Lab techniques

Question 1

Direct Elisa

Answer 1

• Sample containing antigen is coated onto the plate well and the labeled antibody of known specificity is added

Question 2

Indirect Elisa

Answer 2

• 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

Question 3

Sandwich (capture) Elisa

Answer 3

• Plate wells are coated with a capture antibody targeting a specific antigen

Question 4

Inhibition Elisa

Answer 4

• Utilizes competition for labeled antibody between sample and well wall antigen

Question 5

Advantages to Elisa

Answer 5

• Rapid
• Automatic
• Multiple samples
• Objective

Question 6

Disadvantages to Elisa

Answer 6

• Less specific than other assays

Question 7

Immunoprecipitation

Answer 7

• 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

Question 8

Western blotting

Answer 8

• 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

Question 9

Advantages to WB

Answer 9

• Does not require purified antigen for testing

- Allows for determination of fine specificities of antibody responses to multi subunit antigens

Question 10

Disadvantages to WB

Answer 10

• 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

Question 11

Southern blotting

Answer 11

• Like WB but DNA instead of protein

Question 12

Indirect IF

Answer 12

Tag is used to identify antibody on a substrate

Question 13

Direct IF

Answer 13

Tag is used to identify antigen on a substrate

Question 14

Disadvantages to IF

Answer 14

• time consuming
• Labor intensive
• Requires skilled operators
• Subjective

Question 15

DNA microarrays

Answer 15

• 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)

Question 16

Transgenic mice

Answer 16

• 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

Question 17

Gene knockout mice

Answer 17

• Targets specific gene by inserting a foreign sequence that renders the resultant protein non functional, knocking out the gene

Question 18

Most commonly studied mouse model of RA

Answer 18

• 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)

Question 19

New Zealand Mixed Mice

Answer 19

• 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

Question 20

MRL/lpr mice

Answer 20

• 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

Question 21

BXSB mice

Answer 21

• Severe with early onset in males (due to Yaa)

- GN

Question 22

ALeutian disease in mink

Answer 22

• Highly contagious parvovirus
• Immune complex disease that resembles SLE including GN
• Have positive DS dna antibodies

Question 23

g/d mouse

Answer 23

Similar to MRl/Lpr with defect in fas ligand and the process of apoptosis

Question 24

Tight mouse (tsk)

Answer 24

• Fibrillin 1 gene mutation

- Causes autosomal dominant scleroderma like disease with skin and in some cases visceral fibrosis

Question 25

Model of myositis

Answer 25

- Murine polymyositis like disease is associated with infection with viruses such as coxsackie

Question 26

Model of spondyloarthripthy

Answer 26

- B27 transgenic rat