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Biological Analytical Techniques > ELISA > Flashcards

ELISA Flashcards

0
Q

What is ELISA?

A

A biochemical / biological test used to detect presence of components in a sample.

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1
Q

Four types…

A

Sandwich.
Indirect.
Direct.
Competitive.

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2
Q

ELISA is…

A

Qualatitve and very specific.

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3
Q

The molecule of interest can be analysed by….

A

Exploiting the antibody antigen interaction.

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4
Q

ELISA is a selective test, the antigen / antibody can be….

A

Detected specifically in complex samples.

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5
Q

ELISA plates are read by a…

A

Spectrophotometer. This measures absorbance.

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6
Q

How’s does a spectrophotometer work?

A

Measures colour intensity by passing light of specific wavelength through the plate well. Absorbance is measure and this is directly related to concentration.

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7
Q

Using a spectrophotometer makes ELISA a….

A

Quantitive technique.

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8
Q

ELISA is a method of choice for detecting….

A

Biological molecules. Eg proteins.

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9
Q

And adv of ELISA:

A

Samples can be analysed quickly and accurately is a short time at lower cost.
Process can be automated and analyse many plates at once.

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10
Q

Enzymes are used to catalyse reactions. The enzyme is specific for….

A

A particular substrate.

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11
Q

A compound bound by an enzyme helps the…

A

Substrate to react and produce another compound.

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12
Q

The second compounds is usually coloured. The colour change is measured to…

A

Quantify the amount of antibody / antigen present in the sample.

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13
Q

Sandwich ELISA is used as a screening test for….

A

Syphillis, to identify people at higher risk who should be sent for diagnostic test.

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14
Q

A screening test is done before a diagnostic test because….

A

It is cheaper and faster and may also have less risk than a diagnostic test.

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15
Q

Four disadvantages of ELISA:

A

It’s labour intense and complex.
It requires sample prep.
Destructive.
Specific for only one type of toxin.

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16
Q

Can be used as a trace chemical analysis, may be used to detect…

A

Nandrolone, however SPE is preferred over ELISA for this because the sample compound is complex.

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17
Q

Adv over GC-MS…

A

Cheaper and portable.

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18
Q

Positive and negative controls should be tested on the plate at the same time because…

A

It ensures test is contaminated and is functioning as intended.

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19
Q

A polyclonal antibody…

A

Binds to more than one specific epitope on antigen. Allows for the max amount of antigen to be bound from the sample.

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20
Q

Adv of polyclonal antibody…

A

Captures as much antigen as possible.

21
Q

A monoclonal antibody is…..

A

Used as a detection antibody.

22
Q

An adv of a monoclonal antibody..

A

It is highly specific, therefore a specific recognition can be achieved.

23
Q

Four uses for ELISA:

A

Downs testing.
Bacteria and virus.
Food contamination.
Drugs in body fluid.

24
Q

The affinity is the…

A

Strength of binding.

25
Q

Why must plate wells be rinsed?

A

If not, a false positive may occur from any unbound antibodies remaining.

26
Q

Two common enzymes used in ELISA:

A

Horseradish peroxide and glucose peroxide.

27
Q

What is an epitope?

A

A specific part of the antigen with a specific chemical group.

28
Q

How do antibodies bind to antigens?

A

By weak non covalent bonds.

29
Q

Antigens are commonly…

A

Proteins, DNA and RNA.

30
Q

Antigens have a

A

Unique structure and bind with only one epitope.

31
Q

Antibodies are commonly protein molecules and bind to….

A

Specific epitopes on antigens.

32
Q

Antibodies are made by plasma cells in the immune system. They recognise and

A

Destroy harmful toxins.

33
Q

Antibodies are…

A

Immunoglobins.

34
Q

Advantage of sandwich ELISA over direct and indirect…

A

Up to five times more sensitive.

35
Q

How does a sandwich ELISA work?

A

Primary polyclonal antibody is attached to to the well. Sample is added which antigen will bind to p-antibody if it is present. A monoclonal, detecting antibody is added, attached to an enzyme. This binds to the antigen. Substrate is added and results in a colour change if antigen is present.

36
Q

Sandwich ELISA can detect proteins in…

A

Complex samples such as blood and urine.

37
Q

What does a sandwich ELISA detect?

A

An antigen that is bound between two antibodies, if present in the sample.

38
Q

A disadvantage of direct ELISA ….

A

Not very sensitive and therefore not widely used.

39
Q

How does direct ELISA work?

A

Antigen of interest is adsorbed to the plate. Antibody-enzyme is added, which binds if the antigen is present. Substrate is added and results in a colour change is antigen is present.

40
Q

Adv of indirect ELISA over direct ELISA?

A

More sensitive, can detect lower concentrations.

41
Q

How does indirect ELISA work?

A

Antigen is adsorbed to plate. Antibody is added. Secondary antibody containing an enzyme is added. This binds to first antibody. Substrate is added. Colour change observed if antigen is present.

42
Q

Indirect ELISA is used for…

A

HIV testing.

43
Q

Competitive ELISA is used to…

A

Detect infections.

44
Q

There are two types of competitive ELISA:

A

One detects antigens, the other detects antibodies.

45
Q

With competitive ELISA, the more colour change observed, the

A

LESS of target is present. This is inversely proportional.

46
Q

How does competitive Antibody work?

A

AG is bound to well surface. Sample with Ab of interest is mixed with known amount of ab-enzyme. Ab-enzyme and sample ab compete for antigen. Substrate is added.

47
Q

How does competitive antigen work?

A

AB is bound to well surface. Sample with Ag of interest is mixed with known amount of ag-enzyme. Ag-enzyme and sample ag compete for antibody. Substrate is added.

48
Q

Competitive ELISA: a lot of bound enzyme =

A

A lot of colour produced = there is little sample sample to compete with the added ab/ag.

49
Q

Colour change is inversely proportional ….

A

The amount of substrate reacting is lower when concentration is high.

50
Q

Competitive antibody is used for….

A

Syphillis testing.

51
Q

Competitive antigen is used for detecting…

A

Various infections, eg brucellosis.

Biological Analytical Techniques (5 decks)

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