COMPLEMENT FIXATION TEST

Question 1

• Detects the presence of specific antibodies in a patient’s serum.

• This test is considered an indirect test because it does not detect the antibody itself, but rather relies on the consumption of complement as an indicator of an antigen-antibody reaction.

Answer 1

COMPLEMENT FIXATION TEST

Question 2

COMPLEMENT FIXATION TEST

Stage 1
Stage 2

Answer 2

Stage 1: Complement Fixation

Stage 2: Indicator System

Question 3

Stage 1: Complement Fixation

: The patient’s serum is heated to 56°C for 30 minutes.

: A known antigen is added to the heat-inactivated serum. If the serum contains antibodies specific to this antigen, an antigen-antibody complex will form.

: A defined amount of complement is added to the mixture. If antigen-antibody complexes are present, they will activate the complement cascade, leading to the “fixation” or consumption of the added complement.

Answer 3

• Heat Inactivation

• Antigen Addition

• Complement Addition

Question 4

Stage 2: Indicator System

: Sheep red blood cells (RBCs) pre-coated with antibodies against sheep RBCs are added to the mixture. These sensitized RBCs serve as the indicator.

Answer 4

Sensitized Red Blood Cells

Question 5

Stage 2: Indicator System

Observing for Hemolysis:
: If the complement was consumed in Stage 1 due to the presence of antigen-antibody complexes, there will be insufficient complement remaining to bind to the sensitized RBCs.
This prevents MAC formation, and the RBCs remain intact, resulting in no visible hemolysis.

: If there are no antibodies in the patient’s serum, or if the antibody titer is too low to fix all the complement, there will be free complement available. This complement will bind to the sensitized RBCs, activate the complement cascade, and form the MAC, ultimately lysing the RBCs.

Answer 5

Positive Result (Antibodies Present)

Negative Result (Antibodies Absent)

Question 6

• Designed for antigens and antibodies that may be small in size or present in very low concentrations.

• The presence of such antigens or antibodies is determined indirectly by using a labeled reactant to detect whether or not specific binding has taken place.

Answer 6

LABELED IMMUNOASSAYS

Question 7

• The defining feature of this format is the competition between labeled and unlabeled analytes (the substance
being measured) for a limited number of binding sites on the reagent antibody.

The more patient analyte is present, the less labeled analyte can bind.

Answer 7

COMPETITIVE IMMUNOASSAYS

Question 8

• This competitive binding scenario leads to an inverse relationship between the signal from the label and the concentration of the analyte in the patient sample

Answer 8

Competitive immunoassays

Question 9

_________
Also known as sandwich assays or capture assays, utilize an excess of antibody bound to a solid phase.

This excess ensures that all analytes present in the sample can be captured.

Answer 9

NONCOMPETITIVE IMMUNOASSAYS

Question 10

First, the analyte in the sample is allowed to bind to the capture antibody.

After washing away unbound molecules, a second antibody, labeled with a detectable tag, is added.

This second antibody binds to a different epitope on the analyte, forming a “sandwich”.

• The signal generated is directly proportional to the concentration of the analyte in the sample.

• A higher analyte concentration leads to more labeled antibody binding and a stronger signal.

Answer 10

NONCOMPETITIVE IMMUNOASSAYS

Question 11

• Require a separation step to differentiate between bound and free reactants.
• This separation often involves immobilizing the capture antibody or antigen on a solid phase.
• The signal is then measured and correlated with the analyte concentration.

Answer 11

HETEROGENOUS IMMUNOASSAYS

Question 12

• do not require a separation step.
• The reaction occurs in a single liquid phase, and the signal generated is directly measured
• While homogenous assays are faster and simpler, they tend to be less sensitive than heterogenous assays.

Answer 12

HOMOGENOUS IMMUNOASSAYS

Question 13

A fixed amount of enzyme-labeled antigen competes with unlabeled antigen (the analyte in the patient’s sample) for a limited number of antibody-binding sites.

Answer 13

COMPETITIVE ENZYME IMMUNOASSAYS

Question 14

COMPETITIVE ENZYME IMMUNOASSAYS

Principle:

Answer 14

• If the concentration of the unlabeled (patient) antigen is high, it will occupy more binding sites, leaving fewer sites available for the labelled antigen.

• This results in a lower signal, as the enzyme linked to the labelled antigen has fewer opportunities to react with the substrate.

Conversely, a low concentration of the target antigen in the patient sample results in a higher signal, as more labelled antigen is bound.

Question 15

NONCOMPETITIVE ENZYME IMMUNOASSAYS
•_________ or _______ , because the enzyme-labeled reagent does not participate in the initial antigen-antibody binding reaction.

Answer 15

Indirect immunoassays, or so-called
indirect enzyme-linked immunosorbent
assays (ELISA)

Question 16

The enzyme-labeled reagent only
binds after the initial antigen-antibody reaction has taken place.

Answer 16

NONCOMPETITIVE ENZYME IMMUNOASSAYS

Question 17

• Often referred to as sandwich ELISAs or capture assays, use an excess of capture antibody immobilized on a solid phase.

This excess ensures that all of the analyte in the sample can bind to the capture antibody.

• After washing away unbound molecules, second antibody, conjugated with an enzyme, is added.

• This secondary antibody binds to a different epitope on the target antigen,
forming a “sandwich” complex

Answer 17

CAPTURE IMMUNOASSAYS

Question 18

• ELISA leverages the specificity of antibodies to detect and quantify target
molecules in a complex mixture.

• The assay utilizes an enzyme-labelled antibody or antigen conjugate, with the enzyme’s activity serving as a measurable indicator of the antigen-antibody reaction.

The intensity of the signal generated, often a color change, is directly proportional to the concentration of the target molecule in the sample

Answer 18

ENZYME-LINKED
IMMUNOSORBENT ASSAY (ELISA)

Question 19

• The assay uses a radioactive substance as a label.
• Radioactive elements have nuclei that
decay spontaneously, emitting matter and energy.
• Several radioactive labels have been
used, but 125l has been the most
popular.

Answer 19

RADIOIMMUNOASSAY

Question 20

• It is easily incorporated into protein molecules and emits gamma radiation, which is detected by a gamma counter.

• RIA is an extremely sensitive and precise technique for determining trace amounts of analytes that are small in size.

Answer 20

RADIOIMMUNOASSAY

Question 21

• This technique was one of the earliest methods developed to measure total serum IgE.
• In RIST, radiolabelled IgE competes with a patient’s IgE for binding to anti-lgE antibodies immobilized on a solid phase.
• The amount of bound radioactivity is inversely proportional to the concentration of total IgE In the patient’s serum

Answer 21

RADIOIMMUNOSORBENT TEST (RIST)

Question 22

• This test is used to detect allergen-specific IgE antibodies in a patient’s serum.
• In RAST, specific allergens are immobilised on a solid phase.
• If the patient’s serum contains lgE antibodies against that specific allergen, they will bind to the immobilised allergen.
• Atter washing away unbound molecules, radiolabelled anti-lgE antibodies are added, which bind to the patient’s lgE.
• The amount of bound radioactivity corresponds to the concentration of
allergen-specific IgE in the serum.

Answer 22

RADIOALLERGOSORBENT TEST (RAST)

Question 23

• IFA relies on the principle that antibodies can be tagged
with fluorescent molecules, called fluorophores, without affecting their ability to bind specifically to their corresponding antigens.

• When excited by light of a specific wavelength, these fluorophores emit light at a longer wavelength, enabling the visualization of antigen-antibody
complexes under a fluorescence
microscope

Answer 23

IMMUNOFLUORESCENCE ASSAYS (IFA)

Question 24

Antibody that is conjugated with a fluorescent tag is added directly to
unknown antigen that is fixed to a
microscope slide.

• Atter incubation and a wash step, the slide is read using a fluorescence microscope.
• Antigens are typically visualized as bright apple green or orange-yellow objects against a dark background
• Best suited to antigen detection in tissue or body fluids.

Answer 24

DIRECT IMMUNOFLUORESCENCE ASSAYS

Question 25

• Involve two steps.
• First step: patient serum is incubated with a known antigen attached to a solid phase.

Second step: The slide is then washed
and an anti-human immunoglobulin
containing a fluorescent tag is added.

This immunoglobulin combines with the first antibody to form a sandwich, which localizes the fluorescence.

• Such assays are especially useful in
antibody identification.

Answer 25

INDIRECT IMMUNOFLUORESCENCE ASSAYS