Our Technology

MIPSA

Molecular Indexing of Proteins by Self-Assembly

How MIPSA Works

Antibody Reactome Profiling Using Molecular Indexing of Proteins by Self-Assembly (MIPSA)

The antibody “reactome” is the set of possible binding interactions between antibodies and antigens. Defining each antibody reactome involves interrogation with comprehensive antigen libraries.

The antibodies in the specimen bind to their target antigens, and the binding is then analyzed to determine the presence and levels of each antigen-reactivity. This information can be used to understand disease mechanisms and develop better diagnostic tests, treatments, and vaccines.

There are several methods for performing antibody reactome profiling, including microarray and other molecular display-based methods. However, previous methods have been limited by their library content, low throughput, accuracy, and/or high cost. MIPSA is a powerful new molecular display-based method that uses self-assembly to provide the greatest flexibility in library content and the highest data quality, at significantly lower cost.

How does Infinity Bio ‘read’ antibody reactivities?
MIPSA employs self-assembly to create a library of in-solution DNA-barcoded antigens. Once bound by an antibody and then captured onto beads, sequencing of the DNA barcodes maps the repertoire of antibody reactivities.

Antibody profiling with MIPSA involves three key steps:

Step 1

MIPSA library creation

Gene Synthesis and Cloning: Expressing desired proteins or peptides
MIPSA is used to express two types of libraries for antibody profiling:

Libraries of cloned Open Reading Frames (ORFs) that encode full-length proteins
Synthetic DNA oligonucleotide libraries that encode peptides designed to tile across proteins.

Both types of libraries can be used simultaneously in the same reaction for comprehensive antibody profiling. VeraCode™ ensures that only unmutated antigen library member DNA sequences are associated with the barcodes used for downstream analytics.

Self Assembly: Attaching the DNA barcode to the protein
The self-assembly step of MIPSA technology results in the covalent attachment of a DNA barcode to the protein or peptide library member via the HaloTag system. This process is accomplished in three steps:

A. Creation of the barcoded RNA library
The MIPSA process begins with assembly of DNA sequences that include the following key elements:

An RNA polymerase transcriptional start site
A barcode sequence flanked by universal polymerase chain reaction (PCR) primer binding sites
A translation start site
An N-terminal HaloTag protein sequence
The sequence encoding the individual antigen library member

The in vitro transcribed RNA consists of the unique barcode, the sequence encoding the HaloTag protein and the sequence encoding the library member.

B. Reverse transcription
Reverse transcriptase synthesizes the cDNA barcode using a reverse transcription (RT) primer that is conjugated with a HaloLigand.

C. Translation of HaloTagged antigen library
The HaloTag protein domain is translated first, followed by the translation of the antigen protein or peptide library member. The nascent HaloTag protein domain has a cleft that covalently attaches to the HaloLigand, thereby linking the DNA barcode and the antigen library protein or peptide during its translation in a process of “self-assembly”. The products of translation comprise the MIPSA library reagent, which is now ready for use in the detection of antibody reactivity.

Step 2

Antibody-antigen interaction

An antibody epitope, also known as an antigenic determinant, is the part of the antigen that is specifically recognized by an antibody.

Conformational epitopes: formed by the 3D folding of the antigen molecule, frequently involving discontinuous (non-contiguous) amino acid sequences that are brought together in space. Detection of antibodies targeting conformational epitopes typically requires larger fragments or full-length proteins.
Linear Epitopes: tend to lack significant 3D structure and are determined by the primary sequence of contiguous amino acids. Detection of antibodies targeting linear epitopes is often accomplished using peptides.

The MIPSA library reagent is incubated with samples containing antibodies or buffer only (mock immunoprecipitations that determine background binding) for a defined period of time. Antibodies find and bind their target antigen library members in solution during this time.

Step 3

Decoding of information

A. Antibody-Bound MIPSA Library Members are Captured Using Magnetic Beads
After antibody-antigen complexes are formed in solution, magnetic beads are then used to capture the antibodies along with each bound MIPSA library member and its attached cDNA barcode.

B. DNA Sequencing and Analysis
After unbound MIPSA library members are washed away, PCR is used to amplify the MIPSA barcodes. Many samples can be analyzed together on a sequencing instrument by using sample-specific barcoded primers for the PCR step. High throughput DNA sequencing is used to count the number of MIPSA library member molecules captured in each sample. Proprietary software pipelines convert the sequencing count data into interpretable assay results.

MIPSA Advantages

Comprehensive Antigen Library Coverage
Each library contains the most comprehensive antigen library content available.
Antigen Library combination
For each single antibody capture type (e.g., IgG or IgE), multiple antigen libraries can be combined to optimize coverage and cost.
Peptide and Full-Length Antigens
Ability to create peptide AND full-length protein antigens – each can provide a different level of information and resolution and can be combined in one single assay.
Custom Project Antigen Library Development
Our technology allows for fast and flexible content customization. Any protein sequence from any genome and from any species can be turned into a peptide or full-length antigen.

The VeraCode™ System

After MIPSA library assembly has been completed, we use long-read DNA sequencing to determine which set of base-balanced DNA barcodes are randomly associated with each library member encoding DNA sequence.

In this way, the DNA barcodes are associated with the library members in the form of a lookup table we refer to as the MIPSA dictionary. This dictionary is filtered to include only (i) mono-associated DNA barcodes, and (ii) only unmutated versions of the DNA sequences that encode each of the antigen library members.

Our proprietary VeraCode™ system is a unique feature of MIPSA that ensures the accuracy of your data.