Immuno 16 Ab Based Dx

Front 1

what is serology and what is it used for

Back 1

AB:AG specific non covalent interacetion

used to monitor AB levels, primary/secondary infections, acute vs chonic,
Dx of disease

Front 2

what allows us to determine if there is a 1 or 2 IR?

Back 2

Serology

IgM or IgG

**can also be used to monitor the progression of a disease

Front 3

what allows us to watch the rise and fall of infection

Back 3

serology

**measure the amt and type of AB over the course of a disease

Front 4

how can multiple myloma be dx

Back 4

MM causes excessive secretion of a specific AB, this can be detected via serology

Front 5

how is serology used in RA

Back 5

pts with RA make RF, this means that there is IgM with specificity for IgG

Front 6

if you are using immunodiagnostic assays can you detect AG or AB

Back 6

either but ONE MUST BE KNOWN!!

Front 7

what are the 4 main diagnostic assays/

Back 7

1. Nephelometryy: detect immune complex in a liquid w/o ppt

2 PPT rxns: SOLUBLE AG and SOLUBLE AB react and form ppt that can be visualized

3. Agglutination rxns: soluble AB reacts with AG that is attached to something, this clumps and forms ppt

4. Labeled Immunoassay: labet that emits measureable signal (radioacticity/color change) is attached to KNOWN AB or AG anf then the interaction can be visualized

Front 8

what is the diagnostic assay that detects immune complexes in liquid w/o ppt

Back 8

nephelometry

Front 9

what is the diagnostic assay that takes soluble AG and AB and ppts it out of soln for visualization

Back 9

ppt rxns

Front 10

what is the diagnostic assay that uses a solube AB and an AG bound to something, when the AB:AGprotein bind they clump out (can be soluble AG and AB complexed to something)

Back 10

Agglutination rxns

Front 11

what is the disgnostic assay that uses a label that emits something measureable attached to AB, ABlabel then binds to AG and can be measured
(the AG can be labeled too)

Back 11

labeled immunoassay

Front 12

how is nephelometry done?

Back 12

Measure Immune Complex in Soln, NO PPT

1. shine a light, the amt scattered is the same amt of AG:AB complex

2. compare results against std curve to determine actual amt

**used for IgM, IgG, adn IgA. detect rheumatoid factor

Front 13

Nephelometry is done to measure what Ig's and what disease

Back 13

IgM, IgG, IgA

**shine light and measure scatter.scatter is proportional to the amt of complexes

**detect rheumatoid factor

Front 14

hoe does a ppt rxt work? what kind of AG must it be

Back 14

soluble AG and soluble AB mix and form ppt

*AG must be multivalent or polyvalent (2 or more copies of the same epitope, dif epitopes that react with polyclonal AB) so that a lattice will form

Front 15

what is multivalent, wht is polyvalent, what assay requires AG be polyvalent or multivalent

Back 15

Multi: 2 or more of SAME epitope
Poly: dif epitopes that react with a mixed populatio nof AB (polyclonal AB)

**needed for ppt reaction, soluble AG and soluble AB to form PPT, must need several binding abilities so that they will form the lattice

Front 16

what allows maximal ppt in ppt rxns, what is this called?

Back 16

optimal conc of AG and AB, this is seen at the equivalence zone

**get equivalence zone by letting samples diffuse in a matrix

Front 17

how can you reach the equivalence zone for a ppt rxn

Back 17

let the sample diffuse in a matrix

Front 18

an immunodiffusion is an example of what type of assay

Back 18

ppt rxn

ex radial immunodiffusion, double immunodiffusion

Front 19

what is a radial immunodiffusion? the sample to be measured is placed where

Back 19

type of ppt rxn.

*used to detect the amt of AG or AB

**the sample to be measured is placed in a central well and allowed to diffuse though an agar matrix full of the "known"

**the known is mixed into the agar before plating.

**area of ppt ring is proportional to the conc of unknown

Front 20

in a radial diffusion how do we know the conc of the unknown

Back 20

the unknown is in the well adn allowed to diffuse, the area of ppt that forms is proportional to the conc of the unknown

**recall the agar has known in it.

**recall the most ppt will be formed at the equivalence zone where there is optimal conc of ab and ag

Front 21

what are radial immunodiffusions used for commonly

Back 21

Dx viral infections and asses Ig isotypes

**usually we want to measure or detect AG

Front 22

what are double immunodiffusions used for

Back 22

dx of fungal diesase

Front 23

how are double immunodiffisuion tests done

Back 23

simliar to radial but this time both the AG and AB are placed in a well and there is NO AB or AG in the agar

**the stuff is added into seperate wells and the stuff diffuses toward one another, a ppt is formed at the equivalence zone

**keep in mind that both radial and double immunodiffusions are types of PPT rxns

Front 24

what is agglutination

Back 24

when AB cross link the surface AG of target cells or AG coated particles. an aggregation of particles can be observed

Front 25

what is the best AB for agglutination? why? what is agglutination

Back 25

IgM, secreted form. pentamer. lots of binding abiilty

**whtn AB cross link on the sirface of the bug and cause it to clump out

*one component is soluble (AB) and the other is not (insoluble AG attached to a particle)

Front 26

what is it called when a soluble AB crosslinks AG that is bound to something and the whole things clumps out

Back 26

agglutination

Front 27

what types of things can AG be bound to in agglutination rxnx

Back 27

latex beads, RBC, bacterial cells

**the soluble AB binds the insoluble AG and the whole thing clumps out

**if its in a RBC its called hemagglutination

Front 28

what is hemagglutination

Back 28

when AG is boung to RBC nad a soluble AB binds and the whole thing clumps out

Front 29

what is it called whn there is an agglutination in which the AG is bound to RBC

Back 29

hemagglutination

Front 30

how are ppt rxns and agglutination rxns similiar

Back 30

they both require optimal concentrations

**area of optimal conc is called equivalence zone

Front 31

so in a ppt rxn to get hte optimal conc of AB/AG we used a well and let the stuff diffuse and then the equivalence pt was found. how is it dont for Agglutinations

Back 31

serial dilutions

Front 32

what are hemagglutination rxns commonly used for

Back 32

ABO typing.

**RBC mixed with antisera to A or B. when lots of RBC are found we have AG to that AB

if its just a little red bead we have no AG

Front 33

so a pts sample is mixed with A and B antiserum and nothing happens what is the blood type? what if hemagglutination happens in each thing?

Back 33

NO rxx: type O, lack BOTH B and A AG on RBC

RXN in both: person is type AB, reaction to both indicated pt has both A and B AG on surface of RBC

Front 34

what does it mean if a persons RBC is mixed with A and B antisera and only A reacts

Back 34

the person is A blood type

*hemagglutinaation

Front 35

how are titers done

Back 35

with hemagglutination

Determine the presence of AB in the sera of pts against RBC or precoated RBC

Front 36

what helps determine the presence of AB in the sera of pts against RBC or precoated RBC

Back 36

Hemagglutination, TITERS

Front 37

how are hemagglutinations for titers done

Back 37

sera is measured and serially diluted in many dif wells, AG coated RBC is then added to each well, when we get agglutination we know we have that AB. in this case the AG is known and the AB is unknown

**also used for viral infection and autoimmunity

Front 38

so hemagglutination can be used for what 3 things

Back 38

titers
dx of viral infection
dx of autoimmune

Front 39

what is the conc of AB titer equal to

Back 39

the inverse of the greatest dilution (lowest conc) of a pts sera that casuses a detecable rxn

Front 40

what is bacterial agglutination? what is it used for?

Back 40

agglutination rxn in which the AB is soluble nad the AG on bacteria is insoluble

**used to dx bacterial infections: salmonela, campylobacter, ecoli

Front 41

so a virus can use what agglutination? what about bacteria

Back 41

Virus: hemagglutination
bacteria: bacterial Agglutination

Front 42

so what is a latex agglutination used for? how is it done

Back 42

in this case either the AB or AG can be bound to the latex and the other is soluble (in others the AG was bound)

**used to test for: MRSA, menigitis, strep, toxoplasms

Front 43

what type of assay is a western blot, what does it commonyl test for>?

Back 43

labeled

HIV in pts sera

Front 44

what does labeling allow

Back 44

if we label the AB or AG we can visualize or quantify the abotu of AB AG rxn

Front 45

what are some common labels for immunoassays that use labels

Back 45

radioactive isotopes: I, S, titrum. used in RIA: radioimmunosorbant assay

Enzymes: horeradish peroxidase, alkaline pi. used in elisa and immunoblots (western blot)

Flourecent cmpds. (FITC, rhodamine). direct and indirect immunoflourrscnet assays. flow cytometry

Front 46

the elisa and immunoblots use what as labels

Back 46

horeseraddish peroxidase
alkaline phosphatase

Front 47

what are RIA

Back 47

a type of labeled assay
"radioabsorbant assays"

Front 48

what does flow cytometry use as a label

Back 48

flourscent cmpds

*FITC, rhodamine

Front 49

how are RIA's done? where do the AG:AB come from? what does it commonly test? what is a common example of this test

Back 49

RadioImmunoAssay

AB:AG detected using a radiolabeled SECONDARY AB

*the 1 AB is from pts serum, the AG and 2 AB are in teh kit

Often used for AB titers

RAST: tadioallergosorbent test. used for allergies specific to IgE

Front 50

RAST is used to detect what>? how does the test work

Back 50

its a type of RIA (radioimmunoassay, labeled assay)

the 1 AB in pts serum is mixed with a labeled 2 AB adn AG from the kit. (ANTI IgE is labeled in this case)

The RAST is used to detect allergen specific IgE

Front 51

so whts the interaction in a RIA

Back 51

well the AB is in pts serum. its mixed with a kit that has AG nad some type of labeled 2 AB

in the case of RAST. The pt has IgE AB it binds to an AG from the kit. There is then a labeled Anti IgE that binds to the AG:AB complex

Now we can visualize the AG:AB complex by means of a 2 AB

Front 52

what is an ELISA

Back 52

enzyme linked immunoabsorbant assay

*enzyme cong to AB: reacts with something colorless to form some thing colored

Front 53

what radiolabeled assays can be used to detect titers

Back 53

1. RIA: use labeled 2 AB to visualize. AG is knowd, AB is added. Labeled 2 AB is added and bings to the AB:AG comlpex for visualization

2. ELISA: AG is known, AB is added. when AB and AG are complexed they react with horseradish peroxidase or alkialins phosphatase to create a color change that can be measured

Front 54

what are the common labels for ELISA

Back 54

horseraddhsh peroxidase
alkaline phosphatase

**these enzymes bind to an AB:AG complex and crease a measureable color change

**used to detect HIV nad PSA

Front 55

what label creaste a color when an enzyme is added

Back 55

ELISA

**AB:AG (known AG) and then add the substarte (horseraddish peroxidase or alkaline phosphatase) and theer is a measureable color change

used for HIV nad PSA

Front 56

what is a western blot? what does it commonly detect

Back 56

used for HIV

1. HIV protein (KNOWN AG) is seperated via electrophoresis

2, the seperated proteins are transferred to a membraine

3. pts sera are then incubated on teh lanes (the AG is known and we are adding AB)

4. then add antiHuman IgHRP, a secondary AB that is linked to an enzyme

5. the substrate for the enzyme is added and there is a color change

Front 57

what is the name of the procedure thta goes like..

1. a bugs prteins are run on electrophoresis

2. the AG is then serperated and put on a membraine

3. Pts sera (AB, unknown) is then added to the AG protein on the membrane

4. if ther is AB binding occurs

5. abother AB with an enzyme is added nad will bind to AB:AG complex

6. we then add a substrate that will change the color of of the enzyme and its measured

Back 57

western blow

*immunoblotting, a type of labeled assay

Front 58

what more sensitive an elisa or western blot?what can BOTH be used for

Back 58

western blot

both are used for HIV detection of AB in pts serum

Front 59

how does a home preganncy test work

Back 59

ELISA: AG and AB bind, a secondary enzyme linked AB then binds. substrate is added and the color changed

**AB bind to hCG

Front 60

how does an ovulation tetsing kit work?

Back 60

ELISA

AB bind to LH

Front 61

what is the basis of the at home HIV 1 test kit

Back 61

pt AB to HIV are detected by immunoblot

Front 62

how does the at home hep c test workl

Back 62

anti HCV ELISA screen

Recombinant AG immunoblot assay

**4 AG from HCV are used, if you have AB to 2 or more its a + hep C

Front 63

what are immunoflourscent assays? wht is tagged the AB orthe AG

Back 63

AB is tagged with flourscent dye

Front 64

what is direct innimoflorescence

Back 64

labeled 1 AB binds to the AG directly

used for rabies, HSV

Front 65

what is an indirect immunoflourscent assay

Back 65

labeled 2 AB (recall in direct it was a 1 AB that was labeled)

so there is a AB:AG complex and then a 2 labeled AB binds to that complex

**uised for syphalis and some autoimmunities

Front 66

what is the dif btwn direct and indirece immunoflourscents?

Back 66

direct: the 1 AB binds to the AG. the 1 AB is labeled (rabies, HSV)

indirect: the AB and AG bind and then a 2 labeled AB will bind (syphalis, some autoimmunities)

**radioimmunoassays use 2 AB

Front 67

name the labeled assays that use a 2 AB for detection

Back 67

RAST: IgE AB binds AG, then a 2 AB binds that can be detected

ELISA: AG is known, AB binds. then a 2 AB linked with enzyme is added, substrate is added andhte enzyme turns colors

Western Blot: HIV Ag is seperated with gel and transferrred to membrane. Pts AB is added. Then a 2 AB linked with enzyme is added. Substrate is added to see if rxn has occured

Indirect Immunoflourrsecen: 2 AB is labeled iwth flourochorme or something that glows

Front 68

what is being used when you add labeled AB to a cell marker in order to seperate cell popualtions

Back 68

flow cytometry

flourescent technique. the dif wavelentghts emitted will spereate

Front 69

what can be used for the Analysis of T cell subsets in HIV patients

Back 69

flow cytometry- flourencent labels

put labeled AB for specific cell markers. ie CD4 CD19 etc

Front 70

how does flourscent flow cytometry work

Back 70

dif labels are put on AB that are specific for cell surface markers (CD4 CD19)

cells then pass through a laster that seperated them based on wavelength,

now we ahve populations of cells that all have the same unique cell surface marker

Front 71

how can we seperate CD4 from CD8

Back 71

flourscent labeled flow cytometry

labeled AB specific to a cell marker

Front 72

what is a titer? how is it determined?

Back 72

measure of the relative strength of antiserum (how much AB is in serum)

determine as the recripricol of the last dilution of antiserum causeing a measureable effect

Front 73

what is determined as the rescripricol of the last dilution of antiserum that causes a measuranle effect in immunoassay

Back 73

titer, the relative strength of antiserum

Front 74

know how to calc titers for pts sample

Back 74

????????????
recripricol of last dilution of antiserum that causes a measurable effect in an immunoassay

Front 75

what is nephelometry?
what does it measure, how is it quantified

Back 75

measure immune complex in soln

**read by spechtometry

Front 76

what are ppt rxns? what is the solubility of AG and AB, whehn does it ppt out

Back 76

souluble AG
soluble AB

*PPt at equivalence pt

ex radial immunodiffusions adn double immunodiffusions

Front 77

so in a radial dissusion BOTH the ab and ag are soukble, what rxn has one soluble and one insoluble

Back 77

agglutinations

AB or AG is insoluble nad the other is soluble
Agglutination occurs at equivalence

bacterial, latex or hemo agglutinatinos

Front 78

what are the 3 kinds of labeled assays

Back 78

radioisotopes: RIA, WB
ENzymes: ELISA and WB
Flourochromes: IFA, DIF and FC

Front 79

the label is in a secondary AB in what labeled assays

Back 79

RIA
ELISA
WB western blot

**used to detect AG or 1 AB in a pt sameple

Front 80

WB use 2 AB labeled with what

Back 80

1. enxyme
2. radioisotope

Front 81

where is the label for IFA DFA and FC (flourcent)

Back 81

either 1 or 2 AB

**sed to detect AG or AB in pt sample

**FC can also sort by cell type

Front 82

what assay lets us sort celly by cell type

Back 82

flow cytometry