Diebel- Immunologic Tests Flashcards by Sarah Carlson

What is direct agglutination?

Use whole organisms to look for serum antibodies

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What do antibodies bind to in direct agglutination?

Whole organisms–RBC

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What do you visualize in direct agglutination?

Clumping

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When do you visualize a reaction in direct agglutination?

AFTER the Fab portion of the antibody has bound antigen

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What is passive agglutination?

Attaching an antigen to a particle and then running an agglutination

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What do the antibodies bind to in passive agglutination?

Ab is bound to latex then mixed w/ a target. The Ab then binds the target.

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What do you visualize in passive agglutination?

the latex beads

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Why can you visualize a rxn in passive agglutination?

The Fc portion of the Ab was bound to colored latex bead so that when the Fab portion of the Ab binds to antigen the beads clump together, but what you see are the beads bound to the Fc portion of the antibody.

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What starts out on the plate in a direct elisa?

antibody to virus

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What are you detecting in a direct elisa?

antigen form the virus

colored product- antigen concentration

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How are you detecting the product from the direct elisa?

antibody containing conjugated enzymes + substrate for enzyme–> colored product

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What are you washing away during the washes

excess antibody/ conjugated enzyme

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What starts out on the plate in an indirect elisa?

antigen from disrupted virus particles (HIV)

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What are you detecting in a indirect elisa?

pt antibodies to virus (HIV)

colored product = antibody concentration

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How are you detecting the result of an indirect elisa?

Anti-IgG antibodies conjugated to enzyme + substrate for enzyme→ colored product

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What are you washing away in an indirect elisa?

Anti-IgG Ab conjugated to enzyme

What starts out on the plate in a competitive elisa?

pt specimen containing antigen w/ known amt of antigen-specific ab

What are you detecting in a competitive elisa?

Colored product is inversely proportional to the antigen conc (pt’s serum)

How are you detecting antigen in a competitive elisa?

Anti-IgG antibodies conguated to enzyme + substrate for enzyme→ colored product

What are the two things you are washing away in a competitive elisa?

unbound antibody

unbound anti-Ig

What does it mean if you can detect IgM antibodies in a newborn and adult’s sera (collected at the onset of illness)?

Newborn positive: in utero (congenital) infection (TORCH)

Adult positive: primary or current infection

Adult negative: no infection or past infection

What does a positive IgG test result mean for sera collected 2-6 wks after onset?

+: fourfold rise or fall in titer between acute and convalescent sera tested at the same time in the same system

What does a negative IgG test result mean for sera collected 2-6 wks after onset?

no current infection or past infection, or pt is immmunocompromised and can’t mount a humoral Ab response, or specimen is collected before increase in IgG (lyme)

What does a positive IgG test mean for sera collected between onset and convalescence for an adult?

Adult- evidence of infection at some unknown time except in certain cases in which a single high titer is diagnostic (rabies)

What does a positive IgG test mean for sera collected between onset and convalescence for a newborn?

positive- maternal Ab that crossed the placenta negative- pt hasn't been exposed to microorganism or pt has congenital/acquired immune deficiency or specimen collected before increase in IgG (Lyme disease)

What is a hemagglutination inhibition assay?

Some viruses bear multivalent proteins or glycoproteins on surface that interact w/ macromolecules on RBC surface--> RBC agglutination

What are you testing for in the presence of the three different assays?

Blood cell typing- antibody Flu virus Ab Flu virus

How does the readout differ when testing for the presence of a virus Ab rather than a test used to detect the actual virus?

Readout for virus Abs: First wells: High conc of flu virus and pt serum, the flu will bind Ab and keep them in suspension and the RBC will form a pellet at the base of the well. Last wells: As pt serum decreases, Flu virus is free to bind RBC, and RBC remain on the surface.

What is HAT?

A medium used to grow fused myeloma and B cells. If normal B cells are fused to HGPRT negative or TK negative myeloma cells, the B cells provide the necessary enzymes so that the hybridoma cells can grow in HAT medium.

Describe the functions of HAT (hypoxanthine, aminopterin, thymadine)

Aminopterin blocks the activation of tetrahydrofolate and inhibits the synthesis of purines in this pathway. Aminopterin treated cells can use an alternative pathway to synthesize purine nucleotides if hypoxanthine is supplied externally. The enzyme present in cells that catalyses the alternative pathway is hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Thymidylate can also be synthesized if thymidine is supplied externally.

What is hypoxanthine?

Precurosor for GTP but in different pathway that uses scavenging. The myeloma cells can’t do this but normal B cells can. The HGPRT exchanges the H and converts it to IMP, by adding a ribosugar (attaches the base to the ribo sugar) and takes guanine to GMP

What is aminopterin?

* Selecting agent. * Blocks the synthesis of TTP and GTP. * Specifically blocks dihydrofolate reductase, needed to make new nucleotides * Myeloma cells don’t have DHFR so can’t make TTP/GTP by themselves. If you want to grow them by themselves need to supply these pre-cursors

What is thymadine?

* Deoxy sugar with thymine base, but missing phosphatases | * B cells have what they need to convert this into TTP