16/17 - Immunoassays Flashcards
For a DIRECT assay
Interference causes a ________ in Signal
Direct = linear response to signal with analyte
DECREASE
Less analyte would be bound, so less signal would be shown
Adv & Disadvantages of
Immunometric Elisa
VERY PRECISE / ROBUST / SENSITIVE
neg:
Analyte / Antigen** has to be **>6000da MW
to have 2 antigenic sites (haptens)
Cross Reactivity
Response by antibody to substances
OTHER THAN THE ANYLYTE
similar in structure / family member
larger antigens = folding patterns / AA sequences
POLYCLONAL ANTIBODIES
main culprit
Causes for INTERFERENCE
not from cross reactivity
-
Detection System
- wrong substances giving the signal
-
Seperation Issues
- incomplete linkage / poor linkages of AB-enzyme/antigen
- Variation over batches = Heterogenesity
-
Alteration
- of AB / Enzyme / Ag
-
Displacement
- of analyte by other biochemical substances (hormones / FAs)
-
Blockage
- of analyte by binding proteins (albumin etc)
Most Important Antibody Properties
STERIC ALIGNMENT
NON-Covalent Interactions
What type of Assay?

INDIRECT Assay
Competitive Immunoassay
Immunometric Assay Steps
Heterogeneous Assay
#ABs & What is Labeled?
DIRECT ASSAY
-
2 ANTIBODIES
-
Unlabeled AB bound to SOLID phase
- recognizes 1 of the anylyte binding sites
-
LABELED AB is free
- recognizes OTHER binding site
-
Unlabeled AB bound to SOLID phase
-
1 Antigen = Analyte
- with 2 different binding sites
-
we measure the labeled AB which in turn measures the
-
Analyte Concentration
-
POSITIVE expenential curve,
- more analyte = greater signal
-
POSITIVE expenential curve,
-
Analyte Concentration
EMIT
Enzyme Multiplied Immunoassay Technique
HOMOgeneous assay that
Uses ENZYME
that has covalently attached to drug of interest
measure the amount of Free Drug in sample
does not use radioisotopes, uses colored product
Used for:
Assaying THERAPEUTIC DRUGS
URINE TESTS
Clinical uses of Western Blots
Detection of VIRAL PROTEINS
Hep B / C
HIV
lyme disease / prion disesase
athletic doping w / erythropoietin
How Pregnancy Tests work
We detect for HCG in URINE(human chorionic gonadoropin)
uses Monoclonal Antibodies
-
2 Antibodies
- 1* AB = NOT immobilized
- binds to HCG, and has DYE
-
2* AB = immobilized
-
downstream of 1*, also binds HCG
- binds the HCG + 1* dye complex
-
downstream of 1*, also binds HCG
- 1* AB = NOT immobilized
- 2* AB retains the complex of DYED 1*AB + HCG
- –> forms 1st blue line = pregnant
-
2nd blue line = control
- simply binds to the 1* AB no matter what
-
2nd blue line = control
- –> forms 1st blue line = pregnant
MONOclonal Antibody
MonoSPECIFIC AB that is
produced by a single plasma cell
or single CLONE of plasma cells
- ADV:
- Identical protein / same IG Class (usually IgG)
- highly specific –> recognize same epitope
- reduces: cross-reactivty
- UNENDING Supply
Low MW molecule/compound
that is not immunogenic itself
It still can bind to the Antibody, but it does not induce a response
Becomes immunogenic & induces an antibody response
after conjugation to a larger carrier/protein
Hapten
Immunometric ELISA
What Antibodies / Antigens?
What is Measured?
“Sandwich” / DIRECT
-
2 AB’s
- 1 is bound to solid phase
-
2* AB is enzyme-labeled
- enzyme reacts with the substrate
- –> product/color development is MEASURED
- enzyme reacts with the substrate
-
1 substrate
- that reacts with the enzyme
-
1 Hapten
- with 2 binding sites for each AB
Any FOREIGN material specificallly bound by:
_antibody or lymphocyte_s;
does not have to induce an immune response
Antigen
Ag
IRMA
ImmunoRadioMetric Assay
uses a Radioisotope Label on the
SECOND Antibody (not Ag like RIA)
easier to label AB vs Ag
Direct & Indirect Varients
Precipitation/Agglutination Assays
Types / Uses
2 Ag binding sites = double ended AB
+
Soluble Antigen (ag) or Epitope
- Various Types:
- Precipitin Assay
- Blood Typing / Coombs Test
- Hemagglutination assay for HIV
- Nephelometry/turbidimetry
Flexible Hinge
Hinge + 2 Ag-binding sites (Fv)
can ROTATE along various axis
Allow for CROSS-LINKING
of 2 different bacterium or virus
Allow for the most important antibody property:
STERIC AIGNMENT
& Non-covalent interactions
Which ImmunoAssays use ENZYMES?
EMIT
Enzyme Multiplied Immunoassay Technique
typically uses G6P-DH
ELISA
Enzyme-Linked ImmunoSorbent Assay
4 formats
Iodine Radioisotopes
125I
131I
attach to Tyr on proteins/peptides
HETEROgeneous mixture of ABs
with diverse affinities/regions towards an ANTIGEN
produced by a LARGE # of plasma cell clones
polyclonal response -> CROSS-REACTIVITY
Ex. Whale Myoglobin
from various Pharma Farms: goats/sheeps/cows
POLYclonal Antibody
Hemagglutination Assay for HIV Antibodies
2 Antibodies –> Sandwich
-
1* HIV AB
- AB for HIV (produced by the BODY if you had HIV)
- 2* AB
- Ytip Fab Region = anti-RBC = binds to RBC
- Fc region has HIV-ANTIGEN
- 2*AB will bind all over the RBC’s
-
If 1* HIV Antibody is present
- –> will bind to HIV Antigen on 2* AB
- –> Cause AGGLUTINATION!
- = POSITIVE
- –> Cause AGGLUTINATION!
- –> will bind to HIV Antigen on 2* AB
How are BLOOD TYPES determined by immunoassays?
COOMBS Test
More Effective than Agglutination Assay
Uses 2 Antibodies = Coombs reagent
Avoids issue of repulsion due to ZETA potential
- 1* AB
- normal, binds to surface of RBC
- 2* AB
- recognizes the TAIL = Fc region of 1*AB
-
crosslinkage of 2 RBC’s via the tails
- –> Agglutination
-
crosslinkage of 2 RBC’s via the tails
- recognizes the TAIL = Fc region of 1*AB
ImmunoRadioMetric Assay
uses a Radioisotope Label on the
SECOND Antibody (not Ag like RIA)
easier to label AB vs Ag
Direct & Indirect Varients
IRMA
5 Antibody Types
G A M E D
Differentiated by the type of Heavy Chain
- IgG
- main type produced in immune response, kind most used in most immunoassays
- IgA - mucus membranes
- IgM - ME FIRST - first responder in immune response
- IgE - AlEEErgic reactions
- IgD - autoimmunity protection, D-Unknown






