Immunochemistry Lab BIOC 446

34 Pages·2013·0.77 MB·English

by  Mohammad Alghazzawi

King Abdulaziz University
Faculty of Science
Department of Biochemistry
Girls Section

Immunochemistry Lab
BIOC 446

Organized by lecturers:
Omima Niazy and Wedam Alghazzawi
Contents

Lab # Experiment Page
Immunochemistry history
1 2,3
Immunological Tests
2 Human Blood groups 5
3 Standard Curve of Immunoglobulin 7
Quantitative Perception Test
4 9,11
Immunochemical Assays
5 Determination of Fibrinogen 14
Purification of Immunoglobulin by Ion Exchange
6 17
Chromatography
hCG Pregnancy test and Human Immunodeficiency Virus
7 24,25
Test
8 Enzyme-Linked Immunosorbent Assay 26
Ouchterlony Double Diffusion for Antigen and Antibody
9 29
Patterns
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Definition:
Immunochemistry (chemoimmunology) is studying the chemical reaction between
antigens and antibodies that occur in immune system.

History of immunoassays:
 In 1884, Ilya Metchnikoff suggested that cells involved in defense of the body.
 In 1940, evolution of diagnostic tests begun with measurements of enzymes in
biological fluids by colorimetric using agglutination reaction and chemistry
methods.
 In the 1950s, Rosalyn Yalow and Solomon Berson developed the radio-
immunoassay (RIA).
 In the 1960s, using enzyme instead radio-isotopes for color generation. It
called enzyme immunoassay (EIA).

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It is involve the measurement of one or other of the components of the antibody-
antigen system, namely, antibody, antigen and the immune complex.
There are a very large number of assays possible with a given combination of
antibody and antigen and some examples of those involving the quantitation of the
immune complex are given below:

1) Recovery of immune complex
A. Assay by weight
B. Assay of volume (Immunocrit)
C. Measurement of protein or nitrogen

2) Precipitation reactions in gel:
A. Immuno single diffusion
B. One dimension (Oudin )
C. Two dimension (Single radial immunodiffusion )
- Immuno double diffusion (Ouchterloni)
- Immuno electrophoresis

Immuno double diffusion ---- Voltage  Countercurrent electrophoresis

Single radial immunodiffusion ---- Voltage Rocket electrophoresis

3) Complement fixation
4) Labelled antigen or antibody
A. Radioimmunoassay
Radioallergosorbent test :RAST
Measure antigen-specific IgE in radioimmunoassay

B. Agglutination Cells
Haemaglutination Bacteria
Latex

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5) Enzyme linked immunoabsorbent assay
Enzyme-linked immunoabsorbent Assay: ELISA
Uses antigens & antibody
Advantages:
1. Sensitive.
2. Economic.
3. Large numbers of tests are done in short time.

6) Light scattering
A. Immunonephelometry
B. Immunoturbidimetry
C. Flow cytometry

7) Other
A. Changes in viscosity
B. Changes in sedimentation
Direct
C. Immunofluorescence
Indirect

Uses: detection of autoantibodies and antibodies to antigens of tissues & cells.
Disadvantages: Cumbersome
Advantages:
A. Identify antibodies to several deferent antigens in a single test.
B. Identify antigens on live cells.

Fluorescent Activated Cell Sorter: FACS
Measure fluorescence intensity of each (live stained) cell which are separated
accordingly to their particular fluorescent brightness.

The major differences between the assays are the sensitivities and the time taken to
carry them out. As expected, the most sensitive methods are usually the most
difficult technically and therefore the ones that can give the greatest error.
Immunochemical are now used extensively in the biological sciences and some
examples of those currently in use are given in the following series of experiments.
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Principle:
Blood consists of different types of cells floating in plasma. The types of cells are the
red blood cells contain hemoglobin binding with oxygen; the white blood cells
defending the body against infection; the platelets help the blood clotting and the
plasma contains dissolved protein and salt.

The differences in human blood type or blood group are based on the presence or
absence of specific protein molecules called antigens and antibodies on red blood
cells. Antigens are located on the surface of the red blood cells and the antibodies
exist in the blood plasma. The blood groups in persons depend on what the persons
inherited from their parents.

In blood transfusions, the process of receiving blood from another type leads to the
antibodies bind to the foreign antigens, causing dangerous clumping or agglutination
of the blood. Therefore, the successful transfusion depends on matching antigens on
the surface of the red blood cells between two persons.

Rhesus factor (Rh factor). It was first discovered in the blood of Rhesus monkeys.
The persons who have the antigen in their blood are called Rh positive and the
persons who lack that protein are called Rh negative. For example, the Rh factor is an
important in mother whose is Rh negative and her fetus whose blood is Rh positive
that lead to the mother blood attack the fetus blood.

AB0 blood grouping system
There are four different kinds of blood groups: A, B, AB or 0 (null).

Blood group A
The person has A antigens on the surface of red blood cells and B
antibodies generated in blood plasma.
Receive from A and O blood groups
Give to A and AB blood groups
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Blood group B
The person has B antigens on the surface of red blood cells and A
antibodies generated in blood plasma.
Receive from B and O blood groups
Give to A and AB blood groups

Blood group AB
The person has A and B antigens on the surface of red blood cells
and no A or B antibodies in blood plasma.
Receive from all blood groups
Give to AB blood group

Blood group 0
The person does not have A and B antigens on the surface of red
blood cells and A and B antibodies generated in blood plasma.
Receive from O blood groups
Give to all blood groups

Rh+ blood
The person has Rh+ antigens on the surface of red blood cells
and no Rh- antibodies generated in blood plasma.
Receive from Rh+ and Rh- blood groups
Give to Rh+ blood group

Rh- blood does not have antigens on the surface of red blood
cells but it has Rh+ antibodies generated in blood plasma.
Receive from Rh- blood group
Give to Rh+ and Rh- blood groups

Material:
 Clean microscopic slide
 Blood drops
 Marker
 Ani-A, Anti B and Anti-D antibodies reagents

Methods:
1. Mark one end for A, other end for B, and the last end for D on the slide.
2. Add one drop of blood on each position of the marked.
3. Add one drop of Ani-A, Anti B and Anti-D antibodies reagents on each side
respectively.
4. Mix each side with a toothpick and spread each mixture slightly.
5. Observe if any agglutination for red blood cells.
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Principle:
Immunoglobulins (Ig) are a glycoprotein produced by B-cells in immune system in
response to bacteria, viruses, or cancer cells. The five major types of
Immunoglobulins are IgA, IgG, IgM, IgE, and IgD. Gamma globulin (IgG) is a type
of immunoglobulin can be isolated from human blood and injected for passive
immunization against some diseases like hepatitis. Here in this experiment, we will
quantify the protein by Biuret reagent. Increasing quantities of γ-globulin are assayed
by measuring the extinction at 540 nm.

Materials (Preparation for 100 students):
 Isolated γ-globulin.
 D.W.
 Biuret reagent
 Spectrophotometer
 Vortex
 Incubator
 Five test tubes
 Pipette (1ml)

Procedure:
Tube Blank 1 2 3 4
γ-globulin (ml) - 0.25 0.5 0.75 1
H O (ml) 1 0.75 0.5 0.25 -
2
Biuret (ml) 1.5 1.5 1.5 1.5 1.5
Vortex all tubes and incubate at 37°C for 15 min.

Read the absorbance of each tube at 540 nm.
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Result Sheet

Lab partners: -------------------------------

Standard concentration: _____________

A. Calculate the different concentrations of proteins and read the absorbance at 540
nm for each one.
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________

B. Fill the below table
Concentration
Tube # Absorbance
(mg/ml)

C. Plot a standard curve of the absorbance against the concentration of γ-globulin.
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Functional assays:
Biological fluids contain a complex mixture of proteins. There are many assay
systems for assaying a specific protein by reacting with that protein such as
antibodies that react with specific protein so that immunochemical techniques are
accurate and highly specific.
Currently there are a number of assay systems depend on the formation of an immune
complex between the antibody and the antigen.
All antigen-antibody reaction is reversible and a typical 'titration curve' refers to
increasing amount of antigen added to a fixed concentration of antibody as below
figures. Be clearly to identify the antigen excess region if the results are not to be
misinterpreted.

Figure 1: A precipitation curve for a system of one antigen and its antibodies.

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