Immuno Test 2 (Part 3) Flashcards
Which labeling is best?
1) Radioactive:
- MOST SENSITIVE
- Represent HEALTH HAZARDS
2) Fluorescent Labeling:
- LESS SENSITIVE
- NOT HAZARDOUS
Most current lab techniques use Fluorescent Labels:
1) Fluorescein Isothiocyanate (FITC, Green Light)
2) Phycoerythrin (PE, Red Protein from Cyanobacteria)
Indirect Immunostaining
- First Ab binds to Antigen on slide
- Second Ab (Anti-Ab) binds to the Fc portion of the First Ab and has a fluorescent label that will release a color when the Antigen is detected
Basis of Agglutination
- Agglutination is a REACTION between ANTIGEN-ANTIBODY
which, due to COLLOIDAL INSTABILITY, may lead to PRECIPITATION!!!! - The formation of the complexes lead to an INCREASE IN LIGHT SCATTERING
- Can be observed with the NAKED EYE or Monitored PHOTOMETRICALLY using TURBIDIMETRIC or NEPHELOMETRIC Detection!!!!!
Requirement: Have to be MULTIPLE SITES OF INTERACTION on both Antigen and Antibody
Divalent Abs: At Least TWO AG-BINDING SITES
POLYVALENT AG: A molecule with MULTIPLE EPITOPES and is capable of reacting with several Ab molecules!!
CROSS LINKING between Polyvalent Ag occurs
- As the Crosslinking progresses, THE COMPLEX IS LESS STABLE and AGGLUTINATION takes place
- Eventually the COMPLEX PRECIPITATES
Heidelberger-Kendall Curve for Immunoprecipitaiton
- Increasing amounts of C-reactive protein are added into a solution of Alpha CRP Abs
3 areas on curve:
I: [Ab] > [Ag]
II: [Ab] = [Ag] - THE OPTIMUM RATIO
III: [Ab]
- Polyclonal ANTISERUM is usually USED FOR IMMUNOAGGLUTINATION
- Monoclonal Ab IS NOT EFFECTIVE unless there are several identical epitopes on the macromolecule
- MAXIMUM SPECIFIC AGGLUTINATION rate is around NEUTRAL pH:
- Below 6 pH, many proteins are SELF-AGGLUTINATE (pI- isoelectric point)
- Need neutral pH because proteins can precipitate at their isoelectric point
- Below 6 pH, many proteins are SELF-AGGLUTINATE (pI- isoelectric point)
- Reaction is TEMPERATURE DEPENDENT- the rate of Ag-Ab reaction is enhanced by INCREASING the reaction TEMPERATURE
Direct Agglutination Assay
- Direct Assay is used only with POLYVALENT AGS (protein and bacteria)
- Ideally, the reaction should be MEASURED IMMEDIATELY after mixing sample with Reagent
- Detection is PERFORMED by using either TURBIDIMETRIC or NEPHELOMETRIC monitoring
- The DIRECT ASSAY has the potential for an ERROR of High [Ag], Ag EXCESS or [Ab]
Indirect Agglutination Assay
You will ALWAYS DETECT!!!!!!
- Started with Optimal Commercially provided Ag and the Ab starts to break apart!!!!!
- A COMPETITIVE ASSAY base on a POLYVALENT AG and AB reagents provided in the kit:
- [Ab] = [Ag] - The present of TEST Ag INHIBITS Agglutination because:
- [Ab]
Ouchterlony Immunodiffusion
- IMMUNODIFFUSION is one of the earliest and simplest METHODS of STUDYING AB-AG REACTIONS
- Involves the MIGRATION of AB and AG towards each other in a SEMISOLID AGAR GEL!!!!
- A VISUAL PRECIPITATE is formed where the CONCENTRATIONS become EQUIVALENT!!!!
Steps:
1) Antiserum (Abs) is placed in the Central Well
2) Each Ag is placed in the surrounding wells
3) Place the Slide flat in a humid contained and INCUBATE OVERNIGHT at Room Temperature
- Ags and Abs will migrate towards each other. They will start with the low concentration and diffusion of Ab and Ag will take place. They will diffuse more and more until the CONCENTRATIONS are EQUAL, so then the AB and AG are NOT MOVING and forma 3D structure aka a COMPLEX!!!
Ouchterlony Immunodiffusion Three Characteristics
1) Identity:
- (1) = (2)
- There are NO LINES jutting out of the Complex
- These Ags are the same
2) Partial Identity:
- There is ONE LINE jutting out of the Complex toward one of the wells with different Ag
- There is a partial match in the Ags in these wells
3) Non-Identity:
- There are TWO LINES jutting out of the Complex towards each well
- There is no match between the Identities of these Ags
Techniques For Immunoelectrophoresis
- “Rocket” Imuno-Electrophoresis is used for Rapid Ag QUANTITATION
- Agarose gel contains ANTISERUM (1%)
- Into WELLS punched at one end, Load AG SOLUTIONS
- RUN GEL with wells closest to the NEGATIVE ELECTRODE
- As the AG PROTEINS enter the gel, they form a CONCENTRATION GRADIENT, which at some point gives the proper concentration for PRECIPITATION with the Ab IN THE GEL
- The result is that each sample gives a “ROCKET” THE LENGTH of which is PROPOTIONAL TO THE CONCENTRATION OF Ag in the sample!!!!!!
- Precipitate CAN BE STAINED with Coomassie Blue R-250!!!
- The PRECIPITATE APPEARS as a ARROWHEAD or ROCKET
-
* SEMIQUANTITATIVE*!!!!!!!!!!!!!!!
- Semiquantitative estimate of how much protein (Ag) is in the sample
Immunoelectrophoresis
2 Stage Process:
1) Electrophoresis is conducted in the first stage
2) Immunoprecipitation using Abs against specific proteins in the second stage
Step I:
- Electrophoresis
- Serum divided into 5 fractions:
- Alpha1 Globulins
- Alpha2 Globulins
- Beta Globulins
- Gamma Globulins
- Albumin
- A major PURPOSE of IEP is to Identify MONOCLONAL IMMUNOGLOBULINS (M-COMPONENT) associated with Myeloma (Plasma Cell Neoplasia)
- M- COMPONENTS are identified by an ABNORMAL BAND in the GAMMA- GLOBULIN REGION (Occasionally in the Beta)!!!!!!!!!!
- Resolution is IMPROVED STAINING the PROTEINS
Step II:
- Electrophoretic SEPARATION of the SAME SAMPLE is conducted in MULTIPLE LANES along with CONTROL normal serum
- Anti-Ig; Anti-IgA, and Anti-IgM antiserum is placed in Preformed troughs cut parallel to each lane
- The gels are allowed to INCUBATE for 24-48 hours, while the Antisera and Ag diffuse into the GEL
- PRECIPITATE DEVELOPS, fixing the Immunoglobulins in the FORM OF AN ARC!!!!!!
Classical IEP
- Have patient and control serum in well next to each other
- There is a T ZONE between the two wells that is filled up with either IgG, IgA, or IgM!!!!!
- Precipitate lines form between the T Zones and the C and P Lanes!!!!
- A HEAVIER LINE denotes that there is EXCESS of that IMMUNOGLOBULIN in the PATIENT relative to the Control
Hemagglutination (Coombs) Assays
AGGLUTINATION with the Help of RBC!!!!
- Can be used to DETECT RBC Ags or Abs present in the sample again RBC Ags
- By COUPLING EITHER Ags or Abs to RBC, they can be used as LABELS
- CROSSLINKING occurs when RBCs are mixed with Abs DIRECTED AGAINST AG on RBCs
- The AGGLUTINATE will PRECIPITATE at the bottom of the REACTION WELL
- Can be READILY DETECTED as a DARK RED, bright precipitate, the Assay is called a DIRECT COOMBS TEST!!!
- This approach is used for ABO BLOOD GROUPING!!!
Enzyme- Linked Immunosorbent Assay (ELISA)
QUANTITATIVE MEASURMENT OF CONCENTRATION OF SOLUBLE AGS!!!!!!!!!
- Perfomed in a 96 well microplane
Steps:
1) Wells are coated with a CAPTURE MONOCLONAL Ab, which is SPECIFIC for the AG of INTEREST
2) Test samples with UNKNOWN amounts of Ag are ADDED to the WELLS. TO other wells, a STANDARD of KNOWN CONCENTRATION is ADDED
3) Ag is CAPTURED by AB attached to the well
4) Sample/ Standard is REMOVED by WASHING and DETECTION mAB is ADDED
5) HRP-coupled AB is added and the COLOR REACTION is developed by addition of a SUBSTRATE HRP
6) REACTION IS QUANTIFIED BY READ ON ON A PLATE READER (Spectrophotometer)
DIRECT ELISA: Only 1 Ab binds to the Ag
INDIRECT ELISA: One Ab binds to the Ag, and 2nd AB binds to the 1 Ab
SANDWICH ELISA: Monoclonal AB is in well, then AG is added to bind to Monoclonal Ab. Then 2nd Ab is added to well, to bind to Ag
- CAN HAVE LOW AMOUNT OF AG FOR THIS EXPERIMENT
ELISA Step by Step
1) Bind first Antibody to well of micrometer
2) Add varying among of Antigen
- AG taken in LOW CONCENTRATION and everything else taken in EXCESS (ABs)!!!
3) Remove unbound Ag by washing
4) Add labeled SECOND AB specific for Nonoverlapping Epitopes of Antigen
5) Remove unbound Labeled Second AB by washing; MEASURE AMOUNT of SECOND ANTIBODY BOUND
6) DETERMINE AMOUNT OF BOUND SECOND ANTIBODY as a function of the Concentration of Antigen added (Construction of Standard Curve)
Flow Cytometry
CD Antigens are involved in Cell FUNCTION on the SURFACE
- Specific Expression of CD MOLECULES allows them to be USED AS MARKERS of particular cell types:
1) ALL LEUKOCYTES = CD45!!!!!!!!!!!!!
2) ALL T CELLS = CD3
3) ALL B CELLS = CD19
Markers of Immune Cells
CD = Clusters of Differentiation
CD3 = T Cells
CD4 = T Helper Cells
CD8 = Cytolytic T Cells
CD14 = Monocytes
CD19 = B Cells
CD25 = T Regulatory Cells
CD56 = NK CELLS
CD Antigens in Cell Separation
- A particular type can be used to identify, to count, to separate out cells from a picture of different cells (Ex: Blood)
FLUORESCENCE-ACTIVATED CELL SORTING (FACS)
Fluorescence Activated Cell Sorting
- Cell suspension is first labeled with one or more Abs (like ANTI-CD3 and ANTI-CD4) that have been coupled with Fluorescent Molecules
- Commonly Antibodies are CONJUGATED with Fluorescein Isothiocyanate (ANTI-CD3- FITC, GREEN light) or with Phycoerythrin (ANTI-CD4-FITC, RED protein from Cyanobacteria)
- The Mixed population of cells is labeled with Fluorescent Ab and then put through the Laser, Detector, and Electrical field
- The Electrical Field causes the Cells that are DIFFERENTLY labeled, to go into DIFFERENT TUBES - Then a computer ANALYZES the products by expression of CD3, CD8, or CD4 for example
Gut Associated Lymphoid Tissue
M Cells:
- Take and Deliver Ags from the outside of the lumen and bring it into the area of Payers Patch where we have DCs, T Cells, and B Cells
- With a supply of Ag they can produce an Immune response LOCALLY!!!!!
- Glycocalyx: Produced mucus so that bacteria cant bind to the epithelial cells
- ACTIN-RICH MICROVILLAE Extensions
- Epithelial cells TIGHT JUNCTIONS
- Apical attached and SECRETED MUCINS that form Glycocalyx
- Production of various ANTIMICROBIAL PEPTIDES
- M (Microfold) Cells overlie Payer’s Patches and Lymphoid Follicles to FACILLITATE LIMUNAL SAMPLING.
- M Cells exhibit MUCIN Secretion and have modified APICAL and BASOLATERAL surfaces to PROMOTE UPTAKE AND TRANSPORT OF LUMINAL CONTENTS - Professional APCs that inhabit the Subepithelial Dome (SED) of the Peyer’s Patches and Lymphoid Follicles
- Specialized DC SUBSETS can also extend Dendrites between the tight junctions of Intestinal Epithelial cells to SAMPLE LUMINAL CONTENTS
Peyer’s Patch:
1) SED (Subepithelial Dome)
2) TDA (T Cell Dependent Area)
Ag Recognition by CD4+ Cells
1) Ag enters through the M Cells and transfer to LOCAL DENDRITIC CELLS
2) DCs might present Ag to T Cells in the PEYER’s PATHC
3) Mainly, Ag-loaded DCs gain access to a DRAINING LYMPH NODE
4) ACTIVATION OF T CELLS occur in the MLNs (Mesenteric Lymph Node)
5) Ag CAN ALSO ENTER THROUGH THE EPITHELIUM covering the villus Lamina Propina. Ag loaded DCs then migrate to the MLNs
6) ENTEROCYTES migth express MHC Class II and act as local APCs
7) Ag-activated CD4+ T Cells leave the MLN in the EFFERENT LYMPH and enter the BLOODSTREAM through the thoracic duct. They exit not the MUCOSA through vessels in the lamina propane. They MAY DISSEMINATE into the Lamina Propina via the PERIPHERAL IMMUNE SYSTEM
8) Ag might also gain Direct access TO THE BLOODSTREAM from the GUT!
9) Blood Ags interact with T Cells in PERIPHERAL LYMPHOID TISSUES
Major Anti-Inflammatory Immune Mechanisms in GI
1) IMMUNE EXCLUSION: limiting the colonization by pathogens; mediated by SECRETORY IgA (sIgA mainly) and sIgM!!!
2) ORAL TOLERANCE: Food proteins and the microbiota suppress Th2 (IgE), Th1 (DTH and IgG), and Th17 responses
Immune Exclusion
- T and B cells are activated by Dendritic Cells in the Lymphoid follicle and then travel to the MESENTERIC LYMPH NODE (AG RECOGNITION!!!!!)
- The T and B cells leave the Lymph Node through the Thoracic Duct to the Effector Site (RESPONSE!!!!!!)
- B cells make IgA, IgM, and IgG while T Cells become CD4+ and CD8+
- IgA and IgM is secreted and goes to the surface of the intestinal mucosa for protection against bacteria
- Oral Tolerance inhibits IgG, IgE, DTH, CD4, and Macrophages
**THIS WHOLE PROCESS TAKES DAYS*!!!!!!!!
Immune Exclusion Steps
1) Naive B and T cells enter GALT via HEVs
2) After being PRIMED to become memory/ effector B and T cells, they MIGRATE to PERIPHERAL BLOOD for subsequent extravasation at MUCOSAL EFFECTOR SITES
3) The migration is DIRECTED BY the local profile of Vascular ADHESION MOLECULES and CHEMOKINES, the endothelial cells thus exerting a local gatekeeper function for Mucosal Immunity
4) The gut LAMINA PROPRIA contain few B Lymphocytes but many J-chain Expressing IgA (dimers) and IgM (pentamers) PLASMA CELLS
5) Secretory sIgA and sIgM in the intestinal mucosa are ACTIVELY TRANSPORTED INOT THE LUMEN
6) The INTRAEPITHELIAL CD4+, CD8+. and gamma/delta T Cells also present
