अधिक संवेदनशील, जटिल matrices में प्रतिलिपि प्रस्तुत करने योग्य, स्वचालित परिणाम के लिए VIBE मंच पर कस्टम assays के विकास में वर्णित है. सार्वभौमिक कारतूस assays आसानी से कस्टम assays के साथ उपयोग के लिए अनुकूलित अनुमति देता है. इस बहुमुखी प्रतिभा के तेजी से विकास और उपन्यास assays या मौजूदा पुस्तिका assays के स्वचालित संस्करणों, इस वीडियो में उदाहरण के सत्यापन के लिए सक्षम बनाता है.
Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale’s proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.
An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale’s biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.
To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.
Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.
पूर्वगामी उदाहरण AMMP जटिल नमूने में बेहद संवेदनशील मात्रात्मक परिणाम प्रदर्शित करता है. इस प्रदर्शन, जब प्रणाली में अभिकर्मकों स्वरूपण के आराम और नियंत्रण के साथ दिया आवेदनों की एक किस्म के लिए सक्षम है. एक लक्ष्य के लिए उन अनुप्रयोगों विशिष्ट Gadd34 में के रूप में हो सकता है ट्यूमर में, या लक्ष्य के रूप में मेजबान कोशिका प्रोटीन और नमूने है कि शुद्धि धारा पार में पता लगाने quantitation, स्पेक्ट्रम के लिए सामान्य. कम उपरि खिंचाव मंच अत्यंत लचीला AMMP के साथ संयुक्त महत्वपूर्ण assays देने के लिए एक शक्तिशाली उपकरण प्रदान करता है.
The authors have nothing to disclose.
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
NHS-Chromogenic-biotin or NHS-Chromogenic Biotin Labeling kit | ||||
If NHS-Chromogenic-biotin is purchased as a reagent the following additional materials will be required | ||||
Water-miscible organic solvent such as dimethylsulfoxide (DMSO) or dimethylformamide (DMF) | ||||
Phosphate buffered saline (PBS) or other amine free buffer (pH 7 – 9) | ||||
Desalting columns or dialysis units for buffer exchange | ||||
Spectrophotometer able to measure 354 and 280 nm – for measuring protein and biotin concentrations | ||||
Biotinylated antibody (2-5 biotins/Ab) | ||||
Streptavidin magnetic beads | BioScale | (10 mg/mL) ~108 beads/mg | ||
Magnet for microcentrifuge tubes | New England BioLabs | cat # S1510S | ||
Biotinylated BSA for blocking | ||||
Phosphate buffered saline (PBS) | ||||
PBS 0.1% Tween 20 | ||||
Vortex | ||||
Microcentrifuge holder for Vortex or tube rotator | ||||
Antibody or protein to be labeled | ||||
NHS-Fluorescein or NHS-Fluorescein Labeling kit | ||||
If NHS-Fluorescein is purchased as a reagent the following additional materials will be required | ||||
Water-miscible organic solvent such as dimethylsulfoxide (DMSO) or dimethylformamide (DMF) | ||||
50 mM borate, pH 8.5 or other amine free buffer (pH 7 – 9) | ||||
Desalting columns or dialysis units for buffer exchange | ||||
Measurement of protein and fluorescein concentrations requires a spectrophotometer able to measure 493 and 280 nm. | ||||
Diluent | BioScale | to dilute beads and FlAb | ||
Sample Diluent | BioScale | to dilute samples and calibrators | ||
Running Buffer Additive | BioScale |