Immune Netw.  2011 Apr;11(2):114-122. 10.4110/in.2011.11.2.114.

Characterization of Monoclonal Antibodies against Human Leukocyte Common Antigen (CD45)

• Shin HM
• Cho WD
• Lee GK
• Lee SH
• Lee KM
• Ji GY
• Yoon SS
• Koo JH
• Lee HC
• Lee KH
• Song HG

• KMID: 2150699
• DOI: http://doi.org/10.4110/in.2011.11.2.114

Abstract

BACKGROUND
The leukocyte common antigen (CD45) is a transmembrane-type protein tyrosine phosphatase that has five isoforms.
METHODS
We generated seven murine mAbs against human CD45 by injecting cells from different origins, such as human thymocytes, PBMCs, and leukemic cell lines. By using various immunological methods including flow cytometry, immunohistochemistry, and immunoprecipitation, we evaluated the reactivity of those mAbs to CD45 of thymus as well as tonsil lysates. Furthermore, we transiently transfected COS-7 cells with each of gene constructs that express five human CD45 isoforms respectively, and examined the specificities of the mAbs against the transfected isoforms.
RESULTS
In case of thymocytes, lymphocytes, and monocytes, all the seven mAbs demonstrated positive reactivities whereas none was reactive to erythrocytes and platelets. The majority of immune cells in formalin-fixed paraffin-embedded thymus and tonsil tissues displayed strong membranous immunoreactivity, and the main antigen was detected near 220 kDa in all cases. Among the mAbs, four mAbs (AP4, DN11, SHL-1, and P6) recognized a region commonly present in all the five isoforms. One mAb, YG27, recognized four isoforms (ABC, AB, BC, and O). Two mAbs, P1 and P14, recognized the isoforms that contain exon A encoded regions (ABC and AB).
CONCLUSION
In this study, we confirmed that AP4, DN11, SHL-1, YG27 and P6, are mAbs reactive with the CD45 antigen whereas P1 and P14 are reactive with the CD45RA antigen.

Keyword

Leukocyte common antigen (CD45); CD45RA; Monoclonal antibody; Isoform

MeSH Terms

Animals
Antibodies, Monoclonal
Antigens, CD45
Blood Platelets
Cell Line
COS Cells
Erythrocytes
Exons
Flow Cytometry
Humans
Immunohistochemistry
Immunoprecipitation
Leukocytes
Lymphocytes
Monocytes
Palatine Tonsil
Protein Isoforms
Protein Tyrosine Phosphatases
Thymocytes
Thymus Gland
Antibodies, Monoclonal
Antigens, CD45
Protein Isoforms
Protein Tyrosine Phosphatases

Figure

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  Figure 1 The structures of the five human CD45 isoforms derived from cDNA cloning.

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  Figure 2 Reactivity of AP4 on formalin fixed thymus. All T cells of cortex (C) and medulla (M) were reactive with AP4. but, squamous cells of Hassall's corpuscle (H) were not reactive with AP4.

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  Figure 3 Reactivity of P14 on formalin fixed tonsil. All lymphocytes of germinal center (G), mantle (M) and interfollicular (IF) area were reactive with P14.

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  Figure 4 Immunoprecipitation of thymus lysates with AP4, DN11, SHL-1, YG27 and P6 mAb. Lane 1; AP4 lane 2; DN11 lane 3; SHL-1 lane 4; YG27 lane 5; P6. Cells were lysed in lysis buffer containing 1% NP-40. precleared cell lysates were immunoprecipitated with AP4, DN11, SHL-1, YG27 and P6 mAb. The immunoprecipitates were analyzed by SDS-PAGE (6%) under nonreducing condition.

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  Figure 5 Immunoprecipitation of tonsil lysates with P1 and P14 mAb. Cells were lysed in lysis buffer containing 1% NP-40. Precleared cell lysates were immunoprecipitated with P1 and P14 mAb. The immunoprecipitates were analyzed by SDS-PAGE (6%) under nonreducing condition.

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  Figure 6 Transfection study of DN11 mAb. Flow cytometric analysis of DN11 using COS-7 cells expressing the individual human CD45 isoforms. DN11 bound to all five isoforms (the ABC isoform, AB isoform, BC isoform, B isoform and O isoform).

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  Figure 7 Transfection study of P1 mAb. Flow cytometric analysis of P1 using COS-7 cells expressing the individual human CD45 isoforms. P1 bound to two isoforms that include exon A encoded sequences (the ABC isoform and AB isoform).

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