Source: MISSISSIPPI STATE UNIV submitted to NRP
IDENTIFICATION OF COMMERCIALLY AVAILABLE ANTIBODIES THAT WILL EFFECTIVELY LABEL ZEBRAFISH AND KILLIFISH LEUKOCYTES FOR FLOW CYTOMETRY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
ANIMAL HEALTH
Reporting Frequency
Annual
Accession No.
0231536
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Sep 1, 2012
Project End Date
Aug 31, 2014
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
MISSISSIPPI STATE UNIV
(N/A)
MISSISSIPPI STATE,MS 39762
Performing Department
College Of Veterinary Medicine
Non Technical Summary
Much of the basic research on inducible components of innate and acquired immunity was done using in vitro research models with isolated or cultured cells. These types of studies are useful for dissecting specific mechanistic pathways but are artificial, in that cellular interactions that occur in the whole animal are removed from the system. With the historical production of murine monoclonal antibodies, mutants and specific knock out mice, components of the immune system could be dissected in the whole animal model. The use of SCID and recombination activation gene 1 (rag1) mutant mice allowed the investigation of the specific contribution of innate defenses in many infectious diseases. Mutants have normally functioning macrophages, natural killer cells and neutrophils, but lack T and B lymphocytes (Dorschkind 1990, Mombaerts et al. 1992). Similar mutants can now be used in fish immunology research. Zebrafish have been produced that carry a point mutation in the rag1 gene that causes a premature stop codon in the catalytic domain of rag1 (Wienholds et al. 2002). A functional rag1 protein is required for V(D)J recombination when generating functional immunoglobulin and T cell receptor (TCR) genes (Schatz et al. 1989). As in other vertebrates, there is only one functional rag1 gene; loss of function at this locus results in a complete block of immunoglobulin gene assembly and subsequent immunodeficiency (Wienholds et al. 2002). In characterization studies, these rag1-/- zebrafish have been found to lack functional B and T cells and therefore rely solely on their innate immune system (Petrie-Hanson et al.). The College of Veterinary Medicine at MSU maintains a zebrafish colony, and is one of three academic institutions world-wide that maintain rag1 mutant zebrafish and the killifish N. furzeri. The importance of wild-type and mutant zebrafish and the short-lived killifish as biomedical models and the need for monoclonal antibodies to their leukocytes is clear. The availability of a panel of these antibodies will poise these fish to be two of the best models for immunology research.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31108101090100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
0810 - Finfish;

Field Of Science
1090 - Immunology;
Goals / Objectives
To screen commercially available antibodies that are predicted to react with teleost leukocytes and identify those that can be used as specific markers for zebrafish and killifish leukocytes in flow cytometry. Immunological findings gained by using these newly defined tools will be applied to aquaculture species such as channel catfish and tilapia. Continued use and characterization of the zebrafish immunology and infectious disease model requires the establishment of a panel of leukocyte-specific monoclonal antibodies. Only a few zebrafish leukocyte monoclonal antibodies are available. We have recently acquired two hybridoma cell lines developed against two catfish leukocyte markers that also bind specifically to their respective receptors in zebrafish. Our proposal to augment our panel is simple in concept and approach: 1) by means of a personal request or commercial purchase, obtain antibodies to carp, catfish, zebrafish and mummichog (a different genus of killifish) and 2) evaluate them by flow cytometry and immunohistochemistry. We expect to identify critical basic antibodies allowing flow cytometric evaluation of immune responses in zebrafish and killifish.
Project Methods
Part 1: We will request antibodies to carp, catfish, zebrafish and mummichog leukocytes directly from researchers and we will purchase antibodies that show a high probability of utility in zebrafish and killifish. There are two companies that market a broad spectrum of antibodies that may be useful for zebrafish and killifish flow cytometry research. One is AnaSpec, Inc. (www.anaspec.com) which markets a wide variety of rabbit polyclonal antibodies raised to specific zebrafish peptides. These antibodies are shown to have specificity to the peptide antigen, some have been shown to have reactivity in Western blots but few have been evaluated by flow cytometry. Peptides included in this list are IgM heavy chain, IgZ heavy chain, T cell receptor, MHCII, CD4, and CD8. The second company, Aviva Systems Biology (http://www.avivasysbio.com) offers antibodies to more than 7,000 different protein targets and releases more than 200 new antibodies every month. They provide predictive reactivity data for species with genomic sequence information available including many that are predicted to be cross reactive with zebrafish. Part 2: Monoclonal antibody screening and characterization Leukocyte specific staining will be initially evaluated by observing staining characteristics in lymphocyte deficient (Rag 1-) and wild type zebrafish and evaluating morphological characteristics of labeled cells. Fish rearing Rag1-/- zebrafish, wild-type zebrafish and killifish will be propagated and reared following our protocols, and will be maintained under SPF hatchery housing until sampled. Flow cytometry Fish will be anesthetized, the kidney removed, placed in 0.5 ml tissue culture media, dissociated (as described (Hohn et al. 2009, Petrie-Hanson et al. 2009), stained with the antibodies to be tested and evaluated on the flow cytometer. After antibodies with group specificity are identified they will be used in cell sorting experiments where stained cells are collected, prepared by cytospin, Wright's stained and evaluated morphometrically. Because the morphology differentiating the T and B lymphocytes has not been done with zebrafish, for the lymphocyte specific antibodies we will also evaluate their specificity using rtPCR for immunoglobulin and T-cell receptor to distinguish T and B lymphocytes on sorted cells from normal zebrafish using the methods previously reported (Hohn et al. 2009, Petrie-Hanson et al. 2009). We will also perform initial electron microscopic evaluations on the subpopulations identified by different monoclonals.

Progress 09/01/12 to 08/31/14

Outputs
Target Audience: The targeted audience on this project was fish immunology researchers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One graduate student was trained in immunological techniques and flow cytometry analysis techniques. How have the results been disseminated to communities of interest? Results have been disseminated to commuities of interest by professional meetings and emails. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The major goals were to determine if commercially available leukocytes to conserved leukocyte surface markers would cross-label and identify Natural Killer cells in fish. Antibodies that were tested: 9C9, 5C6, human CD3, CD4, CD5, CD14, CD16, CD19, CD20, CD25, CD56. Fish that were tested: zebrafish, South African killifish, channel catfish, Atlantic sturgeon. The monoclonal antibody 5C6 labeled a subpopulation of zebrafish and channel catfish leukocytes. They did not label any leukocyte populations of the SA killifish.

Publications


    Progress 01/01/13 to 09/30/13

    Outputs
    Target Audience: The target audience is fish immunology researchers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? The determination that commercially available antibodies for leukocyte markers do not cross-label fish leukocytes. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? Accomplishments Antibodies that were tested: 9C9, 5C6, human CD3, CD4, CD5, CD14, CD16, CD19, CD20, CD25, CD56. Fish that were tested: zebrafish, South African killifish, channel catfish, Atlantic sturgeon. The monoclonal antibody 5C6 labeled a subpopulation of zebrafish and channel catfish leukocytes. They did not label any leukocyte populations of the SA killifish. Other commercial monoclonal antibodies tested included: 1) FlowCellect Mouse viable Treg characterization kit (Millipore): CD25-PE, CD4-PerCP/Cy5.5. The FlowCellect Mouse viable Treg characterization kit antibodies bound to human peripheral blood but didn’t cross-react with any of the tested fish leukocytes. 2) FITC anti-human Lineage Cocktail (BioLegend): CD3, CD14, CD16, CD19, CD20, CD56. FITC anti-human Lineage Cocktail antibodies bound to human peripheral blood but didn’t cross-react with any of the tested fish leukocytes. 3) FlowCellect Human FOXP3 Treg characterization kit (Millipore): CD3-PE/Cy5, CD4-FITC. All antibodies bound to human peripheral blood but didn’t cross-react with any of the tested fish leukocytes. 4) FlowCellect Human Lymphocyte ZAP-70 characterization kit for CD5-FITC, CD19-APC. All antibodies bound to human peripheral blood but didn’t cross-react with any of the tested fish leukocytes. 5) FlowCellect Human NK cell characterization kit for CD3-APC, CD16-FITC, CD56-PE. All antibodies bound to human peripheral blood but didn’t cross-react with any of the tested fish leukocytes. None of these labeled any leukocyte populations of zebrafish, South African killifish, channel catfish, or Atlantic sturgeon.

    Publications


      Progress 01/01/12 to 12/31/12

      Outputs
      OUTPUTS: Monoclonal antibody 5C6 is binding to lymphocytes. The recommended dilution of mAB 1:10 results in a good signal. There is a gradual decrfease of Median Fluorescent Intensity (MFI) with dilution. In the lymphoid gate, wild type fish have an average of 502 positive cells out of 10,000 cells. The T and B cell deficient mutants have an average of 214 positive cells out of 10,000 cells. Vaccinated mutants have an average of 144 positive cells out of 10,000 cells. Nothobranchius furzeri is a South African fish that inhabits small ponds that dry seasonally. While different populations of N. furzeri show large variations in longevity, the laboratory strain, "Gona-re-Zhou" (GRZ) has a life span of three months when raised at 28C. It has been suggested that within that time period, the physiological processes of these fish progress through the natural changes associated with aging. This senescence would make this fish a critical laboratory model for investigations involving the aging immune and neuro-endocrine systems. Additionally, their unique habitat and reproductive requirements are extremely difficult to mimic in a laboratory setting. We have successfully spawned and reared these fish under different temperature regimes. Differential counts of peripheral blood leukocytes revealed younger fish had higher lymphocyte counts compared to older fish, 68% to 16%, respectively. The neutrophil was the predominate phagocyte. Flow cytometric analyses of kidney hematopoietic tissue additionally demonstrated a decreasing lymphocyte population and increasing phagocyte population with age. PARTICIPANTS: Dr. Lora Petrie-Hanson, Dr. Claudia Hohn, Ralph Tran, VMS graduate student TARGET AUDIENCES: Fish industry and Immunologists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

      Impacts
      The monoclonal antibody 5C6 can be used effectively for labeling some lymphocyte-like cells in the zebrafish. In killifish, differential counts of peripheral blood leukocytes revealed younger fish had higher lymphocyte counts compared to older fish, 68% to 16%, respectively. The neutrophil was the predominate phagocyte. Flow cytometric analyses of kidney hematopoietic tissue additionally demonstrated a decreasing lymphocyte population and increasing phagocyte population with age.

      Publications

      • No publications reported this period