Vishy- Ag/ Ab reactions Flashcards
IgM have ____ binding sites for antigen. ____ refers to minimal conc of antibodies that can succ bind to antigens and cause a detectable test reaction.
10; Titers
Describe agglutination reactions
- Direct agglutination: Belong to IgM; form lattice. If they react with cell surface antigens, they aggregate and form lattice.
- Indirect agglutination: Participating Ab belong to IgG –> monomers. 2 binding sites for Ag. They dont aggregate as well and dont form lattice. Have to add secondary Ab –> anti Ab that bind to primary Ab and form lattice.
what are the two types of Ag/Ab interactions
- Agglutination reactions
2. Precipitation reactions
Describe the single diffusion types of Precipitation reactions
- Single diffusion: When Ab bind to soluble Ag in liquids. They form immune complexes and precipitate. Ab are included along with molten agar poured on a glass plate. When agar solidifeids, a well is created and Ag are introduced into the well and keep diffusing in agar where it meets Ab. There is formation of precipitate ring. The farther away the precipitate ring is away from well = conc of Ag is higher. If precipate ring is closer to well, the conc of Ag is low. This technique is quantative.
What is equivalence zone
The conc of both Ab and Ag are optimal and there is formation of a detectable precipitation reaction
what is zone of ab excess
no detectable precipiation reaction bc of excess Ab
what is zone of Ag excess
no detectable precipitation reaction bc of excess Ag
Describe double diffusion
Molten agar poured on plate. Multiple wells are created. In some wells ag is introduced in some wells Ab are introduced. Once Ag diffuse and they bind Ab they form a precipitate ring, if there is cross reactvity, there iwll be partial binding and precipiate rings. This is qualitiative! Ab
What is immunoelectrophoresis
Measures serum proteins. Proteins present in serum go towards anode/cathode dep. on charge. In the wells, ab are introduced and ag and ab diffuse. If Ab are directed towards Ag there will be preciptate rings.
What is radio immunoassay
used to measure levels of hormone in human blood. Based on competition bet radio labeled antigen and unlabeled Ag in human blood sample for Ab.
Less radioactivity means ____hormones in patients sample. High radioactivity means _____hormones in patient’s sample
more; less
What is ELISA
Measure the levesls of Ag or Ab. Secondary Ab is commercial and conjugated with an enzyme which could be alkaline phosphatase or horse radish peroxidase. It binds to primary Ab. Wash to remove unbound Ab. When you add substrate, there will be color formation. Positive color formation means positive test reaction
What are advantage of ELISA
- Sensitive: nanogram levels or lower
- Reproducible
- Minimal reagents
- Qualitative and Quantitative
- Greater scope: Wells can be coated with Ag or Ab
- Suitable for automation –> high speed
- NO radiation hazards
Describe Western Blotting
Proteins are run in electrophoresis and undergoe resolution based on molecular weight. High on top, low on bottom. Transfer all proteins to a membrane and incubate it with patients serum and add secondary Ab and add substrate. You will then be able to see bands.
What is immuno flourescence
Qualitative! Detects the expression of Ag on cell surface of human cells or bacterial cells. ab are conjugated with flouresence dye and observe under flouresece microscope. If flourescene under microscope = +
The flourescene markers PE (red) or flourescein (green)
Ab can be covalently linked to these flourescence markers.
Describe direct and indirect immunoflourescence
Direct: Primary Ab conjugated with flourescence molecule
Indirect: secondary Ab are conjugated with flourescence dye.
What is FACS
Used to quantify the number of CD4 + T cells. When there is loss of these types of cells = person at risk for tuberculosis infection. Take immune cells from body and stain for CD4 marker with FITC and pass cells through fax machine.
How can you determine phagocytic efficiency of monocytes
if you see intracellular organisms the macrophages have normal ability to ingest microorganisms.
How would you determine if lymphocytes have normal capacity to multiply in response to Ag
collect peripheral blood mononucleal cells. Throw Ag or mitogen. If Ag then monocytes will present Ag to T cells or B cells then …add radioactive thymidine. Once T cells multiply radioactive thymidine get incorporated in DNA. High radioactive counts amounts means cells have normal capacity to multiply in response to Ag or mitogen
how do you det cytotoxic ability of NKs
take target cells that have low exp of MHC molecles, add NKs and label target cells with radioactive chromium. RC gets incorporated into cytoplasmic proteins of target cells. IF NK have normal bailty to kill target cells they will release perforin and granulysin and this will result in rupture of target cells which will release radio active chromium into supernatant. Collect supernatant and measure radioactivity. High radioactivity means NK have normal ability to kill target cells that are abnormal
People with A BG will have ____ antibodies. People with B BG will have ____ antibodies. People with AB wont have either Ab. People with O blood group will have ____ and ____ antibodies.
B; A; AB
What is blood group antigens
carbohydrate molecules exp on cell surface of RBcs and WBC’s and is commonly referred to as H antigen.
People with O blood group have Nac glucosamine, ____ and ______
galactose; fucose
