ELISA lab

what holds together the antigen binding sites heavy and light chains?

disulfide bridges

antibodies are also called

glycoproteins

antibodies are a vital part of the…

immune system

where are antibodies found?

in the blood and other bodily fluids

antibodies are produced by…

b-cells

what do antibodies belong to?

a class of proteins called immunoglobulins (Ig)

main function of antibodies

recognize and initiate removal of foreign substances in the body (ex. bacteria or viruses)

antibodies consist of four _________________

polypeptide chains

what types of polypeptide chains make up antibodies?

two identical heavy chains and two identical light chains

what does the structure of antibodies form?

a y-shaped protein molecule

what are the 5 antibody isotype classes?

Ig: D, E, A, M, G

isotype

related antibodies but variations in heavy chain

isotypes vary _______ the antibody class

within

what is the same in isotype classes?

the two heavy and light chains

variations in isotypes occur within the

Ig

variations are due to differences in the __________ region

variable

what part of the antibody is extremely variable?

the region at the tip

what do isotypes allow for?

• millions of antibodies to exist

• many antigens to be recognized

antigens

foreign substances or materials that do not belong in the body (bacteria or viruses)

explain antibody recognition of antigen

recognizes on specific region of antigen called epitope

what is the region called that is recognized on the antigen? 

epitope

explain antibody (epitope) recognition

specific “induced fit” between the antibody variable regions

what can antibodies bind to?

only their binding antigen

when the antibody binds to the antigen

the antigen is tagged to be destroyed by immune system

antibody survival

must undergo activation to survive

activation

• causes rapid proliferation of B-cells

• makes more copies of antibodies

how often does the body make antibodies?

continuously

what happens to antibodies that do not recognize an antigen?

they are destroyed

what are ELISA tests based on?

immune system antibody molecules

what does ELISA stand for?

Enzyme Linked Immunosorbent Assay

what does ELISA utilize?

enzymes (enzyme-linked)

enzyme linked

• enzymatic reastion produces colorimetric changes

• linked (attached) to an antibody

immuno

based on immune system “component”

sorbent

antibody or antigen must be “affixed” to surface

assay

qualitative and quantitative investigative procedure

ELISA determines

the presence of a substance

what substance is usually being determined in ELISA?

an antigen, in a liquid sample or wet sample

what kind of tool is ELISA

a common health diagnostic tool

what is ELISA performed on?

ELISA plates that contain multiple “wells”

ELISA is based on

antigen/antibody interactions

previous assays required

radioactivity

ELISA developed year

1960

radioactive substance linked to

antibodies or antigens

what did sensitive equipment detect in previous ELISA?

the emitted radioactive signals

what’s the problem with radioactivity?

radioactivity kills

why did they change from old ELISA?

a safer, non-radioactive, signal was desired

some enzymative reactions can produce _______

color

what is the enymatic reason equation?

enzyme + substrate —enzyme substrate complex—> product

• lumbar puncture procedure to obtain cerebrospinal fluid (CS)

• analyte (CSF) placed into well

• proteins in analyte bind to plastic wells

• detergent wash to remove unbound antigen

• block remaining surface of wells

• primary antibody binds to specific antigen

• common wash steps

• unbound primary antibody is washed away

• enzyme linked secondary antibody (bioconjugate) binds to primary antibody

• common wash steps

• unbound secondary antibody is washed away

• enzyme substrate is added

common ELISA enzyme

peroxidase

common ELISA substrate

3,3, 5,5 tetramethylbenzidine (TMB)

peroxidase/TMB reaction

enzymme (peroxidase) + substrate (TMB) —> colorimetric product

what does it mean to add the enzyme to the antibody?

it is enzyme linked, taking advantage of color changes

when was modern ELISA born?

1971

what are the three types of ELISA?

• competitive

• direct/sandwich

• indirect

explain the 5 steps of direct ELISA

1. the ELISA plate it coated with the primary antibody (specific to antigen)

2. 2. non-reacting protein added to block any plastic surface remaining uncoated by the antigen (wash buffer)

3. antigen introduced to well and bind to the antibody if recognized

4. secondary enzyme linked antibody is introducted, specific for antigen of interest and binds to antigen

5. substrate TMB added to reaction

explain the 5 steps of indirect ELISA

1. analyte is added to each well where proteins adhere to the plastic (charging interactions of plastic and antigens)

2. non-reacting protwin is added to block and plastic remaining uncoated

3. primary antibody introduced and binds to bound antigens if recognized

4. secondary enzyme linked antibody introduced and binds to primary antibody

5. substrate TMB added to reaction (colorimetric)

ELISA test results are both ________ & _______________

qualitative and quantitative

the colorimetric change is directly proportional to the ______

amount of anitgen