1
Q
For a DIRECT assay
Interference causes a ________ in Signal
A
Direct = linear response to signal with analyte
DECREASE
Less analyte would be bound, so less signal would be shown
2
Q
Adv & Disadvantages of
Immunometric Elisa
A
VERY PRECISE / ROBUST / SENSITIVE
neg:
Analyte / Antigen** has to be **>6000da MW
to have 2 antigenic sites (haptens)
3
Q
Cross Reactivity
A
Response by antibody to substances
OTHER THAN THE ANYLYTE
similar in structure / family member
larger antigens = folding patterns / AA sequences
POLYCLONAL ANTIBODIES
main culprit
4
Q
Causes for INTERFERENCE
A
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)
5
Q
Most Important Antibody Properties
A
STERIC ALIGNMENT
NON-Covalent Interactions
6
Q
What type of Assay?
A
INDIRECT Assay
Competitive Immunoassay
7
Q
Immunometric Assay Steps
Heterogeneous Assay
#ABs & What is Labeled?
A
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
8
Q
EMIT
Enzyme Multiplied Immunoassay Technique
A
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
9
Q
Clinical uses of Western Blots
A
Detection of VIRAL PROTEINS
Hep B / C
HIV
lyme disease / prion disesase
athletic doping w / erythropoietin
10
Q
How Pregnancy Tests work
A
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
11
Q
MONOclonal Antibody
A
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
12
Q
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
A
Hapten
13
Q
Immunometric ELISA
What Antibodies / Antigens?
What is Measured?
A
“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
14
Q
Any FOREIGN material specificallly bound by:
_antibody or lymphocyte_s;
does not have to induce an immune response
A
Antigen
Ag
15
Q
IRMA
A
ImmunoRadioMetric Assay
uses a Radioisotope Label on the
SECOND Antibody (not Ag like RIA)
easier to label AB vs Ag
Direct & Indirect Varients
16
Q
Precipitation/Agglutination Assays
Types / Uses
A
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
17
Q
Flexible Hinge
A
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
18
Q
Which ImmunoAssays use ENZYMES?
A
EMIT
Enzyme Multiplied Immunoassay Technique
typically uses G6P-DH
ELISA
Enzyme-Linked ImmunoSorbent Assay
4 formats
19
Q
Iodine Radioisotopes
A
125I
131I
attach to Tyr on proteins/peptides
20
Q
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
A
POLYclonal Antibody
21
Q
Hemagglutination Assay for HIV Antibodies
A
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
22
Q
How are BLOOD TYPES determined by immunoassays?
COOMBS Test
A
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
23
Q
ImmunoRadioMetric Assay
uses a Radioisotope Label on the
SECOND Antibody (not Ag like RIA)
easier to label AB vs Ag
Direct & Indirect Varients
A
IRMA
24
Q
5 Antibody Types
A
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
25
**_EMIT_**
## Footnote
**Homogeneous or HETEROgeneous?**
**What is Immobilized?**
***_Homogeneous_***
*nothing is immobilized*
faster & more convenient
_only other homogeneous is **FPIA**_
*we are measuring the enzyme + substrate =*
**PRODUCT** (typically colored)
26
**Hapten**
***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**
27
**Uses for ELISA?**
**_Immunocapture / Indirect_**
PRIMARY ASSAY for detection of:
**AB --\> Pathogen**
**_SPECIFIC ANTIBODIES_**
_Antiviral IgG_
_IgM_ against HEP A
Measels / mumps etc.
28
**Competitive Immunoassay**
Heterogeneous Format
**#ABs & What is Labeled?**
***Indirect Assay***
* **_1 Antibody_**
* **__****ATTACHED** (by **Fc** region) to **Solid Phase**
* **_2 Analytes_**
* **1 is LABELED**, other *unlabeled*
* After Binding, we wash **away the *unbound*** ***materials***
* **measure signal** from the **_bound-labeled analytes_**
* As analyte concentration increases,
* *signal strength decreases*
* *regative exponential slope*
29
**Other Radioisotopes**
**Proteins**
**Fluorescein**
## Footnote
fluorophore
**GFP**
green fluorecent protein (whole protein)
30
**Which immunoassays are COMPETITIVE?**
Competitive = ***_indirect_***
**RIA**
**Competitive ELISA**
***Indirect IRMA***
FPIA
measures the ***Unbound***
***inversely proportional*** to amount of Ag
31
**Monoclonal Antibodies**
**ADVANTAGES**
Highly **SPECIFIC** = recognize **exactly the same EPITOPE**
**Identical** protein molecules
Same **Ig Class** (usually **IgG**)
**Unending supply**
*reduce background binding*
***less cross reactive binding***
32
**RIA**
**R**adio **I**mmuno **Assay**
**heterogeneous competition** immunoassay that uses
radiolabeled **_antigens_** or **_antibodies_**;
in the original liquid-phase format, the assay
would require a **precipitation step** to
separate antibody-antigen complexes;
Used for **Small molecule analytes** (steroids / cytokines / peptides)
*Not well suited for LARGE analytes (whole proteins*
33
**RIA Uses & Their Issues**
**RadioImmunoAssay**
Currently used for **_Small Molecule Anayltes_**
(Steroids / Cytokines / Peptides)
*not well suited for **large analytes** = whole proteins*
Radioactivity is a MAJOR drawback:
Hazard / waste disposal / decay
Licensing / unable for automation
34
**Common ENZYMES for immunoassays**
that use enzymes
_EMIT_
Commonly uses **_Glucose 6-Phosphate Dehydrogenase_**
adding NAD + GlC6P --\> we monitor NADPH formation by fluoresence
*_ELISA_ also uses enzymes*
Other enzymes:
**_Horseradish Peroxidase_**
**_Alkaline Phosphatase_**
35
**ELISA**
**_Enzyme-Linked ImmunoSorbent Assay_**
Uses **ENZYMES** to detect/quantify **_HAPTENS_**
*has 4 different formats*
also is **_HETEROgeneous;_**
uses **microtiter plates** w/ _**bound** **AB** or **bound Ag**_
to form **sandwich**
36
**Antigen**
**Ag**
Any **FOREIGN** material specificallly bound by:
_antibody or lymphocyte_s;
*does not have to **induce an immune response***
37
**Medical Uses for**
**EMIT****?**
Enzyme that covalently attaches to drug of interest
**_HbA1C Levels_**
**_URINE TESTS /**_ _**Free Drug Ratio_**
**_Pregnancy_** (plasma / serum tests)
**Hydrocodone**
Other **Therapeutic Drugs**
38
What type of Assay?
**DIRECT** **ASSAY**
sandwich / immunoMETRIC
39
**Radiolabel**
**=**
**Radioactive Label**
A _Label_ that uses
**_Unstable Isotopes_** that spon. transform
into a **more stable state**
--\> emitting **ENERGY** in the form of **Particles / EM pulses**
Ex. **_RIA_** = RadioImmunoAssay
40
**Name the SPECIFIC Immunoassay**
**_DIRECT_**
**_IRMA_**
ImmunoRadioMetric Assay
Positive correlation
We count the **BOUND 2nd antibody**, which is proportional to the **Ag**
41
For a ***INDIRECT*** assay
## Footnote
**Interference causes a ________ in Signal**
**_INCREASE_**
Indirect = *inverse signal response to more analyte*
Since less analyte would detected,
we would see a GREATER signal
42
**_IRMA_**
**Homogeneous or HETEROgeneous?**
**What is Immobilized?**
**HETEROgeneous** assay
**Both Direct & *Indirect*** :
**1\* Antibody is immobilized**
Direct measures the labeled 2\* AB, which is proportional to Ag
*Indirect measures the unbound labeled 2\* AB, inverseley proprotional*
43
**_RIA_**
## Footnote
**Homogeneous or HETEROgeneous?**
**What is Immobilized?**
**_HETEROgeneous_**
*Nothing is bound*
44
**Labels for Antibodies Vs Antigens?**
* ***ANTIGENS:*** *quite **small*** (MW \<600)
* ***difficult to find a site to attach a LABEL***
* *******without compromising AB recognition*
* ***__*****_Antibodies:_** **LARGE** proteins (MW ~150,000)
* **MANY** possible sites to attahch labels
* Ex. **_Radioisotopes_** (IRMA)
* **_Enzymes_** (ELISA)
* **GFP** = green fluorescent protein
* **EMIT** - use **enzymes!**
45
**Haptens / Homopolymers / Polysaccharides**
**as ANTIGENS**
* Hapten:
* *Does NOT elicit a immune response on its OWN*
* **can bind, but *not likely if only ONE***
* Polysaccharide/Homopolymer:
* Consist of **many epitopes**
* all have the SAME specificity
* ***NOT VERY IMMUNOGENIC***
46
**Heavy Chain**
Antibody Structure Components
**Longer** *darker* **Chain**
2 of them, identical to one another
linked together by **DiSulfide bonds** (cys-residues)
contain the **_Fc Region_**
Linked to ***light chain*** by **Di-sulfide bonds**
47
**Variable Region**
Contain the **Tips of the "Y"**
where **_binding/recognition of the antigen occurs_**
**Large variety of AA sequences**
for **AB Specificity & Affinity!**
**F-v Region**
48
**What are often the BEST ANTIGENS?**
**Why?**
**_PROTEINS_**
Lots of **Variability** in **AA Sequences**
More **Differences** = **More Antigens recognized**
MORE **CHANCES to ELICIT a RESPONSE**
49
**F-c** **Region**
Antibody Structure Component
**Base of the chunky "Y"**
consist of the end of the:
**2 dark chains**
c=first part crystallized, abc
50
**Name this Assay**
**_IMMUNOMETRIC ASSAY_**
**Sandwich Assay**
**_Direct Assay_**
more Analyte --\> Greater Signal
Signal is on the 2\* ANTIBODY
51
**Main Enzyme RadioIsotopes**
**HRP**
## Footnote
Horseradish Peroxidase
**G6P-DH**
glucose 6-phosphate dehydrogenase
**ALP**
alkaline phosphatase
52
**Longer** *darker* **Chain**
2 of them, identical to one another
linked together by **DiSulfide bonds** (cys-residues)
contain the **_Fc Region_**
Linked to ***light chain*** by **Di-sulfide bonds**
**Heavy Chain**
Antibody Structure Components
53
**HOMOgeneous Assay**
Analyte is labeled,
*Does **_NOT REQUIRE SEPERATION_***
of **bound & free antigen**
Faster & Convenient
We simply collect the **product = colored**
**_Ex._** **_EMIT_**
54
**F**-**ac** **Region**
Antibody Structure Component
**"V"** part of the **chunky Y**
Contains the **Variable Region**
both the _light and dark chain_
*does not include the Fc region*
55
**Source of POLYclonal ABs**
* Pharma Farms:
* _Goats / Sheep / Cows / Horses_
* ******Ag INJECTED --\>** into an animal
* animal's **B-Cells** produce **Antibodies to the Ag**
* **Various Antibodies** *with slightly different AA's are formed***
* **=\> POLYCLONAL RESPONSE**
56
_Any substance_ with a
**measurable property** that can be **attached** to an
**_Ag's / AB's_**
**_any other binding substance_**
(biotin / protein A / Avidin)
includes:
_Chromophores / Fluorophores_
_Radioactive Isotopes / Enzymes_
**Label**
57
Response by antibody to substances
***OTHER THAN THE ANYLYTE***
similar in _structure / family member_
_larger antigens_ = folding patterns / AA sequences
**_POLYCLONAL ANTIBODIES_**
main culprit
**Cross Reactivity**
58
**Epitope**
The **Specific Portion** of an **_antigen_** that
**_Binds to an ANTIBODY or T-cell receptor_**
*antigenic determinant*
59
**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**
**MONOclonal Antibody**
60
**EMIT**
**2 Ways the Antibody can *INHIBIT*** **the enzyme**
**_STERIC INTERFERENCE_**
Antibody PHYSICALLY gets in the way of the binding
**_INDUCED CONFORMATIONAL CHANGE_**
*keeps the substrate from binding*
61
**FPIA**
**Fluoresence**
**Polarization**
**Imunno-**
**Assay**
62
**Isotope**
**Atomic species** that
_in the **nucleus**_
have the **_SAME # of PROTONS_**
*but a **different # of neutrons***
_radioisotopes:_
_used to radioactively label Ag's or AB's_
63
**Antibody Structure**
64
**Interference**
**_ANY FACTOR_** causing **BIAS** in an assay result
*that is **NOT** in the presence of a*
***_Cross-Reacting Substance_***
* Numerous Causes:
* **Detection** system / **Seperation** problems
* **Batch Variation** / **Enzyme Alteration**
* **Analyte Displacement / Blocakage**
65
Analyte is labeled,
*Does **_NOT REQUIRE SEPERATION_***
of **bound & free antigen**
Faster & Convenient
We simply collect the **product = colored**
**_Ex._** **_EMIT_**
**HOMOgeneous Assay**
66
**Western Blot**
**Assay**
Used AFTER **_Protein Gel Electrophoresis_**
cut channel/column and lay on _polymer sheet_ (support)
**1\* AB** -\> specific to 1 protein
**2\* AB** **w/ DYE** or **enzyme** -\> recognized **fc region of 1\*ab**
Signal detects protein bands that bound to primary AB
**_illuminate / fluorecence_**
Used for:
**HIV INFECTION** / **Viral Proteins** (hep B/C) / **Doping**
67
**Agglutination (aggregation**) vs Precipitation
Using Antibodies
**_Aggregation = Agglutination_** Using ABs
when the **Antigen = LARGE / INSOLUBLE**
ex. Whole Cell
68
**Name This Assay**
**_COMPETITIVE ASSAY_**
***Indirect Assay***
Adding more *unlabeled Analyte (x)* --\> *lower signal (y)*
more analyte is competing with LABELED analyte (with signal)
69
***Light Chain***
Antibody Structure Components
**2 identical *shorter chains***
*not linked together*
linked to the **heavy chain by DiSulfide Bonds** (cys-residue)
70
**Uses for Immunometric=Sandwich ELISA?**
Good for **large antigens/proteins \>6000da**
**INSULIN**
**TSH / Thyroxine**
**Cortisol / ACTH**
71
**Immunoassay**
a _procedure_ for **_detecting or measuring_**
specific **proteins/substances** through their
**_properties as Ag's or AB's_**
72
**HETEROgeneous Assay**
Format that uses **_TWO PHASES_**
typically **liquid + solid** to
seperate **bound/reacted** from ***unbound/unreacted***
Ex.
**_ELISA**_ & _**IRMA_**
both **_direct & *indirect*_**
(Immunometric / sandwich) & *(competitive)*
73
Used AFTER **_Protein Gel Electrophoresis_**
cut channel/column and lay on _polymer sheet_ (support)
**1\* AB** -\> specific to 1 protein
**2\* AB** **w/ DYE** or **enzyme** -\> recognized **fc region of 1\*ab**
Signal detects protein bands that bound to primary AB
**_illuminate / fluorecence_**
Used for:
**HIV INFECTION** / **Viral Proteins** (hep B/C) / **Doping**
**Western Blot**
**Assay**
74
**_ANY FACTOR_** causing **BIAS** in an assay result
*that is **NOT** in the presence of a*
***_Cross-Reacting Substance_***
* Numerous Causes:
* **Detection** system / **Seperation** problems
* **Batch Variation** / **Enzyme Alteration**
* **Analyte Displacement / Blocakage**
**Interference**
75
**Monoclonal Antibody Production**
* Inject mouse with **Ag**
* collect **Spleen Cells** (contain **B-cells** that produce ABs)
* infuse with ***HAT resistance***
* ADD: **Immortal Myeloma** Cell line + **HAT medium**
* **PEG** --\> causes the cells to FUSE
* *Unfused cells will DIE*
* **Fused spleen cells = IMMORTAL** (*hat resistance + myeloma)*
* continuously producing **ANTIBODIES**
* **DILUTE** into wells then, **SCREEN** for **Desired Cell**
* Isolate desired **_Monoclonal Spleen cell_**
* that produces _MONOCLONAL ANTIBODIES_
76
Antibody Structure Component
**Base of the chunky "Y"**
consist of the end of the:
**2 dark chains**
c=first part crystallized, abc
**F-c** **Region**
77
**AntiBody**
**AB**
**Proteins** made by the **_B-Cells_** of the immune system
They serve to identify **Antigens** in the body by binding them
78
Agglutination (aggregation) vs **Precipitation**
Using Antibodies
***Precipitation Using ABS***
When the **Ag is**
***SMALL &*** **_SOLUBLE_**
ex. PROTEIN
79
**_G A M E D_**
Differentiated by the type of **Heavy Chain**
* Ig**G**
* ****main type produced in _immune response_, kind most used in **most immunoassays**
* Ig**A -** mucus membranes
* Ig**M** - **M**E FIRST - first responder in immune response
* Ig**E -** Al**EEE**rgic reactions
* Ig**D** - autoimmunity protection, **D-Unknown**
**5 Antibody Types**
80
Format that uses **_TWO PHASES_**
typically **liquid + solid** to
seperate **bound/reacted** from ***unbound/unreacted***
Ex.
**_ELISA**_ & _**IRMA_**
both **_direct & *indirect*_**
(Immunometric / sandwich) & *(competitive)*
**HETEROgeneous Assay**
81
**List several different types of LABELS for antigens**
**Radioisotopes**
*dangerous and difficult*
**Fluorophores**
Fluorecein = LARGE dye, for **UV** light
**Whole Proteins**
GREEN fluorescent protein
**Whole Enzyme**
G6PDH - color the ENZYME
82
**Atomic species** that
_in the **nucleus**_
have the **_SAME # of PROTONS_**
*but a **different # of neutrons***
_radioisotopes:_
_used to radioactively label Ag's or AB's_
**Isotope**
83
**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_**
**_EMIT_**
**E**nzyme **M**ultiplied **I**mmunoassay **T**echnique
84
**Proteins** made by the **_B-Cells_** of the immune system
They serve to identify **Antigens** in the body by binding them
**AntiBody**
**AB**
85
**Substances succeptable to CROSS-REACTIVITY**
**_PolyClonal Antibodies_**
Compounds of Similar **Family / Structure**
= **_Steroids_**
Closely related **folding patterns / AA sequences**
**_Large Antigens = Proteins_**
86
A _Label_ that uses
**_Unstable Isotopes_** that spon. transform
into a **more stable state**
--\> emitting **ENERGY** in the form of **Particles / EM pulses**
Ex. **RIA** = RadioImmunoAssay
**Radiolabel**
**=**
**Radioactive Label**
87
**HCG Test for Pregnancy Photo**
88
**Name this SPECIFIC immunoassay**
***INDIRECT***
**_IRMA_**
ImmunoRadioMetric Assay
***Negative correlation***
We REMOVE and count the ***unbound 1st antibody***
which is ***inversely proportional to _Ag_***
89
**Immunogen Vs Antigen**
**_Immunogen_**:
is a substance that is
_capable of inducing a_
**_IMMUNE RESPONSE_**
* Antigen does NOT have to induce an IMMUNE RESPONSE*
* it just has to specifically bind*
90
How are **BLOOD TYPES** determined by **immunoassays?**
**_Blood Type Agglutination Assay_**
**Blood type** = **_Type of ANTIGEN it HAS_**
* "Eldoncard" agglutination assay:
* Each Antibody **_Crosslinks RBC_** --\> bridges Ag's together
* causes **aggregation/agglutination** if it has
* that **specific antigen**
* **4 Pads:**
* **Anti-A** = ****has antibody for A
* **Anti-B** = has AB for B
* **Anti-D**
* ****has AB to the most common **Rhesus Factor Antigen**
* **CONTROL**
* *****should not aggregate at all!*
91
**POLYclonal Antibody**
**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
92
**heterogeneous competition** immunoassay that uses
radiolabeled **_antigens or antibodies_**;
in the original liquid-phase format, the assay
would require a **precipitation step** to
separate antibody-antigen complexes;
Used for **_Small molecule analytes_** (steroids / cytokines / peptides)
*Not well suited for LARGE analytes (whole proteins)*
**RIA**
**RadioImmunoAssay**
93
**Common Isotopes used in Radiolabels**
**_RIA = RadioIsotope Labeled Antigens_**
**Iodine** = 125I or 131I to **Tyr** on **Peptides/Proteins**
**Tritium** = 1H exchange to 3H
**Phosphorylate** = 32P-Containing-**ATP**
94
**F-v Region**
**Variable Region**
Contain the **Tips of the "Y"**
where **_binding/recognition of the antigen occurs_**
**Large variety of AA sequences**
for **AB Specificity & Affinity!**
95
a _procedure_ for **_detecting or measuring_**
specific **proteins/substances** through their
**_properties as Ag's or AB's_**
**Immunoassay**
96
**Competitive ELISA**
**Adv / Disadvantages**
Useful for ***_LOW MW Analytes_***
does NOT have to be a **hapten**
w/ 2 DISTINCT immunogenic sites
*Neg:*
***NOT as SENSITIVE as immunometric ELISA***
97
Antibody Structure Component
**"V"** part of the **chunky Y**
Contains the **Variable Region**
both the _light and dark chain_
*does not include the Fc region*
**F**-**ac** **Region**
98
**Competitive ELISA**
What **ABs / Ags?**
What is **Measured?**
Enzyme-Linked ImmunoSorbent Assay
***_Indirect_***
* **1 Antibody**
* bound to solid phase
* **2 Analytes**
* _1 analyte is **labeled w/ ENZYME**_
* **__**We **_measure the amount of bound enzyme_**
* **1 Substrate**
* reacts w/ ENZYME --\> color development
99
**2 identical *shorter chains***
*not linked together*
linked to the **heavy chain by DiSulfide Bonds** (cys-residue)
***Light Chain***
Antibody Structure Components
100
**Which Immunoassays are** **IMMUNOMETRIC?**
Immunometric = **_DIRECT_**
If 2/ 2 sites = **_Sandwich_**
**Direct IRMA**
**Immunometric ELISA**
Cortisol / ACTH / TSh / Thyroxine
Measure the **bound labeled AB**
which is **directly proportional** to the amount of Ag
101
**Western Blot Definition**
**Membrane-based assay** in which **proteins are separated**
by **electrophoresis**,
followed by **transfer to a membrane** and
**_probing with a labeled antibody_**
102
**Hydrogen & Phosphorous**
**Radioisotopes**
**Tritium** **3H**
## Footnote
exchange 1H to 3H
**32P**
**phosphoralated-ATP**
103
The **Specific Portion** of an **_antigen_** that
**_Binds to an ANTIBODY or T-cell receptor_**
*antigenic determinant*
**Epitope**
104
**_ELISA_**
## Footnote
**Homogeneous or HETEROgeneous?**
**What is Immobilized?**
**_HETEROgeneous_**
**AB** is bound
105
**Label**
_Any substance_ with a
**measurable property** that can be **attached** to an
**_Ag's / AB's_**
**_any other binding substance_**
(biotin / protein A / Avidin)
includes:
_Chromophores / Fluorophores_
_Radioactive Isotopes / Enzymes_
