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Monoclonal anti-lambda 5 antibody FS1 identifies a 130 kDa protein associated with lambda 5 and Vpre-B on the surface
F Shinjo1, R R Hardy, J Jongstra
1Toronto Hospital Research Institute, Ontario, Canada.
Abstract:
mAbs specific for mouse lambda 5 protein were prepared by fusion of spleen cells from a hamster immunized with recombinant lambda 5 protein synthesized in bacteria and the mouse myeloma cell line SP2/0-Ag14. Here we report the characteristics of the antibodies produced by the FS1 hybridoma. FS1 antibody stains a variety of mouse pre-B cell lines but not B cell lines or T cell lines. The staining of the pre-B cell lines A-1 and C-7 by phycoerythrin (PE)-conjugated FS1 (FS1-PE) can be blocked by preincubation of these cells with unconjugated FS1 antibody or with affinity purified polyclonal lambda 5 specific Ig but not with normal hamster or mouse IgG or with affinity purified polyclonal anti-Mb-1 Ig. From these experiments we concluded that FS1 specifically recognizes lambda 5 protein. We used FS1-PE to probe for surface (s) lambda 5+ cells in normal BALB/c mouse bone marrow. Such cells were undetectable when total bone marrow or FACS sorted subpopulations were analyzed. However, when B220+, CD43+, s lambda 5-bone marrow cells were cultured for 4 days on the stromal cell line FLST2 in the presence of IL-7, s lambda 5 expression became apparent. Further expansion of these cells in IL7 alone augmented the s lambda 5 expression to readily detectable levels. This modulation may indicate that s lambda 5 expression on normal bone marrow cells in vivo is transient and that at any given moment only a small fraction of bone marrow cells expresses low levels of lambda 5 protein on the surface.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Monoclonal antibodies (mAbs) targeting the mouse lambda 5 protein were developed. The FS1 hybridoma antibody specifically recognizes lambda 5 protein, aiding in the study of pre-B cell development.
Area of Science:
- Immunology
- Cell Biology
- Hematopoiesis
Background:
- The lambda 5 protein is a component of the B cell receptor (BCR) found in early B cell development.
- Understanding the expression and function of lambda 5 is crucial for elucidating B cell differentiation pathways.
Purpose of the Study:
- To characterize monoclonal antibodies (mAbs) specific for the mouse lambda 5 protein.
- To investigate the expression of surface lambda 5 (sλ5) on normal mouse bone marrow cells.
Main Methods:
- Generation of hybridoma cell lines producing mAbs against recombinant lambda 5 protein.
- Flow cytometry analysis using FS1-PE conjugated antibody to detect lambda 5 expression on cell lines and bone marrow cells.
- In vitro culture of bone marrow cells with stromal cell lines and cytokines (IL-7) to study sλ5 modulation.
Main Results:
- The FS1 hybridoma produced mAbs that specifically recognize mouse lambda 5 protein.
- FS1 antibody stained pre-B cell lines but not mature B or T cell lines.
- Surface lambda 5 expression was undetectable on normal mouse bone marrow cells but could be induced in vitro, suggesting transient expression.
Conclusions:
- The FS1 antibody is a specific reagent for detecting mouse lambda 5 protein.
- Lambda 5 expression on normal bone marrow cells is likely transient and at low levels in vivo.
- Cytokine-supported culture conditions can induce detectable surface lambda 5 expression on developing B cells.
