Immuno Assay Flashcards

1
Q

Each cell involved in the adaptive immunity has specialised receptors that can bind to clusters of molecules on the surface of foreign cells – known as

A

epitopes

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2
Q

One group of adaptive immune cells are known as B Lymphocytes or B cells. They secrete their receptors in large amounts – and the secreted receptors are called

A

Antibodies

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3
Q

The foreign invader is known as an

A

antigen

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4
Q

Immunoassay deals with what part of the immune system

A

Adaptive immune system

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5
Q

Facts about immunoassay

A

Scientists working in many fields are interested in this process. The human body has a way of specifically identifying and attacking anything foreign to the body. This can be exploited by biochemists/biomedical scientists/HCS/pharmacologists/pharmaceutical Scientists for many reasons e.g ;
vaccinations - one of the ways the immune system is exploited

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6
Q

Define immunoassay

A
immuno = immune system e.g. antibodies and antigens
assay = determine how much of something is present

Therefore an immunoassay exploits the specific binding affinity an antibody has for a specific antigen to help to quantify how much of either the antibody or antigen is present.

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7
Q

State two major requirements for an immunoassay

A

An antibody

An detection system

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8
Q

Structure of an antibody

A

has two heavy and two light chains variable region gives antibody it specificity
5 types of constant region
Antibodies are immune system-related proteins called immunoglobulins. Each antibody consists of four polypeptides– two heavy chains and two light chains joined to form a “Y” shaped molecule.

The a.acid sequence in the tips of the “Y” varies among different antibodies. This region (110-130 a.acids) gives the antibody specificity.

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9
Q

The constant region of the antibody determines-

A

The constant region determines the mechanism used to destroy antigen. Antibodies are divided into 5 major classes, IgM, IgG, Iga, IgD and IgE, based on their constant region structure and immune function.

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10
Q

How is diversity created on antibodies variable sites?

A

(a) By having multiple gene sequences
(b) Inaccurate and multiple joining – random component
(c) Different heavy and light chain combinations
(d) Somatic mutation – occurs 1 million times more often in variable regions of antibodies compared to other amino acid chains.

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11
Q

State how to obtain a polyclonal antibody

A
  1. Immunise animals with an antigen
  2. reimmunize the animal to produce a secondary response
  3. take a blood sample and test to see if it has made the antibody
  4. can continue to take blood sample from the animal during its life - a continuous source of polyclonal antibody

animal that stays alive
big animal used eg rabbit

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11
Q

State how to obtain a polyclonal antibody

A
  1. Immunise animals with an antigen
  2. reimmunize the animal to produce a secondary response
  3. take a blood sample and test to see if it has made the antibody
  4. can continue to take blood sample from the animal during its life - a continuous source of polyclonal antibody

animal that stays alive
big animal used eg rabbit

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12
Q

State how to obtain a monoclonal antibody

A

a mouse is used

  1. innoculate animal
  2. reinnoculate to get a secondary response
  3. sacrifice animal to remove the spleen
  4. isolate lymphocyte and grow on a cell culture to see if antibody is produced
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13
Q

What happens to the removed lymphocytes in the process of obtaining a monoclonal antibody

A

Remove B-cells from the spleen of an animal that has been challenged with the antigen. These B-cells are then fused with myeloma tumor cells that can grow indefinitely in culture (myeloma is a B-cell cancer).
The fused hybrid cells (called hybridomas) will multiply rapidly and indefinitely (since they are cancer cells) and will produce large amounts of antibodies.

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14
Q

List how monoclonal antibody is different from polyclonal antibody

A
Restricted to mouse 
Can develop and grow cell culture (quick and cheap)
Life long culture
Constant antibody source
Little purification needed
Most often used in immunoassays
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15
Q

how polyclonal antibody is different from monoclonal

A

Many animals can be use (as long as big enough).
Need animal houses – strict regulations
Finite source – as animal will eventually die of old age and the source changes each time a new animal is used.
Must purify antibody’s from all other cell components
Best for large antigens who’s epitopes may change e.g. bacteria

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16
Q

What animal should you try and induce antibody production in?

A

For monoclonal antibodies you are restricted to mice

For polyclonal antibodies you need a large animal e.g a rabbit, goat, sheep

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17
Q

In choosing the animal you must consider;

FCG

A

Foreignness – Antibody will only be produced if the antigen is foreign to the animal (i.e. not similar to some natural substance in the body).
Convenience – need animals easy to buy and look after – that don’t take up too much space
Genome – even animals of the same species have slightly different genomes. These may respond differently to an antigen – so you may have to use the same strain of animal.

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18
Q

What antigen should be used to inoculate the animal?

A

It is not easy to make an antibody for everything. One must consider the following properties of the antigen
(a) Molecular size – Bigger the better
(b) Composition – Proteins with only a few different amino acids
(c) Heterogeneity – a difficulty is posed if a particular antigen has a number of different forms
For (c) proteins with sugars attached are a good example. The protein is the same but is modified later by sugars, but can have various amounts of sugar added and still act the same way. In this case could cause problems.

(d) Haptens – small non-protein molecules have Antigenicity (can be recognised by the immune system and bind to antibodies) but lack immunogenicity (the ability to initiate an immune response). You can trick the immune system to create antibodies by binding it to a protein – known as a carrier
(e) Solubility of the antigen – Large insoluble antigens are better at causing antibody

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19
Q

How can we promote antibody response to the antigen.

A

Consider the use of an “Adjuvant

20
Q

State how adjuvant works

A

Adjuvants work by;
Prolonging the persistence of the antigen in tissues
Co-stimulating an immune response (getting B cells to rush to the area)
e.g. water in oil suspensions containing some killed Tuberculosis microbacterium is commonly used as a suspension for vaccinations.

21
Q

How do you administer an antigen to an animal?

A

Possible routes include; intravenous (directly into the blood – useful in mice for monoclonal antibody production);
Subcutaneous (under the skin – goes in via the lymph nodes).
Best to inoculate animal at least twice to get 2o response.

22
Q

How much antigen should be administered?

A

Low dose tolerance; if you don’t put enough in – you will not stimulate enough of the immune system

High dose tolerance: If you put too much in you can sometimes get unresponsiveness of the immune system – still not clear why this occurs.

23
Q

State Alternative way to produce antibodies without the use of animals;

A

By using Libraries of antibodies DNA sequences – you can chop up the DNA and put it together in different sequences in the variable region. You can insert the gene sequence into a cell and culture it to produce new antibodies.

24
Q

What is an advantage of using an alternative way to obtain antibodies?

A

overcome problems of foreignness

25
Q

State the disadvantage of using alternative route to obtain antibody

A

each time you want a new antibody you must screen 10^8 antibodies – very complex and time consuming. Not yet a very popular technique.

26
Q

State facts about the detection system

A

An immunoassay uses the specific binding of an antibody to an antigen.
However to know that binding has occurred we need to be able to detect this.
There are two main categories of detection systems that exist;
1. Those which exploit a change in physical properties when they i.e. if it leads to precipitation
2.Those that require the use of a label e.g. a radiolabel or enzyme label that can cause a signal linked to when binding occurs

27
Q

Two physical properties can be exploited in these techniques

A

(a) Precipitation – the correct ratio of antibody to antigen (no more or less) can lead to crosslinking – causing precipitation (ppt)
(b) Agglutination – Similar requirements except crosslinking does not result in ppt – just a change in turbidity of the solution which can be measured using nephelometry (i.e shine light through the solution

28
Q

Both agglutination and precipitation require….

A

Both of these methods require both the antibody and the antigen to be bivalent (i.e the antibody must bind to two antigens – and the antigen must bind to two antibodies);

29
Q

The ppt or agglutination will only occur when…..

A

there is the correct ratio of antibody to antigen. This is important since it is the basis for determining how much antigen is present. But why is this so?

30
Q

Excess antibody Prevents……

A

crosslinking as enough antibody to bind to each individual antigen – therefore statistically unlikely to find the antigen shared by two antibodies –no crosslinking.

31
Q

Excess antigen …..

A

This means that the antigens will bind to all available antibody sites and again this doesn’t facilitate cross linking.

32
Q

ppt of agglutination will occur when….

A

At a specific ratio of antibody to antigen (specific to a set immunoassay) – there is optimum chance of crosslinking between the antibody and the antigen. This is when ppt of agglutination will occur.

33
Q

How to determine the concentration of an unknown antigen

A

A plot of antibody ppt vs amount of antigen added will look like this. This can be used as a standard curve so an unknown antigen concentration can be determined.

34
Q

State Specific examples of immunoassay techniques exploiting precipitation

A
  1. Mancini test (radial immunodiffusion
    Fill a petri dish with gel containing your specific antibody
    Place the antigen in the centre and allow it to diffuse out.
    At the very centre the conc. of antigen is too high, as it diffuses it will eventually reaches the equivalent causing a ring of ppt
  2. Other techniques e.g looking as changes in turbidity of solutions exist
35
Q

Specific examples of immunoassay techniques using labels

A

Radioimmunoassay (RIA)

Enzyme Linked ImmunoSorbent Assay (ELISA)

36
Q

Radioimmunoassay (competitive assay) RIA as a detective system

A
  1. Coat a substiociometric amount of antibody on a solid phase. Add 100% radiolabelled antigen – incubate, wash and measure reactivity (100%)
  2. Repeat using a constant amount of radiolabelled antigen – but different conc, of non-radiolabelled antigen
    Higher Conc. of unlabelled antigen = lower Radioactivity. Use std curve for unknowns
37
Q

Advantages of using radio labels

A

Sensitive;

Very quick to run

38
Q

Disadvantages of using radiolabel

A

Sensitivity limited to a reading above background radiation
Although quick to run – slow to read (on g counter or b counters).
Need specialized equipment/lab.
Radioactive and often unstable reagents
Safety and disposal problems of radioactive material and scintillation fluids.

39
Q

Define Enzyme Linked ImmunoSorbent Assay (ELISA

A

This technique uses an antibody that has been labelled with an enzyme (e.g peroxidases of phosphatases) which either;
Reacts with a particular substrate to produce a colour
Reacts with a particular substrate to cause luminescence
This is so the antibody can be detected

40
Q

List ELISA methods

A

ELISAs methods vary widely.
In basic terms they can be used to;
Assay for an unknown amount of antibody (Method 1)
Assay for an unknown amount of an antigen (Method

41
Q

Assay for an unknown amount of antibody (Method 1

A
  1. An antigen (specific to the antibody) is adsorbed onto a solid phase (excess).
  2. The test antibody is added and binds to the antigen, and the wells washed.
  3. A second enzyme labelled antibody (an antibody of the first antibody) is added and the solution incubated. Any excess enzyme labelled antibody is washed off.
  4. A substrate for the enzyme is then added to the solution and a colour or fluorescence is produced (and is proportional to the amount of test antibody).
42
Q

Method 2: Sandwich ELISA – Assay for antigen

A
  1. The antibody is coated onto the bottom of the well (excess). The test antigen is added and will bind. All other biological material that is not bound is washed off.
  2. A second antibody which recognises an alternative epitope on the antigen is added and sandwiches the antigen between the two antibodies. A third enzyme labelled antibody is then added
  3. Excess enzyme labelled antibody is washed off.
  4. The substrate is added – and a colour change observed which is proportional to the amount of antigen present.
43
Q

list Examples of use of ELISA assays;

A

Pregnancy test (HCG)
Heart disease (CK-MB)
Hypoglycemia (Insulin)
Prostate cancer (prostate specific antigen)
Quantification of some pharmaceutical compounds
Testing athletes blood samples for prohibited substances

44
Q

State the advantages of using ELISA

A

If carried out in a microtiter plate (e.g. 96 well) you can do many tests simultaneously – all in ~ 5secs.
A plate reader is quite cheap
No radioisotopes are needed
Safer than RIA

45
Q

State the disadvantages of using ELISA

A

Disadvantages

Have to incubate at each step, so can take time

46
Q

Alkaline phosphatase and antialkaline phosphatase assay

A

This is a very useful technique used to stain tissues prior to looking at the under an electron microscope to show tissues with a particular antigen of their surface. e.g. sometimes used to look at cancer cells for a tumour to see if the cells have originated from elsewhere (i.e. to indicate metastasis).

47
Q

State the methods used in Alkaline phosphatase and antialkaline phosphatase assay

A

Method:
Treat the tissue of interest with an antibody specific to the antigen you with to look for.
Add a 2nd enzyme labelled antibody which will bind to the constant region on the 1st antibody
After washing tissue – add substrate (fast red) to stain at all places where the enzyme is bound

48
Q

Comparing sensitivities of immunoassays techniques

A

see slide 32 for table