócicas mediante imunoensaio, baseado em aglutinação de látex (RPLA). Os ensaios preliminares com “chantilly” contaminado com 25 ng de enterotoxina estafilocócica A (EEA) indicaram que a cromatografia de afinidade com íon Cu2+ eliminou interferentes que causavam reação falso-positiva em RPLA. A eficiência do procedimento de limpeza foi testada em 15 extratos alimentares (de surtos e/ou com alta contagem estafilocócica), compreendendo vários tipos de alimentos comumente consumidos no Brasil. Onze extratos alimentares apresentaram um pico único de eluição com perfis semelhantes e as frações testadas por RPLA demonstraram EEA (4 alimentos), EEB (1), EEA + EEB (3), EEA + EED (2) e EEA + EEB + EED (1). Os problemas de reações inespecíficas com “kit” RPLA em 2 extratos de bolos recheados foram eliminados, indicando que a adoção desta operação evitaria a reação falso-positiva. CU2+ AFFINITY CHROMATOGRAPHY: ELIMINATION OF FALSE-POSITIVE IN THE DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN Abstract The application of Cu2+ chelate Sepharose affinity chromatography was evaluated to reduce the interference of food constituents in the detection of staphylococcal enterotoxin with reversed passive latex agglutination immunoassay (RPLA). The preliminary assay using chantilly contaminated with 25 nanograms of staphylococcal enterotoxin A (SEA) indicated that Cu2+ ion affinity chromatography eliminated the food matrix constituents responsible for false-positive reaction of RPLA. The efficiency of this clean-up procedure was tested in 15 food extracts (from outbreaks and/or with high staphylococcal count), comprising various types of food commonly consumed in Brazil. Eleven food extracts showed only one elution peak with similar profile and the fractions assayed by RPLA demonstrated SEA (4 food), SEB (1), SEA + SEB (3), SEA + SED (2) e SEA + SEB + SED (1). The problems of nonspecific reactions with the RPLA kit in 2 cake extracts were eliminated, indicating that the adoption of this operation avoids the false–positive reaction.