Lecture 8: Lab Techniques (2)

Question 1

Serum vs Plasma

Answer 1

Serum = blood after clotting factors (serum + blod clot)

Plasma = blood before blood clots (Plasma, WBC’s/platelets, RBC

Question 2

ELISA stands for

Answer 2

enzyme-linked immunosorbent assay

Question 3

ELISA facts

Answer 3

Rely on specific antibodies to bind the target antigen, and a detection system to indicate the presence and quantity of antigen binding.

The key step, immobilization of the antigen of interest, can be accomplished by direct adsorption to the assay plate or indirectly via a capture antibody that has been attached to the plate.

The detection antibodies are usually labelled with alkaline phosphatase (AP) or horseradish peroxidase (HRP).

Question 4

Types of ELISA drawings and explanations AND process (wash steps etc)

Answer 4

Lecture slide

Question 5

3 ways which ELISA data is interpretated

Answer 5

Quantitative
ELISA data can be interpreted in comparison to a standard curve (a serial dilution of a known, purified antigen) antigen in various samples.

2.Qualitative
ELISAs can also be used to achieve a yes or no answer indicating whether a particular antigen is present in a sample, as compared to a blank well containing no a or an unrelated control antigen.

Semi-Quantitative
ELISAs can be used to compare the relative levels of antigen in assay samples, since the intensity of signal will vary directly with antigen concentration.

Question 6

Enzymes in ELISA conjugates

Answer 6

HRP and ALP

ALP and HRP are conjugated (chemically bound) to other molecules (ie Streptavidin is often used for this purpose due to its high affinity for biotin)

ALP and HRP can also be directly conjugated to an antibody, thereby enabling its use as a detection antibody in immunoassays.

Question 7

ELISA substrates for different enzymes

Answer 7

Substrate for HRP:
TMB substrates contain two essential components:
H2O2, which is the substrate for HRP, and
tetramethylbenzidine (TMB), which generates the blue-coloured product.
The enzymatic reaction is stopped by adding an acid such as sulfuric acid. As a result, the substrate product turns yellow with maximum absorbance at 450 nm.

Substrate for ALP
The most common substrate for ALP isp-Nitrophenyl Phosphate (pNPP), which yields a yellow product that absorbs light at 405 nm.

Question 8

What is used to stop ELISA

Answer 8

Stop Solution is 0.16M sulfuric acid

In the presence of HRP enzyme conjugates, TMB and peroxide react to produce a blue by product having maximum absorbance at 605nm.

The colour intensity is proportional to the amount of HRP activity, which in turn is related to the levels of target analyte in an optimized ELISA procedure.

Addition of sulfuric acid stop solution changes the colour to yellow (absorbance maximum at 450nm)

Question 9

Agglutination principle

Answer 9

a reaction between a particulate antigen and an antibody resulting in visible clumping. Depends on the cross linking of polyvalent antigens

Question 10

Types of Agglutination

Answer 10

Direct Agglutination:
agglutination reactions where the antigens are found naturally on a particle. direct agglutination of particulate antigen with specific antibody

Passive agglutination
- employs carrier particles that are coated with soluble antigens (eg Latex, charcoal)

Question 11

Types of direct agglutination

Answer 11

Slide agglutination:
a suspension of unknown antigen is kept on a slide and a drop of standardized antiserum is added (or vice versa). A positive reaction is indicated by formation of visible clumps.

Tube agglutination:
serum is diluted in a series of tubes and standard antigen suspensions (specific for the suspected disease) are added to it. After incubation, antigen-antibody reaction is indicated visible clumps of agglutination

Question 12

Principle of Coombs Test and how does IgG cause aggultination?

Answer 12

In certain diseases or conditions, an individual’s blood may contain IgG antibodies that can specifically bind to antigens on the red blood cell (RBC) surface membrane.

▪ Red cells coated with complement or IgG antibodies do not agglutinate directly when centrifuged.

▪ In order for agglutination to occur an additional antibody, which reacts with the Fc portion of the IgG antibody, or with the C3b or C3d component of complement, must be added to the system.
▪ Because antibodies are gamma globulins, an antibody to gamma globulin can form bridges between red cells sensitized with antibody and cause them to agglutinate.

Question 13

Direct agglutination: Coombs Test Indirect and Direct Method and results

Answer 13

Direct method, the sensitization of red blood cells (RBCs) with incomplete antibodies takes placein VIVO. Cell-bound antibodies can be detected with antiserum against human immunoglobulin is used to agglutinate patient’s RBC.

Indirect method, the sensitization of RBCs with incomplete antibodies takes placein VITRO.Patient’s serum is mixed with normal red cells and antiserum to human immunoglobulin. Agglutination occurs if antibodies are present in serum.

Results:
Negative Result:
No clumping of cells (no agglutination). This means there are no antibodies to red blood cells.

Positive Result:
Clumping (agglutination) of the blood cells during a direct Coombs test means that you have antibodies on the red blood cells and that you may have a condition that causes the destruction of red blood cells by your immune system (hemolysis). Eg: Syphilis, SLE, haemolytic anaemia etc

Question 14

Passive agglutination description and examples of carriers

Answer 14

employs carrier particles that are coated with soluble antigens (ie latex or carbon)

either antibody or antigen is attached to certain carrier

Latex: employs latex particles as carrier of antigen or antibodies. If corresponding antigen or antibody is present in a test specimen, antigen
antibody bind and form visible, cross-linked aggregates.

RBC: RBCs are used as carrier particles. RBCs coated with antigen to detect antibodies in the serum, is called indirect
hemagglutination test

Question 15

Viral Haemagglutination: Unique aspect of viruses and agglutination and describe the viral haemagglutination inhibition test

Answer 15

Many viruses have the ability to agglutinate RBCs without antigen–antibody reactions.
Eg influenza, mumps, and measles

Viral hemagglutination inhibition test
- used to detect antibodies in patient’s sera that
neutralize the agglutinating viruses.
- Patient’s serum is first incubated with a viral preparation (virus).
- Then RBCs that the virus is known to agglutinate are added to the mixture.
- If antibody is present, this will combine with viral particles and prevent agglutination.
- Lack of agglutination indicates presence of antibody in patient’s serum

Question 16

Zone of Equivalence Diagram description

Answer 16

illustrates how the ratio of antigen and antibody affects the amount of precipitation. To achieve the optimal ratio, antigen is slowly added to a solution containing antibodies, and the amount of precipitin is determined qualitatively.

Initially, there is not enough antigen to produce visible lattice formation; this is called the zone of antibody excess.

As more antigen is added, the reaction enters theequivalence zone where both the optimal antigen-antibody interaction and maximal precipitation occur.

If even more antigen were added, the amount of antigen would become excessive and actually cause the amount of precipitation to decline; this is called the zone of antigen excess

Question 17

Describe immunodiffusion

Answer 17

An analytic technique in which reactants diffuse to intermingle with each other and react immunologically.

Generally, the primary reagent will be antiserum and the substance to be analyzed will be antigen;

Immunodiffusion tests are performed in semisolid media, usually gels, to allow reactants to diffuse while preventing convective mixing, and to support accumulation of the antigen–antibody reaction product, usually a precipitate, for observation.

Question 18

What is percipitation

Answer 18

Precipitationreactions are based on the interaction of antibodies and antigens.

They are based on two soluble reactants that come together to make one insoluble product, theprecipitate.

These reactions depend on the formation of lattices (cross-links) when antigen and antibody exist in optimal proportion

Question 19

Ouchterlony immunodiffusion principle

Answer 19

Antisera is added to the middle well and antigens in surround wells

The antibodies and antigen diffuse through the gel, causing a precipitin arc to form at the zone of equivalence. In this example, only the antigen in well 2 contains epitopes to the antibody. The resulting precipitin arc is stable because the lattice is too large to diffuse through the gel

Question 20

Single radial immunodiffusion

Answer 20

In this radial immunodiffusion (RID) assay, an antiserum is mixed with the agar before it is cooled, and solutions containing antigen are added to each well in increasing concentrations (wells 1–4). An antigen solution of an unknown concentration is added to well 5. The zones of precipitation are measured and plotted against a standard curve to determine the antigen concentration of the unknown sample.

Question 21

Define Flocculation

Answer 21

Colloidal particles come out of suspension to sediment under the form of floc. Differs from percipitation because the colloidal particles are merely suspended in the solution and not dissolved (as this is percipitation).

Question 22

Flocculation - RPR principle

Answer 22

This test measures IgM & IgG antibodies to lipodial material released from damaged host cells as well as possibly cardiolipin released from treponems.

If antibodies are present, they combine with lipid particles of the antigen, causing them to agglutinate.

The charcoal particles co-agglutinate with antibodies and show black clumps on white cards.

Question 23

RPR versus VDRL

Answer 23

Specimen for VDRL has to be heat treated before use (inactivates complement)

VDRL is a colourless reagent
VDRL requires a microscope

RPR cant be used for CSF
RPR is macroscopic

The rapid plasma reagin (RPR) test uses the same antigen asVDRL, but the antigen is bound to a carbon particle to allow visualization of the reaction without a microscope

Question 24

Describe the process of lateral flow test

Answer 24

1. Sample is applied to the ABSORBENT PAD and once this is soaked, the sample will move through the test strip via capillary action.
2. The sample migrates to the conjugate pad which contains the conjugate antibodies which have a coloured particle attached (gold). The antibody is specific for the analyte (antigen)
3. The sample moves through the conjugate pad and binds to conjugate antibodies and some conjugate antibodies will not be bound to antigen but will flow up the strip with the sample.
4. Once the sample reaches the test line, any conjugate antibodies bound to antigens will bind to the capture antibodies will are specific for epitopes on the anaylte antigen creating a sandwhich ELISA effect and thus colouration.
5. The sample continues to move up the strip to the control line and any unbound conjugate antibodies will bind to the capture antibodies specific for the antibody conjugate causing colouration.
6. The sample then proceeds to the wick (waste pad)

Question 25

Polyclonal vs monoclonal antibody

Answer 25

Polyclonal:
Heterozygous mixture of antibodies which binds to the same antigen but different epitopes of that antigen.
Produced by different B cell plasma clones

Monoclonal:
Homoxygous mixture of antibodies which binds to the same antigen and one single epitope of that antigen.
Produced by a single cline of plasma B cells.

Question 26

Fluroscent Immunoassay Direct vs indirect

Answer 26

Direct: Antigen - Primary ab with flurphore

Indirect: Antigen - primary ab - secondary ab (flurophore attached)

Question 27

Immunoelectrophoresis Principle
-combines what two tests
-generally how does it work
-Proteins separated by
- Test helps identify what

Answer 27

Combines electrophoresis and immunoprecipitation for identifying and characterizing proteins within complex mixtures.

An antigen mixture is first separated into its component parts by electrophoresis and then tested by double immunodiffusion.

• Protein components are separated by size and electrical charge.
• The test helps in the identification and approximate quantisation of various
  proteins present in the serum.

Question 28

Immunoelectrophoresis Method (3 steps) and how are antigens identified

Answer 28

1. Sample first separated into component parts by electrophoresis.
2. Antibodies then places in trough.
3. Time allowed for antibodies and antigens to
   diffuse into the gel.

Nature/identity of antigens can then be determined

Different antigens (proteins) can be identified based on the intensity, shape, and position of the precipitation lines.

Question 29

Immunoelectrophoresis separates proteins into what 5 groups and based on what

Answer 29

five major fractions by size and electrical charge: serum albumin, alpha-1globulins, alpha-2 globulins, beta 1 and 2 globulin, and gamma globulins.