Lecture 10

Question 1

Immunological Assay Uses

Answer 1

• Evaluate immune status of patient

- Diagnosis of disease

Question 2

Agglutination

Answer 2

Clumping of aggregates visible to naked eye

Question 3

Types of Agglutinations

Answer 3

1. Direct

2. Indirect

Question 4

Direct Agglutination

Answer 4

• Aka DAT
• Agglutination of pathogen cells
• Agglutination of RBCs based on antigens

Question 5

Indirect Agglutinaiton

Answer 5

• 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

Question 6

Leishmaniasis

Answer 6

• DAT
• Analysis of antibody in patient sera through ability to agglutinate parasite
• Antigen - killed promastigote parasites
• Antibody - serial dilutions of patient sera

Question 7

Hemagglutination

Answer 7

• Another DAT example

- Agglutination of bloods due to non-compatible blood types

Question 8

Additional DAT Example

Answer 8

• DAT
• Influenza/Mumps agglutinating
• RBCs infected with virus causes a positive microtiter reaction, but this doesn’t specify WHICH virus

Question 9

Hemagglutination Inhibiting Test

Answer 9

Series of test to identify which virus is infecting the patient. DAT example

Question 10

Beta-Hemolytic Streptococci

Answer 10

• 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

Question 11

Rheumatoid Factor

Answer 11

• 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)

Question 12

Enzyme Immunoassays

Answer 12

• 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)

Question 13

EIA Example

Answer 13

Anti-CD8 Antibody conjugation with enzyme

Question 14

ELISA

Answer 14

• Can be direct, indirect, or capture assay (“Sandwich Assay”)
• Positive control, negative control, and Patients are listed on test.

Question 15

Immunochromatographic

Answer 15

• 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.

Question 16

Flourescent Antibody Techniques

Answer 16

• 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

Question 17

Immunoblots: Western Blots

Answer 17

• 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

Question 18

Cellular Immunity Assays

Answer 18

• 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)

Question 19

Analysis for Neutrophils

Answer 19

• Blood count assessment: absolute and shift to left

- Neutrophil function - oxidative burst and protein analysis

Question 20

Assays for Complement

Answer 20

• 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