Lecture 10 Flashcards
Immunological Assays
Immunological Assay Uses
- Evaluate immune status of patient
- Diagnosis of disease
Agglutination
Clumping of aggregates visible to naked eye
Types of Agglutinations
- Direct
2. Indirect
Direct Agglutination
- Aka DAT
- Agglutination of pathogen cells
- Agglutination of RBCs based on antigens
Indirect Agglutinaiton
- Agglutination caused by antigen or antibody coated beads
- Agglutination with RBCs sensitized with antigen or coated with IgG
Tests for pathogen or antibodies in the patient’s blood
Leishmaniasis
- DAT
- Analysis of antibody in patient sera through ability to agglutinate parasite
- Antigen - killed promastigote parasites
- Antibody - serial dilutions of patient sera
Hemagglutination
- Another DAT example
- Agglutination of bloods due to non-compatible blood types
Additional DAT Example
- DAT
- Influenza/Mumps agglutinating
- RBCs infected with virus causes a positive microtiter reaction, but this doesn’t specify WHICH virus
Hemagglutination Inhibiting Test
Series of test to identify which virus is infecting the patient. DAT example
Beta-Hemolytic Streptococci
- Example of Indirect, Antigen detection
- Lancefield typing
- Groups A-H
- Beads covered with antibodies for the different groups to identify the unknown pathogen cultures group
Rheumatoid Factor
- Example of Indirect, Antibody detection
- Auto-antibody that binds with other antibody (Fc protein)
- 80% of patients of rheumatoid arthritis (Type III Hypersensitivity that effects joints)
Enzyme Immunoassays
- Aka EIA
- Antigen-antibody binding still is focus
- Antibody has enzyme attached to Fc portion
- Looking at a slice of tissue for a reaction
- Substrate for enzymes provides color reaction
- Clinical uses - tissue immunohistochemistry & Enzyme-linked Immunosorbent Assay (ELISA)
EIA Example
Anti-CD8 Antibody conjugation with enzyme
ELISA
- Can be direct, indirect, or capture assay (“Sandwich Assay”)
- Positive control, negative control, and Patients are listed on test.
Immunochromatographic
- Lateral flow tests (strip tests)
- Point-of-care or beside assays
- Easy to perform and easy reaction analysis
- Runs analyte along strip to get antibody connection, capillary flow, and then test line and lastly control line
- Both test and control line must react for test to be a valid positive
- Used for pregnancy tests, STD tests, HIV tests, etc.
Flourescent Antibody Techniques
- Antigens are attached to surface
- Patients serum is run over it (if antibodies are present then they will bind to antigen)
- Secondary antibody added with fluorescent tag to be seen under fluorescent lighting
Immunoblots: Western Blots
- Proteins separated in gel by electric current
- Transferred to nitrocellulose membrane
- Primary antibody used to detect protein of interest
- Secondary antibody tagged with radioactive isotope, fluorophere, or enzyme
Cellular Immunity Assays
- Blood count - absolute or ratios
- Culture lymphocutes from patients: can then expose to different immunogens or assess their activation or functionality (proliferation through mitosis or production/release of cytokines or chemokines)
Analysis for Neutrophils
- Blood count assessment: absolute and shift to left
- Neutrophil function - oxidative burst and protein analysis
Assays for Complement
- Hemolytic activity- antibodies sensitized blood cells = target, defects or absence of 1+ complement leads to a “-“ result
- Complement component analysis - identification of the specific component involved
