Immunologic Tests - Diebel

Question 1

What are some of the factors that can hinder diagnosis of infectious disease by culture?

Answer 1

Inability to cultivate organism
Difficulties getting organism transported to lab
Fastidious nature of some organisms resulting in long incubation periods
Administration of antimicrobial therapy before specimen collection

Question 2

What are some immunologic tests for accomplishing direct and serologic detection?

Answer 2

Immunoprecipitation and agglutination-based assays
Microscopic examination
RIA/EIA (ELISA)
Western Blotting
Determining antibody titers (ELISAs)
Skin Tests

Question 3

Molecular biology or immunologic testing has assays for either….

Answer 3

Antigen Assays or

Molecular Assays

Question 4

What do Antigen Assays look for?

Answer 4

Search for microbial or virus antigens, using fluorescent antibody, EIA…

Question 5

What do Molecular Assays look for?

Answer 5

Search for key genes of pathogen, nucleic acid hybridization, PCR

Question 6

What can you look for in a blood sample?

Answer 6

Search for antibodies using agglutination, RIA, EIA…

Question 7

Slide 6

Answer 7

x

Question 8

How does one make a hybridoma (making an antibody against a specific antigen)?

Answer 8

xx

Question 9

What is a-direct agglutination test?

Answer 9

“Combine serum + antibody to test for particular antigen no clumping/aggregation = negative clumping/aggregation = positive see direct results of antibody-antigen complex ABLE TO VISUALIZE A REACTION AFTER THE Fab PORTION OF THE ANTIBODY HAS BOUND AN ANTIGEN “

Question 10

What is passive (indirect) agglutination?

Answer 10

“Specific antibody + Latex beads added to serum ABLE TO VISUALIZE REACTION BECAUSE THE Fc PORTION OF THE ANTIBODY WAS BOUND TO A COLORED LATEX BEAD SO THAT WHEN THE Fab PORTION OF THE ANTIBODY BINDS TO ANTIGEN THE BEADS CLUMP TOGETHER no clumping = negative beads clumping = positive “

Question 11

What are the antibodies binding to in direct agglutination?

Answer 11

Directly bind to antigen (if positive test).

Question 12

What are the antibodies binding to in passive (indirect) agglutination?

Answer 12

Bind to latex bead + antigen (if positive).

Question 13

What are the 6ÿsteps of the Direct (Sandwich) ELISA test?

Answer 13

“Specific antibodies bound to bottom of plate Add sample serum (with antigen) Wash with buffer Add enzymeÿtagged antibody Wash with buffer Add substrate for tagged antibody color = positive test no color = negative test color change directly linked to the antibody that DIRECTLY recognized the antigen.”

Question 14

What are the 6 steps of the Indirect ELISA Test?

Answer 14

“Specific antigen bound to plate Add sample serum (with antibodies) Wash with buffer Add anti-IgX antibodies with enzyme tag Wash with buffer Add substrate tag color change = positive test no color change = negative test”

Question 15

What starts out on the plate in a Direct ELISA Test?

Answer 15

antibodies

Question 16

What starts out on the plate in a Indirect ELISA Test?

Answer 16

antigen

Question 17

What are you detecting in a Direct ELISA test?

Answer 17

the presence of a particular suspected antigen in a patient’s serum

Question 18

What are you detecting in a Indirect ELISA test?

Answer 18

the presence of a particular suspected antibody in a patient’s serum(i.e. looking for the body’s immune response to a particular antigen)

Question 19

What are you washing away during the washes in a Direct ELISA test?

Answer 19

antigen that is not specific to the plated antibody

Question 20

What are you washing away during the washes in a Indirect ELISA test?

Answer 20

antibodies that are not specific to the plated antigen

Question 21

What does aÿserodiagnosis indicate if IgM is detectedÿin a patient’s sera collected at the onset of the illness?

Answer 21

“Positive in Newborns: in utero (congenital) infection Positive in Adults: primary OR current infection Negative in Adults: no infection (secondary) OR past infection “

Question 22

What does aÿserodiagnosis indicate if IgGÿis detectedÿin a patient’s sera collected 2-6 weeks after onset of the illness?

Answer 22

“Positive: fourfold rise or fall in titer between acute and convalescent sera tested at the same time in the same test system Negative: No current infection or past infection OR patient is immunocompromised and cannot mount a humoral antibody response OR convalescent specimen was collected before increase in IgG “

Question 23

What does aÿserodiagnosis indicate if IgG is detectedÿin a newborn patient’s sera collected betweenÿonset and convalescence (recovery) of the illness?

Answer 23

“Positive in Newborn: maternal antibodies crossed the placenta Negative in Newborn: patient has not been exposed to microorganism OR patient has a congenital or acquired immune deficiency OR specimen was collected before increase in IgG “

Question 24

What does aÿserodiagnosis indicate if IgG is detectedÿin an adultÿpatient’s sera collected betweenÿonset and convalescence (recovery)ÿof the illness?

Answer 24

“Positive in Adult: evidence of infection at some unknown time except in certain cases in which a single high titer is diagnostic (rabies, Legionella, Ehrlichia) “

Question 25

What are the 6 steps of the Competitive ELISA Test?

Answer 25

Mix sample serum (with antigen) + known amount of antigen-specific antibody Add specimen-antibody complex serum to antigen-specific coated plate Wash with buffer (to remove unbound antibody) Add anti-IgX enzyme-tagged antibody Wash with buffer Add substrate for tag color change = negative test no color change = positive test

Question 26

What starts out on the plate in a Competitive ELISA Test?

Answer 26

antigen

Question 27

What are you detecting in a Competitive ELISA Test?

Answer 27

the presence of aÿparticular suspected antigenÿin a patient’s serum

Question 28

What are you washing away during the washes in a Competitive ELISA test?

Answer 28

unbound antibody then laterunbound anti-IgX antibody

Question 29

What are you testing for presence of in the Hemagglutination assay with a constant amount of RBCs + decreasing amount of serumÿin a microtiter plate?

Answer 29

“Antiviral antibodies(+) presence = induces agglutination of the RBCs lattice structure covers well (-) absence = no agglutination of RBCs button, RBCs sink to bottom of well “

Question 30

What are you testing for presence of in the Hemagglutination assay with a constant amount of RBCs + constant antibody + decreasing amount of patient virusÿin a microtiter plate?

Answer 30

“Virus/viral particle(s)(+) presence = button (RBCs sink) virus is bound by antibody (-) absence = RBC-antibody lattice no virus presentÿ “

Question 31

What are you testing for presence of in the Hemagglutination assay with RBC + anti-A/anti-Bÿantibodies?ÿ

Answer 31

“A-antigen/B-antigenused for blood typing (+) presence = clumping type A blood/type B blood (-) absence = no clumping type O “

Question 32

What are the functions of the three essential chemical components found in HAT media?

Answer 32

“H: Hypoxanthine rescues GTP important in scavenger pathway for IMP/GMP requires HGPRT A: Aminopterin blocks nucleotide synthesis by blocking DHFRÿ T: Thymidine rescues TTP Form ofÿartificial selectionÿfor cells containing working TK (thymidine kinase)ÿand HGPRT”