Activated memory B cell supernatants were screened in a high throughput format for neutralization activity using a micro-neutralization assay, as described2 (link). Heavy and light chain variable regions were isolated from B cell lysates of selected neutralizing hits by reverse transcription from RNA followed by multiplex PCR amplification using family-specific V-gene primer sets. For some antibodies, traditional cloning methods were used for antibody isolation, as described2 (link). For other antibodies, amplicons from each lysate were uniquely tagged with multiplex identifier (MID) sequences and 454 sequencing regions (Roche). Single round of replication pseudovirus neutralization assays and cell surface binding assays were performed as described previously2 (link),27 (link),28 (link). Glycan reactivities were profiled on a printed glycan microarray (version 5.0 from the Consortium for Functional Glycomics (CFG)) as described previously29 (link).
Protocol detail
Isolation and Characterization of Neutralizing Antibodies
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
Antibodies
Antibody
Assays
B cell
Cell
Gene
Glycan
Isolation
Light
Memory b cell
Microarray
Multiplex pcr
Replication
Reverse transcription
V primer
Corresponding Organization :
Other organizations : International AIDS Vaccine Initiative, Scripps Research Institute, Ragon Institute of MGH, MIT and Harvard, Seattle University, Theraclone Sciences (United States)
Top 5 similar protocols
Protocol cited in 115 other protocols
Variable analysis
independent variables
- Activation of memory B cells to generate supernatants
- Selection of neutralizing hits from B cell lysates
- Antibody isolation methods (traditional cloning or amplicon sequencing)
- Neutralization activity of supernatants (measured by micro-neutralization assay)
- Heavy and light chain variable regions of selected neutralizing antibodies
- Neutralization activity (single round of replication pseudovirus neutralization assays)
- Cell surface binding activity (cell surface binding assays)
- Glycan reactivity profiles (printed glycan microarray)
- Micro-neutralization assay conditions (as described in reference 2)
- Traditional cloning methods (as described in reference 2)
- Single round of replication pseudovirus neutralization assays and cell surface binding assays (as described in references 2, 27, and 28)
- Printed glycan microarray protocol (as described in reference 29)
- Not explicitly mentioned
- Not explicitly mentioned
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!