Antigen-Specific Plasma Cell Isolation Using ER Markers
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Solution Overview
Problem
Current methods for producing monoclonal antibodies are inefficient and limited, particularly in identifying antigen-specific plasma cells across various animal species due to immune tolerance and the complexity of hybridoma techniques, which hinder the development of effective antibody pharmaceuticals.
Innovation Solution
A method involving the use of fluorescent-labeled antigens and endoplasmic reticulum-specific dyes to identify and isolate antigen-specific plasma cells and plasmablasts, allowing for the production of target antigen-specific antibodies through gene sequencing and recombinant antibody production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the hybridoma method is used to produce monoclonal antibodies, then antibody production capability is achieved, but the process requires extensive cloning and screening time
Solution Approach 1:
The invention extracts and utilizes endoplasmic reticulum-specific markers (such as calreticulin, GRP78, or GRP94) that are uniquely expressed in plasma cells and plasmablasts. By detecting these markers through flow cytometry or fluorescence-activated cell sorting (FACS), the method directly isolates antibody-producing cells without requiring hybridoma formation, cloning, or extensive screening processes
Solution Approach 2:
The invention introduces endoplasmic reticulum-specific markers as intermediary indicators to identify and isolate plasma cells and plasmablasts. These markers serve as mediators that enable direct detection and separation of antibody-producing cells based on their unique endoplasmic reticulum development, bypassing the need for functional screening in traditional hybridoma methods
2Reliability
If the hybridoma method is used, then monoclonal antibodies can be produced, but the method is limited to mouse antibody-producing cells and difficult to apply to other animal species
Solution Approach 1:
The invention applies endoplasmic reticulum-specific markers that are universally expressed in plasma cells and plasmablasts across different animal species. Markers such as calreticulin, GRP78, and GRP94 are conserved endoplasmic reticulum proteins found in mammals, birds, fish, and other vertebrates, enabling the same isolation methodology to be applied universally across species without requiring species-specific adaptations
Solution Approach 2:
The invention changes the identification parameter from species-specific cell surface antigens (used in hybridoma methods) to universal endoplasmic reticulum markers that are consistently expressed in antibody-producing cells across all animal species. This parameter change enables the methodology to be applied to mice, rats, rabbits, dogs, cats, birds, fish, and other species with the same protocol
3Measurement precision
If conventional screening methods are used to identify antigen-specific plasma cells, then some antigen-specific cells can be identified, but there is no guarantee of obtaining clones that produce antigen-specific antibodies
Solution Approach 1:
The invention replaces mechanical screening methods (such as ELISPOT or functional assays) with a biological marker-based identification system. By detecting endoplasmic reticulum-specific markers that are constitutively expressed in plasma cells and plasmablasts, the method directly identifies cells with antibody production capability without relying on functional screening, thereby guaranteeing that isolated cells can produce antibodies
Solution Approach 2:
The invention performs preliminary identification of plasma cells and plasmablasts based on their unique endoplasmic reticulum marker expression before antigen stimulation or functional screening. This preliminary action ensures that only cells with inherent antibody production capability are isolated and further analyzed for antigen specificity, guaranteeing reliable antigen-specific antibody production
4Measurement precision
If the ELISPOT method or other conventional plasma cell identification methods are used, then antigen-specific plasma cells can be identified, but special devices and extremely onerous operations are required
Solution Approach 1:
The invention replaces complex mechanical ELISPOT systems requiring special substrates, incubation chambers, and multiple washing steps with a flow cytometry-based detection system. The endoplasmic reticulum markers are detected using standard flow cytometry or FACS equipment through fluorescently labeled antibodies, reducing operational complexity while maintaining identification precision
Solution Approach 2:
The invention uses fluorescently labeled antibodies against endoplasmic reticulum markers (such as anti-calreticulin, anti-GRP78, or anti-GRP94 antibodies conjugated with fluorophores) to detect plasma cells and plasmablasts. The fluorescent color change enables direct visualization and sorting of target cells using standard flow cytometry equipment, eliminating the need for special ELISPOT devices and simplifying the identification process
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables the rapid and efficient production of antigen-specific monoclonal antibodies from a wide range of animal species, overcoming the limitations of existing techniques and facilitating the development of new antibody pharmaceuticals.
Implementation Method 1
A method involving the use of fluorescent-labeled antigens and endoplasmic reticulum-specific dyes to identify and isolate antigen-specific plasma cells and plasmablasts
Implementation Method 2
A method involving the use of fluorescent-labeled antigens and endoplasmic reticulum-specific dyes to identify and isolate antigen-specific plasma cells and plasmablasts
Data Source
Figure 1A~2B
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AI summary
[Problem] To provide a method that efficiently produces antigen-specific monoclonal antibodies from a wide range of animal species, and to provide a new antigen-specific monoclonal antibody using this technique. [Solution] A nonhuman animal is immunized with a target antigen, lymph fluid or the like is collected from the immunized nonhuman animal, or lymph fluid or the like is collected from a human having antibodies to the target antigen, the collected lymph fluid or the like is combined with (1) a labeled target antigen and (2) a marker that can selectively binds to plasma cells and/or plasmablasts, and cells that have bound to (1) the labeled target antigen and (2) the marker are then selected. The plasma cells and or the plasmablasts that have specifically bound to the target antigen by the method are selected, an gene of an antibody for the target antigen is collected from the selected cells, the base sequence thereof is identified, an antibody or antibody fragment is prepared on the basis of the base sequence of the identified gene, and an antibody or antibody fragment specific to the target antigen is produced.