Methodology used in Immunology

Transcription

1 Methodology used in Immunology

2 Antigen reacts with antibody to form an aggregate or precipitate

3 The Precipitin Reaction Equivalent concentration of antigen & antibody forms a precipitate

4 Blood Typing If the Rh antigen is also present, the person is Rh +, if they do not have the Rh antigen, they are Rh -

5 Coombs Anti-globulin test Hemolytic anaemia Detects anti-rh antibody (IgG) Direct and Indirect

6 human

7 Enzyme Linked Immuno Sorbent Assay

8 Enzyme Linked Immuno Sorbent Assay Antigenic site = epitope = Antigenic determinant 1 Antibody can be polyclonal or monoclonal MUST be Specific binding

9 Colorimetric detection Antibody used for detection is Labeled with biotin Biotin binds to streptavidin that is linked to an enzyme (HRP) HRP catalyzes the substrate (NBT/BCIP) to yield a color.

10 Detection of DIG-labeled nucleotides/antibody is done using anti-dig antibodies conjugated to alkaline phosphatase. When a substrate such as CSPD (dioxetane) is added, alkaline phosphatase breaks down the substrate, which emits a photon of light. Fluorescently-tagged DNA probes or antibodies have the advantage that no enzymatic reaction is necessary for detection. Dye-conjugated nucleotides are incorporated into DNA during labeling eg. PCR labeling.

11 Antinuclear antibodies ANA They are auto-antibodies against nuclear components that can be detected by indirect immunofluorescence on cell nuclei. The diffuse staining pattern is the commonest, associated with SLE The speckled pattern is associated with Mixed Connective Tissue Disease The nucleolar pattern is associated with sclerosis Anticentromere staining is associated with a subgroup of sclerosis

12 The ELISPOT assay Variation of ELISA Quantitates the number of cells producing antibodies Identifies cells producing specific cytokines

13 Western Blot for detection of anti HIV antibodies A positive test shows the presence of p24 AND either gp 120 or gp 160 C + Neg

14 Immunoprecipitation For detection of proteins using specific antibodies Using an antibody to isolate and purify a protein from a whole cell lysate. Normally you will only purify the protein the antibody recognizes. Any additional proteins that co-purify are candidates for interacting proteins.

15 Immunoprecipitation affinity purification based on isolation of Ag-Ab complexes analyze by gel electrophoresis isolation of Ag-Ab complexes Bacterial proteins that bind IgG (Fc): protein A (Staphylococcus aureus) protein G (Streptococcus) binds more species and subclasses

16 Separation of Cells on a Ficoll Hypaque density gradient

17 Separation of cells using magnetic beads Magnetic beads coated with anti CD45 antibody Mouse Spleen Magnetic column Other cells CD45 Cells

18 Immunofluorescence and Immunohistochemistry PBMC stained with Anti IL-2 FITC PBMC stained with Anti IL-2 Peroxidase/DAB IL-2 producing cells Stained in situ

19

20 Flow Cytometry

21

22 Light Scatter Gating Side Scatter Projection Forward Scatter (measures size) Neutrophils Monocytes Lymphocytes Scale Degree Scatter (measures complexity)

23

24 CD45 vs SS

25

26 Normal Loss of CD4+ T cells HIV??

27 Immunophenotype of a patient with CLL Mostly CD45+ Indicating leukocytes CD14 -No monocytes CD5+ and CD20+ Indicating CD5+ B lymphocytes Mostly CD23+ B cells (also excludes mantle cell) CD19+ and Ig kappa + Indicating B cell monoclonality

28 FLOW CYTOMETRY - Lymph Node, FNA EARLY B-CELL CD % PAN B CD % CD20 INTENSITY 2+ PAN T/B CLL CD % CD19+/CD5+ CELLS 41.4 % ACTIVATED B-CELL CD % SIG KAPPA 97.5 % SIG LAMBDA 0.5 % CD % CD19+/CD % CD79b 20.5 % B SUBSET, FMC % CALLA CD % CD19+/CD10+ CELLS 0.5 % T-CELL ANTIGEN CD % INTERPRETATION B-cells express CD5, CD19, CD20, CD23 and monotypic immunoglobulin KAPPA light chain, c/w B-CLL/SLL. FSC-Height CD19 PERCP SSC-Height CD19 PERCP CD5 PE 44.1 % expression of kappa & lambda in CD19+/CD5+ lymphocytes % % 10.4 % CD19 FITC KAPPA FITC LAMBDA PE

29

30 Fluorescence Activated Cell Sorting

31 Some Applications of Flow Cytometry Surface antigens. Lymphocyte sub-sets. Immunophenotyping : peripheral blood & bone marrow. Leukemia diagnosis Stem cell counting. (CD34). Cytokine receptors & intracellular flow cytometry DNA Analysis (Ploidy) Apoptosis

32 Cell Cycle and DNA analysis

33 DNA Ploidy

34

35

36 Detection of Apoptosis using Annexin V Normal cell Apoptotic cell

37 Detection of Apoptosis using Annexin V

38 Detection of Apoptosis by TUNEL

39 Functional assays for lymphocytes

40 Measurement of Cell Proliferation

41 Measurement of Cytotoxicity

42 Hemacytometer: Use 10X objective Count the number of cells in squares 1-4; 1 determine average # cells/square Average # cells/square X 10 4 = # cells/ml

43 Parameter Phagocytosis Lymphocyte subsets Lymphocyte activity Cytokines Immunoglobulin levels Complement CRP, lysozyme and other humoral factors Methods Migration and chemotaxis, detection of respiratory burst Flow cytometry, immunohistochemistry Lymphocyte proliferation test, Cytotoxicity assay, mixed lymphocyte reaction Bioassay, ELISA, PCR Radial immunodiffusion RID, ELISA Hemolytic activity, ELISA ELISA, turbidimetry

44

45 Liquid Bead arrays Immunoassays (e.g., ELISA) Protein-protein interaction assays (e.g., interaction mapping) Enzyme assays (e.g., kinase, phosphatase, and protease assays) Receptor-ligand assays Protein-nucleic acid assays

46 The Luminex machine

47 Luminex Multiplexed Assay Principle 5.6 micron polystyrene beads internally dyed with red and infrared fluorophores. 100 bead sets available, each with different dye ratios and each with a unique spectral signature determined by its red/infrared ratio. Capture reagents (e.g., antibodies, oligos, peptides, receptors) chemically coupled to beads. Small size/surface area and 3-dimensional exposure of the beads allows for nearly solution-phase kinetics. The red laser identifies which assay is carried on the bead, while the green laser quantifies the analyte. UC SF

48 Principles of the Luminex Technology

49 Beadlyte Immunoassay format for the Luminex Machine. After In Free The The A After In Free The assays The biotinylated, assays phycoerythrin immune-complex/ excess primary Microsphere phycoerythrin immune-complex/ another excess another with Microsphere with streptavidin-pe wash primary antibody streptavidin-pe wash high biotinylated, analyte high is is is specific is step analyte a step analyte excited excited Streptavidin numbers binds by microsphere analyte binds binds 5.6mM for to specific Streptavidin numbers binds by microsphere the 5.6mM for to the the to reporter specific to analyte specific polystyrene reporter the specific analyte the excess excess non-specifically PE is non-specifically PE is laser then polystyrene reporter analyte islaser then is analyte isantibody biotinylated biotinylated added added excited and bead excited and to bead no is to the reporter bound emits by is with conjugated crossreactivity added the reporter bound emits by the assay. the assay. a ****** The with conjugated crossreactivity added two biotinylated two to ****** biotinylated fluorescence antibodies fluorescence to fluorescent antibodies the fluorescent the to The laser. biotinylated to assay laser. assay the with biotinylated reporter The bind reporter with bead bead other after reporter The bind which to other dyes which bead bead to the antibody is specific after dyes incorporated surface analytes another reporter antibody is specific quantified quantified Strep-PE Strep-PE emmission antibody leading incorporated surface analytes another by wash by occurs. emmission antibody leading by binds wash occurs. into by into amine step. the a the to nonspecific amplification. reader. quantified different coupling one specific amplification. reader. quantified of of the the manner. four amine step. it to binds non- Luminex a is to it Luminex is signal signal to one different coupling ratios. manner. by reaction by four the the available available luminex luminex sites. and sites. and the the bead bead ratios. reaction identified. identified.

50 TSPE primers are chimeric oligos with a virus-specific sequence and Tag sequence complimentary to anti-tag sequence bound to microbead TSPE Reaction TSPE primers are extended by Taq polymerase and incorporate biotin reporter molecule Biotin Biotin Biotinylated amplicons are detected with a fluorescent SA- Phycoerythrin conjugate and signals are read using the Luminex instrument Tag > Anti- Tag

51 Bead Hybridization

52 UC SF Aβ 40 Aβ 42 BDNF DR-5 EGF ENA-78 Eotaxin Fatty Acid Binding Protein FGF-basic G-CSF GCP-2 GM-CSF GRO α GRO-KC HGF I-TAC ICAM-1 IFN-α IFN-γ Cytokines IL-1α IL-1β IL-1ra IL-1ra/IL-1F3 IL-2 IL-3 IL-4 IL-5 IL-6 IL-7 IL-8 IL-9 IL-10 IL-11 IL-12 p40/p70 IL-13 IL-15 IL-16 IL-17 IL-18 IP-10 JE/MCP-1 KC KC/GROa LIF Lymphotacin M-CSF MCP-1 MCP-1(MCAF) MCP-2 MCP-3 MCP-5 MDC MIG MIP-1α MIP-1β MIP-1γ MIP-2 MIP-3 β OSM PDGF-BB RANTES Rb (pt821) Rb (total) Rb pspt249/252 Tau (ps214) Tau (ps396) Tau (total) Tissue Factor TNF-α TNF-β TNF-RI TNF-RII TNF-SF5 VCAM-1 VEGF

53

54 Immune Cell Function Test Measures the Net State of Immune Function based on lymphocyte proliferation assay. Used to Monitor Therapy Response Immune Function is Independent of CD4 Count

55 Immuknow Incubate hrs. Wash Lymphocyte Stimulation w/pha ATP ATP ATP ATP detection reagents Magnetic separation of CD4 cells. Luminometer Cell lysis to release ATP Measure light intensity J. Britz et. al., In Vitro CMI: Rapid Assay for Measuring Cell-Mediated Immunity, CRC Press, 2002

56 ImmuKnow : Stratification of Immune Response Immune Response (ATP ng/ml) P < Strong Moderate Low 0 Healthy Adults (n=155) Stable Transplant Recipients (n=127) FDA Claim: ImmuKnow is used for the detection of cellmediated immunity in an immunosuppressed population. Kowalski, R. et. al., Clinical Transplantation:17:77-88, 2003

57 Immune Function Correlates With Clinical Outcomes Immune Response (ATP ng/ml) P < P < Strong Moderate Low 0 Healthy Rejection Stable Infection (n=155) (n=39) (n=504) (n=66) Meta-Analysis: NIDDK, UCLA, JHU, UPMC, NZTI, LifeLink, UTMB, UAB, UMD, INOVA

58

59 THANK YOU