Lab techniques Flashcards
Direct Elisa
- Sample containing antigen is coated onto the plate well and the labeled antibody of known specificity is added
Indirect Elisa
- Most common
- Samples added to wells coated with a known antigen
- Antibodies in the sample that bind to the antigen are detected using enzyme labeled anti human IgG
Sandwich (capture) Elisa
- Plate wells are coated with a capture antibody targeting a specific antigen
Inhibition Elisa
- Utilizes competition for labeled antibody between sample and well wall antigen
Advantages to Elisa
- Rapid
- Automatic
- Multiple samples
- Objective
Disadvantages to Elisa
- Less specific than other assays
Immunoprecipitation
- Preciptating a protein antigen out of solution using an antibody that specifically binds to that particular protein
- Can be used to isolate and concentrate a particular protein from a sample containing thousands of proteins
Western blotting
- Protein antigens are separated based on molecular weight by electrophoresis
- The separated antigen is blotted onto a nitrocellulose membrane that is then intubated with patient or control serum
- Bound antibody is identified by use of labeled anti human IgG
- Antibody present in the patient serum is identified by comparison with control serum results
Advantages to WB
- Does not require purified antigen for testing
- Allows for determination of fine specificities of antibody responses to multi subunit antigens
Disadvantages to WB
- Time consuming
- Expensive
- Requires denaturing proteins prior to gel ELectrophoresis
and will therefore not detect antibodies against conformational isotypes - Poor sensitivity to Ro and SCL-70
Southern blotting
- Like WB but DNA instead of protein
Indirect IF
Tag is used to identify antibody on a substrate
Direct IF
Tag is used to identify antigen on a substrate
Disadvantages to IF
- time consuming
- Labor intensive
- Requires skilled operators
- Subjective
DNA microarrays
- Collection of microscopic DNA spots attached to a solid surface
- Hybridizes complimentart DNA or RNA
- Can be used to look for mutations of snps and gene expression (RNA)
Transgenic mice
- Pronuclear injection into a single cells of the mouse embryo containing desired DNA sequence (gene) which randomly integrates into the mouse genome
- Used to insert new genetic information or over express endogenous genes
Gene knockout mice
- Targets specific gene by inserting a foreign sequence that renders the resultant protein non functional, knocking out the gene
Most commonly studied mouse model of RA
- Autoimmune arthritis induced by immunization with emulsion of complete Freund’s adjuvant AND type II collagen
(as opposed to adjuvant arthritis which is injection with M TB and is used more for reactive arthritis)
New Zealand Mixed Mice
- Oldest model of lupus
- Developed severe lupus like phenotype including LAD, splenomegaly, ANA, dsDNA ad immune complex mediated GN leading to kidney failure and death
- Disease is strongly biased in favor of females
MRL/lpr mice
- Develop SLE like phenotype characterized by LAD due to accumulation of double negative CD4-/CD8- and B220+ T cells
- Accelerated mortality rate and both males and females are significantly affected
- High concentrations of circulating immunoglobulins including ANA, ssDNA, dsDNA, SM, RF, resulting in large amounts of immune complexes
- Mutation is in Fas receptors leading to a defect in apoptosis
BXSB mice
- Severe with early onset in males (due to Yaa)
- GN
ALeutian disease in mink
- Highly contagious parvovirus
- Immune complex disease that resembles SLE including GN
- Have positive DS dna antibodies
g/d mouse
Similar to MRl/Lpr with defect in fas ligand and the process of apoptosis
Tight mouse (tsk)
- Fibrillin 1 gene mutation
- Causes autosomal dominant scleroderma like disease with skin and in some cases visceral fibrosis
Model of myositis
- Murine polymyositis like disease is associated with infection with viruses such as coxsackie
Model of spondyloarthripthy
- B27 transgenic rat
