cytokine immunoassays:  new methods to evaluate steller sea lion immune health

Download Cytokine Immunoassays:   New Methods to Evaluate Steller Sea Lion Immune Health

Post on 19-Jan-2016

25 views

Category:

Documents

0 download

Embed Size (px)

DESCRIPTION

Cytokine Immunoassays:  New Methods to Evaluate Steller Sea Lion Immune Health. Mary Bozza, Research Associate Alaska SeaLife Center. Steller Sea Lion Immune Health. Do Steller sea lions bioaccumulate contaminants to toxic levels? How can we measure toxicity? - PowerPoint PPT Presentation

TRANSCRIPT

  • CytokineImmunoassays: New Methods to Evaluate Steller Sea Lion Immune HealthMary Bozza, Research AssociateAlaska SeaLife Center

  • Steller Sea Lion Immune HealthDo Steller sea lions bioaccumulate contaminants to toxic levels?

    How can we measure toxicity?Toxins can cause immune system disruptionMarine contaminants linked to immunosuppression include: Organochlorines, PCBs, Cadmium

  • Steller Sea Lion Immune Health

    Alaska SeaLife Center Contaminants Project: Endocrinology, Immunology and Toxicology S. Aktkinson, B. Middlebrooks, Q. Li Individual Health Assessment of Steller Sea Lions in Alaska Burek, Beckmen, Rea, and Gellat

  • Project Goals:Develop assays to measure Steller sea lion cytokines in serum and from white blood cells in vitroEvaluate immunocompetence:Establish baseline cytokine profiles to assess immune health of individual Steller sea lionsCorrelate cytokine profiles withimmune and overall health parameters body burdens of contaminants to determine toxicity

  • What are Cytokines?

    Proteins secreted by immune cells in response to inflammation or other stress

    Called interleukins, they act as chemical messengers to regulate inflammation

    Routinely used to study other mammalian immune systems including human & mouse

    Commercially available reagents may be available to measure Steller sea lion cytokines if appropriate assays are developed

  • An immunoassay uses an antibody to recognize the protein of interest; the concentration of the antibody-protein complex is measured

    Challenge:Antibodies to Steller sea lion cytokines do not exist!Proteins are not available to generate or validate antibodies

    Solution:Make Steller sea lion protein using molecular cloning techniquesUse the protein to validate existing cross-reactive antibodies to develop an assay

  • Project Goals:Develop assays to measure Steller sea lion cytokines in serum and from white blood cells in vitroII. Evaluate immunocompetence:Establish baseline cytokine profiles to assess immune health of individual Steller sea lionsCorrelate cytokine profiles withimmune and overall health parameters body burdens of contaminants to determine toxicity

  • 6. Validate assay using cross-reactive antibodies1. SSL immune cells cultured in vitro with a bacterial extract (LPS)2. Isolate mRNA

    4. Clone and sequence full-length cytokine genes by PCR3. Construct a cDNA library5. Protein ExpressionGoal I: Develop Assays

  • Step 1. LPS Stimulation of Mononuclear CellsCollect whole bloodPlace cells in culture dish, add LPS (lipopolysaccharide) to stimulate immune responseCentrifuge blood over a Ficoll-Hypaque gradient to isolate mononuclear cellsCell SignalingTranscriptionStep 2. Isolate mRNA

    TranslationSecretion

  • Step 2: Isolate mRNASize fractionation by electrophoresis (1% agarose gel)Total RNA from HS LPS stimulated mononuclear cellsPolyA+ Control RNA (human placenta)

    1 2

  • Step 3. Construct a cDNA LibraryIsolate mRNA transcripts from tissues or cells. Convert transcripts into double-stranded DNA fragments called cDNA.

  • Insert cDNA into plasmid vectors that can replicate in bacteria.Introduce vectors into bacteria.Amplify library.

  • Advantages of a cDNA LibraryContains only protein-coding DNA (genes)Represents the entire collection of genes expressed in cell or tissue typeVector with inserted gene can be used to produce proteinCan amplify and store the library for long term useClone many other genes from stimulated white blood cell cDNA libraryNew libraries from Steller sea lion cells or cell linesNew libraries from other species cells or cell lines

  • 6. Validate assay using cross-reactive antibodies1. SSL mononuclear cells cultured in vitro with a bacterial extract (LPS)2. Isolate mRNA

    4. Clone and sequence full-length cytokine genes by PCR3. Construct a cDNA library5. Protein Expression

  • Step 7. Assay DevelopmentELISA (enzyme linked immunosorbent assay) or RIA (radioimmunoassay)Test commercially available antibodies for binding to proteinValidate assay using purified proteinValidate assay using serum

  • Ways to Use New Assays

  • Ways to Use New AssaysCollect blood samplesCytokine Profiles by RT-PCR(Inflammation, Infection or Disease)Cytokine ELISAs(Inflammation, Infection or Disease)Stimulate WBC in vitro(Immunocompetence)Cytokine Profiles by RT-PCRCytokine ELISAsWhite blood cellsSerum

  • Steller Sea Lion Immune HealthDo Steller sea lions bioaccumulate contaminants to toxic levels?

    How can we measure toxicity?Toxins can cause immune system disruptionMarine contaminants linked to immunosuppression include: Organochlorines, PCBs, Cadmium

  • Whats next?LPS stimulation donecDNA library is being constructedNext step: PCR CloningCollaborationVectors, methods for protein production Which genes to clone, proteins to assay?Additional libraries?

  • AcknowledgementsDr. Shannon AtkinsonDon CalkinsDr. Jo-Ann MellishASLC Mammal Dept.ASLC Veterinary Services Dept.Carol StephensBlood Donors: Woody, Kiska, Sugar, Tina and PenderJon MorelandLizabeth MoundalexisAnnette DAlessandro & Angie Steeves

Recommended

View more >