Paper : 16 Immunology

Module : 13 Immunotechnology-III

Principal Investigator Dr. Sunil Kumar Khare,Professor

Dept. of Chemistry,
I.I.T. Delhi

Paper Coordinator Dr. M.N.Gupta, Emeritus Professor

and Dept. of Biochemical Engg. and
Content Writer Biotechnology, I.I.T. Delhi

Content Reviewer: Dr. Prashant Mishra, Professor

Dept. of Biochemical Engg. and
Biotechnology, I.I.T. Delhi

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 Description of Module

Subject Name Biochemstry

Paper Name 16 Immunology

Module Name/Title 13 Immunotechnology-III

Dr. Vijaya Khader

Dr. MC Varadaraj

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 1. Objectives

• To understand the various modes in which complement activation can be used

to detect antigen/antibody

• How lymphocytes can be characterized by their secretory product

• How the possibility of erythroblastosis fetalis can be detected by Coombs test

• To learn a number of techniques which employ fluorescent labels

2. Concept Map

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 3. Description

Basically, all immunochemical techniques are based upon the recognition of antigen
by the antibody. The formation of the immune complex can be tracked by its
activating complement. It can also be tracked by labelling antibody by a fluorescent
molecule instead of a radiolabel.

The formation of Ag-Ab complex is a specific association. This fact is further exploited
in various techniques which are carried out using spectrophotometry,
spectrofluorimetry, optical microscopy, electron microscopy and fluorescence
microscopy

All these techniques, in a way differ in visualization method which is used to look at
ag-ab formation or its consequences.

Complement Fixation Tests

These tests can detect either antigen or antibody. The test antibody is mixed with the
antigen and a small amount of active complement is added. The immune complex
activates the classical pathway of the complement and “fixes” the complement

Any residual active complement is detected by the lysis of the antibody coated
erythrocytes. If no complement is left, no lysis of RBC will take place. A quantity of
complement is added in this test which is just enough to lyse the RBC. Hence, even a
small amount of test antibody forms the corresponding amount of immune complex
and fixes a significant % of the complement. Complement fixation can detect antibody
at the level of 1 µg/ mL. By using a constant amount of antibody, one can estimate
antigen instead in a given sample.

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 Controls with just antigen and antibody alone are very necessary. Some antisera
(antibody preparation) can fix complement as these may contain some immune
complexes. Some antigen can fix complement alone (by alternative pathway).

Figure 1

The complement fixation test detects antibodies 1. A test antiserum is titred in

doubling dilutions and a fixed amount of antigen is added to each well or tube. If
antibody is present in the test serum, immune complexes will form. 2. Complement is
then added to the mixture. If complexes are present, they will fix complement and
consume it. 3. In the final step, indicator cells (red cells) together with
subagglutinating amount of antibody (erythrocyte antibody) are added to the mixture.
If there is any complement remaining these cells will be lysed; if it was consumed by
the immune complexes there will be insufficient to lyse the the red cells.
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 A quantity of complement is used which is just enough to lyse the indicator cells if
none is consumed by the complexes. The assay is often performed on plastic
plates. By using constant amount of antibody and titrations of the antigen, the assay
can be applied to testing antigens. Appropriate controls are most important in this
assay because some antibody preparations consume complement without the
addition of antigen, for example, if the antibody preparation is serum already
containing immune complexes. Some antigens can also have anti-complement
activity. The controls should therefore include antibody alone and antigen alone to
check that neither fix complement by themselves.

Detection of the lysis of the RBCs

In the above test, the lysis of the target cells (usually RBC as discussed in the
previous slide) can be followed by different methods listed below. The choice can
be based upon a trade off between convenience and desired sensitivity.

 Decrease in “absorbance” of the cell suspension

 Release of hemoglobin which again can be measured (if RBCs are used)

 Release of another enzyme

 Release of an introduced isotope such as 51Cr

 Loss of membrane integrity which leads to failure to exclude dyes such as

trypan blue or eosin

 Failure to take up compounds such as fluorescein diacetate which are

converted to fluorescent compounds like fluorescene by intracellular enzyme.

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 Other assays using complement mediated cell lysis

The antibodies specific for surface antigens on many cells can be estimated. The
antigens can even be coupled to the cell surface so the test design actually is
generic in nature

Figure 2

The test antibody is incubated with the antigen bearing cells. Complement is added
and cell lysis is measured by any of the method discussed earlier. Cell lysis occurs by
classical pathway of complement activation. The degree of lysis is limited by the
concentration of test antibody as only those cells will be lysed which have antibody
bound to them. A standard curve with known antibody concentration can be generated
to plot antibody concentration vs degree of cell lysis. The above test will work only with
the complement activated isotypes of Ig: IgM and some subisotypes of Ig. Other
isotypes can also be detected with an additional step before complement addition. The
cells incubated with t est antibodies are trested with heterologous anti-Ig of the
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 complement activating class. Only these cells will bind these 2nd antibody which had
previous Ig. Only these will be lysed upon addition of the complement.

The Jerne Plaque Assay

Figure 3

The concept discussed so far can be further extended to assay for B-lymphocytes
producing antibodies specific for a cell surface antigen. An excess of antigen
bearing RBC are mixed with the test sample of lymphocyte suspension. The mixed
suspension is placed as a monolayer on a surface. With time lymphocytes release
antibodies. The monolayer is overlaid with the complement. If the test sample of
lymphocytes releases antibodies specific for antigen bearing RBC, these antibodies
bind to the antigen bearing RBC. The subsequently added complement gets
activated on such RBC ONLY. Number of zones or plaques of lysed RBCs reflect

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 the number of correct lymphocytes present in the test sample. A further
confirmation can be obtained by adding free antigen to the lymphocyte suspension.
In this case, no plaque formation confirms the specificity of the lymphocytes.

Reserved Plaque Assays

Figure 4

This is another variant of the plaque assays. This measures the total lymphocyte
population and not just lymphocyte producing antibody against a particular antigen.

In this case anti immunoglobulin bound RBC are used. The mature B cells release
any Ig and binds to these cells. Addition of the complement lyses Ig-anti Ig-RBC
complexes.

The anti-Ig is not specific to antigen binding siutes of Tg. Infact, Protein A which binds
to Fc fragment of the Ig can be substituted for anti-Ig in this reserved plaque assay

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 ELISPOT Assay

Figure 5

B-cells are relatively easier to identify/estimate. These cells secret only antibodies.
T-cells on the other hand have different classes. T-cell receptors are membrane
bound whereas antibodies are secreted in large amounts.

Hence many methods which are used to characterize T-cells can be easily adapted
for characterizing B-cells. ELISPOT is one such assay.

The assay is so named because it is essentially based upon ELISA principle and
counts the coloured spots produced in case of a positive identification.

The plate is coated with the antigen (for characterizing B-cells) or an antibody
against the cytokine (for characterizing T-cells).

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 The lymphocytes are placed on top of this. The lymphocytes secrete the antibody
(B-cells) or the cytokine (T-cells)

A second antigen or cytokine specific antibody (coupled to an enzyme) is added

and if the secretory product is specific to the added antigen/antibody, it binds to
them at the spots near the lymphocytes giving rise to a spot of insoluble coloured
precipitate if the test is positive.

Lymphocyte stimulation test

Figure 6

T-lymphocytes can be detected by stimulation with the presence of antigen.

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 The llymphocyte stimulation test involves lymphocyte separation on Ficoll Isopaque.
The defibrinated blood is diluted and mixed with tissue culture medium and is added to
Ficoll. After centrifugation RBCs and PMNs form pellet. Lymphocytes collect at the
interface of Ficoll (below) and diluted plasma (top).

Lymphocyte population can be further separated from macrophages by transferring the

suspension to a plastic vessel. Macrophages bind to the plastic surface leaving the
lymphocytes in suspension. Alternately, addition of iron filings lead to their uptake by
phagocytes which now can be removed by using a magnet.

Figure 7

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 In the lymphocyte stimulation test, the lymphocytes separated as above are washed to
remove any antigen. These are mixed with antigens and the culture medium. 3H-
Thymidine is added and cells are harvested after 16 hours to measure radioactivity.
Lymphocyte if specific to the antigen get stimulated and start DNA synthesis which leads
to the incorporation of 3H-thymidine.

Coomb’s Test

Figure 8
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 Robin Coomb was the first one to raise anti-Ig antibodies. To recognize him, this test
which uses such Ig to detect antibodies which cause erythroblastosis fetalis was named
after him. To recapitulate, if father is Rh+, Rh- mother makes antibodies against Rh
antigen which are paternally inherited.

These antibodies (Ig) cross placenta, coat fetal RBC. The immune complexes thus
formed are destroyed by liver phagocytic cells. This causes haemolytic anemia in the
fetus or the new born infant. Rh antigens are spaced out on RBC, so anti-Rh antibodies
coated RBC cannot fix the complement in vitro. In the Direct Coombs test, antibodies
raised against maternal Ig agglutinate maternal Ig coated RBC.

In the Indirect Coomb’s test, the anti-Rh antibodies of the nonagglutinating kind can also
be detected. Test serum is incubated with Rh+-RBCs. If antibody against Rh antigen is
present, it binds to these Rh+-RBC. An anti-Ig added agglutinates these cells. The
Coomb’s test can also be used to detect antibodies to drugs that cause haemolytic
anemia.

Immuno blotting (Western Blotting)

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 Figure 9

Presence of a protein in a cell lysate or any crude mixture can be detected by Western
Blotting. The SDS-PAGE of the cell lysate is carried out. Bands of proteins from the gel
are transferred to a nitrocellulose membrane which is a more robust support.

Radio isotope labelled antibody specific to the target protein is added and binds to the
band (if present) of that protein. This general methodology will detect any combination of
antibody and antigen and is used widely, although the denaturing effect of SDS means
that the technique works most reliably with antibodies that recognize the antigen when it
is denatured.

Fluorescent Labels

Fluorescence spectroscopy and fluorescent microscopy have emerged as powerful tools

in biology in general. Many immunochemical techniques exploit the sensitivity inherent in
fluorescence based methods.

Before we discuss these methods, it may be useful to cover few fundamentals and
information of a generic nature.

Fluorescent compounds such as fluorescein, o-phthalaldehyde, etc can be coupled

to antibodies (or antigens especially if these are protein antigens).

If the need arises more than one label can be used with different labels having
different λmax emission. For example, fluorescein is excited at 495 nm and emits at
519 nm to give yellow green fluorescence. Rhodamine, on the other hand is excited
at 550 nm to give a red fluorescence with λmax emission around 573 nm.

Fluorescent Proteins

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 Fluorescent proteins can be divided into seven groups according to their color and
chromophore structure.

Figure 10

Chromophore and color diversity in fluorescent proteins

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 Microscopic Techniques

Many labels can be used to view immune complex formation with various kinds of
microscopes.

125
I labelled antibody enables visualization of the immune complex formation by
autoradiography

Immunohistochemistry

Figure 12

Enzyme labelled antibodies allows visualization under an optical microscope with

the help of a chromogenic substrate.

Ferritin is an iron storage protein which appears as electron dense spots when viewed
through an electron microscope. Haemocyanin, oxygen transport protein from
crustacean haemolymph, latex microspheres and colloidal gold particles are other
electron dense labels for antibodies.

Fluorescence Microscopy

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 Figure 13

Direct Immunofluorescence: The antibody is covalently coupled to any fluorescent label

discussed earlier. The antibody incubated with the cells or frozen tissue sections binds
specifically to the antigen

Indirect Immunofluorescence: The frozen tissue section is incubated with the test
antibody. The immune complex is then visualized by addition of a second layer of anti-
antibody labelled with a fluorescent molecule.

Flow Cytometry

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 Figure 14

Flow cytometry allows cell populations to be identified from the profile of the surface
molecules. Size, granularity and fluorescence can be measured as cells pass through a
flow cytometer in a stream of droplets. For example, cells can be labelled with different
fluorescent antibodies to look at the surface density of different antigens on cells present
in the population.

FACS (Fluorescence activated cell sorter)

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 Figure 15

The cell population is carried in a sheath fluid and droplets are formed.

A laser falls on the droplet which crosses the path.

Detectors measure the granularity by side scatter, cell size by forward scatter and
fluorescent detectors identify cells by specific markers.

Quantification of proportions and phenotype of each sub population can be carried out.
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 Cell samples can be recovered separately for each kind.

Summary

 Complement fixation tests are based upon the ultimate lysis of cells

 Plaque assays based upon complement action are also available

 ELISPOT and lymphocyte stimulation methods are useful for identifying the cell type
among lymphocytes

 Coomb’s tests are indispensible in detecting the possibility of the erythroblastosis

fetalis

 Fluorescent labels in solutions or as part of the histochemistry provide host of newer

breed of immunochemical techniques

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