Immunological Procedures Flashcards
Predominant Specimen For Immunological Testing
And some others
Serum: includes everything excluding coagulation proteins
Other: urine, CSF, tissue
Potential Techniques/Assays That Can Be Performed
4
- Agglutination/precipitation
- ELISAs
- PCR, western/southern blots
- Flow Cyto
Antibodies: The Two Kinds
Monoclonal: purified, cloned from a single cell, bind to specific antigen and specific epitope on that antigen
Polyclonal: a bunch of antibodies from several cells that bind the same antigen, but probably multiple epitopes
Production of Monoclonal Antibodies
- Immunization of mouse, specific antigen
- Harvest spleen cells - combine with myeloma cells with polyethylene glycol = produces hybridoma
- Placed in a culture that allows them to grow
- Screened for presence of antibody
- Permanent and uniform supply of monoclonal antibodies
Note: can also be made recombinantly
Applications for Monoclonal Antibodies
Diagnostic vs therapeutic
Diagnostic
- ID and quantify hormones, other biomarkers
- Typing tissue, blood
- ID infectious agents, tumor antigens, autoantibodies
Therapeutic
- Opsonization to destroy disease causing microbes
- Cancer treatment
- Immunosuppression of organ transplant
Electrophoresis: Basics
what it is, uses, types of sample, examples
Separates molecules according to electrical charge
Use: ID and characterize serum proteins
Sample types: serum, CSF, urine
Examples: serum protein electrophoresis, classical immunoelectrophoresis, immunofixation electrophoresis
Electrophoresis: Serum Protein Electrophoresis
purposes, main plasma proteins (5)
Purpose: ID presence of abnormal proteins, ID absence of normal proteins, determine when different groups of proteins are present in high or low levels
Main plasma proteins: albumin, alpha 1, alpha 2, beta, gamma globulins
Precipitation and Agglutination
What, and for both
Purpose: visible expression of antigens and antibodies
Precipitation: aggregation of soluble test antigens and antibody to produce insoluble complex
Agglutination: aggregation of cell-bound antigens like beads or RBCs with antigen when an antibody is present
important!
Precipitation: In Detail
affinity vs. avidity, how it affects complex
Affinity: initial force of attraction
Avidity: strength of binding
How does that affect complex: higher affinity/avidity = more complexes = more sensitivity
- Depends on relative proportions of antigen and antibody present, optimum precipitation in the zone of equivalence
Zones of Antibody-Antigen Complexes
3
Prozone: antibody excess, no precipitation, false negatives, can dilute antibody serum to fix
Equivalence zone: good ratio of antigen:antibody
Postzone: excess antigen, no precipitation, small clumps
Precipitation: Detection of Precipitation
dependent on what (2)
Fluid matrix and solid matrix
Precipitation Detection: Fluid Matrix Basics
2 things u can measure
- Turbidimetry: measure turbidity of solution, spectrophotometer
- Nephelometry: measures light scattered at an angle from the beam as its passed through the solution
Precipitation Detection: Fluid Matrix - Turbidimetry and Nephelometry
principle, difference, clinical
Basic principle: light enters the turbid cuvette
- Turbidimeter measures amount of transmitted light through solution
- Nephelometer measures amount of scattered light
Difference: nephelometry is more sensitive
Clinical
- Turbidimetry: protein and bacteria
- Nephelometry: immunoglobulins, serum proteins
Agglutination Basics
what, quantifying
Lattice formation from cell-bound antigen
Seroconversion: titer, highest dilution where agglutination occurs
- Too much antibody, does not reach end point
Labeled Immunoassay: Basics
what, types
Antibodies and antigen complexes, generates measureable results
- presence of antigen or antiody can be measured
Types: enzyme imunnoassay, chemiluminescence assay, immunofluorescence assay
