IMMUNOLOGIC METHODS

A. TESTS FOR CELLULAR IMMUNE FUNCTION

1. DELAYED-TYPE HYPERSENSITIVITY measure individual immunocompetence; may indicate a prior

exposure to the microorganism or to chemicals.

PATCH TESTING (E.G. PPD TEST) . Should erythema or wheal appears within 12-18 hours, then it
means immediate hypersensitivity reaction. This is of limited value in infants and in patients with
immunosuppression since their T cells are depleted. Caution should be used in highly sensitized or
previously immunized individuals who may have severe local and systemic reactions.

PPD test is also known as the Mantoux test.

A tuberculin reaction of >= 5 mm of induration is classified as positive in the following groups:

• HIV-positive persons
• Recent contacts of TB case
• Persons with fibrotic changes on chest radiograph consistent with old healed TB
• Patients with organ transplants and other immunosuppressed patients (receiving the equivalent
of >= 15mg/day of prednisone for >= 1 month)

A tuberculin reaction of >= 10mm of induration is classified as positive in persons who do not meet the
preceding criteria but who have other risk factors for TB. These include:

• Recent arrivals (< 5 years) from high-prevalence countries

• Injection drug users
• Residents and employees of high-risk congregate settings: prisons and jails, nursing homes and
other long-term facilities for the elderly, hospitals and other health-care facilities, residential
facilities for AIDS patients, and homeless shelters
• Mycobacteriology laboratory personnel
• Persons with clinical conditions that place them at high risk
• Children < 4 years of age, or children and adolescents exposed to adults in high-risk categories.

A tuberculin reaction of >= 15mm of induration is classified as positive in persons with no known risk
factors for TB.

In general, these guidelines for interpreting skin test reactions should also be applied to persons who may
have occupational exposure to TB (e.g., health care workers or staff of nursing homes, drug treatment
centers, or correctional facilities). Thus, the appropriate cutoff for defining a positive reaction depends on
the employee's individual risk factors for TB, including recent TB exposure, and the prevalence of TB in
the facility.

Patch test for common allergens, using standardized panels, is readily available now in several
countries(T.R.U.E. test or thin layer rapiduse epicutaneous test). This has panels in which allergens are
incorporated, most common of which are:

Nickel , Fragrance, Neomycin, Epoxy resin, Formaldehyde, Black rubber mix and 21 other common
allergens. It is a good test for recurrent dermatitis, contact dermatitis, hand dermatitis, hand and leg
dermatitis, facial dermatitis, atypical allergies.
2. IN VITRO LYMPHOCYTE ASSAYS

a. Basic lymphocyte enumeration and purification, wherein the T and B cells are separated using density
gradient centrifugation. WBC may also be counted by microscopy, using the Coulter Counter. They may
also be measured using flow cytometry, which uses antibodies to cell surface antigens.

b. CD4/CD 8 ratio, which is very important for HIV patients.

c. T cell functional assays

d. B-lymphocyte assays

e. Phagocyte function assays measure the ability of the blood phagocytes to ingest a fixed number of
latex particles in a specific time. It often measures phagocytic killing, chemotaxis,

B. TEST FOR QUALITATIVE AND QUANTITATIVE TESTS FOR ANTIGENS AND ANTIBODIES

1. IMMUNODIFFUSION measures antigen-antibody complexes through the formation of

precipitates. The most common method used is double immunodiffusion(ouchterlony radial
immunodiffusion technique. It is also known as DID (double immunodiffusion).

2. ELECTROPHORESIS is a method of separating proteins in an electric field. Zone electrophoresis

separates proteins based on the surface charge. The method is can quantitate serum proteins
and immunoglobulins. It is useful in the detection of multiple myeloma, Waldenstrom
macroglobulinemia and hypergammaglobulenimia. In immunoelectrophoresis, the antibodies
move around in an electric field.This distinguises monoclonal from polyclonal increases in γ-
globulin. It also detects heavy and light chain paraproteins, making it useful in patients with
plasma cell dyscrasias and autoimmune diseases. It can also detect the reduced or total absence
of immunoglobulins in diseases.

In counterimmunoelectrophoresis, the rapid diffusion of the antigen and antibody in a gel

medium towards each other, leads to semiquantitative measurement of antigen in the
biologic fields. Also called Countercurrent Immunoelectrophoresis or
Counterelectrophoresis.This method is useful in the analysis of CSF, and the diagnosis
of : Cryptococcosis, meningococcal meningitis, H. influenza meningitis,
staphylococcal endocarditis.

3. RADIOIMMUNOASSAY (RIA) can quantitate any substance that is immunogenic or haptenic, and
can be labeled with a radioactive isotope. The technique of radioimmunoassay has revolutionized
research and clinical practice in many areas blood banking and diagnosis of allergies.

A mixture is prepared of radioactive antigen .Because of the ease with which iodine atoms can be
introduced into tyrosine residues in a protein, the radioactive isotopes 125I or 131I are often used.
Antibodies against that antigen are added. Known amounts of unlabeled ("cold") antigen are
 added to samples of the mixture. These compete for the binding sites of the antibodies. At
increasing concentrations of unlabeled antigen, an increasing amount of radioactive antigen is
displaced from the antibody molecules. The antibody-bound antigen is separated from the free
antigen in the supernatant fluid, and the radioactivity of each is measured.

4. AGGLUTINATION is a method whereby the antibodies are mixed with antigen-coated particles.
The antigen-antibody complexes form a visible clump. IgM is more effective than IgG in
agglutination due to its pentameric structure.

a. Latex agglutination uses latex particles as carriers of the antigen. This can be used in
Rheumatoid Factor (RF).

b. Direct agglutination is termed “direct’ because antibody directly agglutinates the target
particle antigen. It consist of mixing an unknown quantity of antibody with a fixed amount
of antigens. The positive result is clumping.

c. Indirect (passive) agglutination requires the coupling of soluble antigen to RBC or inert
particles. It is termed “indirect” since the antigen must be linked to a particle(bead, RBC),
thus allowing visualization of the agglutination reaction.

d. Hemagglutination inhibition is the method of determining the antigen by preventing

agglutination between antigen-coated RBCs and the specific antibodies. This can
determine antigen concentration in serum and other biologic fluids.

e. Coombs test or antiglobulin test measures antibodies which are too low to agglutinate by
themselves. Useful in blood typing, in hemolytic disease of the newborn, and
autoimmune hemolytic anemias.

Direct Coombs test detects serum proteins adherent to RBCs taken from the patient.
Indirect Coombs test detects incomplete antibodies in the serum, which is then incubated
with RBCs. The antibody-coated cells are then agglutinated by a Coombs antiglobulin
serum.

5. IMMUNOFUORESCENCE

This detects antibodies or antigens in tissue or fluids. It is important in the detection of SLE and
dermatologic diseases.

a. Direct immunofluorescence identifies antibody bound to tissue. An fluorescence labeled

antihuman immunoglobulin is overlaid on the tissue.

b. Indirect immunofluorescence tests for antibodies in the serum or plasma of the

patient.Normal tissueis overlaid on the serum, then fluorescence-labelled antihuman
immunoglobulin is overlaid on the tissue.

These are good in the detection of SLE, pemphigus, bullous pemphigoid, and Henoch-Schonlein
vasculitis

6. Complement assays determine complement levels in their serum.

 7. Complement fixation is a method to measure the levels of antibody and antigen by the
consumption of complement. Once the antigen0-antobody complexes fix complement, RBC are
added to bind with the unfixed complement. This is useful for HBsAg determination, antiplatelet
antibodies, Ig , and DNA.

8. Monoclonal antibodies refers to the production of antibodies with a single, identical specificity.
Monoclonal antibodies are the most widely used form of cancer immunotherapy at this time.

Monoclonal antibody therapy is a form of passive immunotherapy because it uses antibodies

made in large numbers outside the body (in the lab) rather than by a person's own immune
system. These treatments often do not require the person's immune system to take an "active"
role in fighting the cancer.

The first monoclonal antibodies were made in the lab by fusing a myeloma (a type of bone marrow
cancer) cell from a mouse with a mouse B cell that makes a specific antibody. The cell that results from
this fusion is called a hybridoma.

The combination of a B cell that can recognize a particular antigen and a long-lived myeloma cell makes
the hybridoma cell a kind of perpetual antibody-making factory. Because the antibodies are all identical
clones made from a single (mono) hybridoma cell, they are called monoclonal antibodies (sometimes
abbreviated as MoAbs, or MAbs).

Monoclonal Antibodies Used to Treat Cancer

MAb Name Trade Name Used to Treat: Approved in:

Rituximab Rituxan Non-Hodgkin lymphoma 1997

Trastuzumab Herceptin Breast cancer 1998

Acute myelogenous leukemia

Gemtuzumab ozogamicin* Mylotarg 2000
(AML)

Chronic lymphocytic leukemia

Alemtuzumab Campath 2001
(CLL)

Ibritumomab tiuxetan* Zevalin Non-Hodgkin lymphoma 2002

Tositumomab* Bexxar Non-Hodgkin lymphoma 2003

Colorectal cancer 2004

Cetuximab Erbitux
Head & neck cancers 2006

Colorectal cancer 2004

Bevacizumab Avastin
Non-small cell lung cancer 2006

Panitumumab Vectibix Colorectal cancer 2004

9. POLYMERASE CHAIN REACTION can identify the presence of DNA in a sample likeblood ,CSF,
and tissue specimens. DNA in the sample is first purified,and a synthetic DNA primer is added
 complementary to the pathogen DNA. Precursors of protein synthesis, nucleotides and
polymerases are then added, causing a replication of the DNA gfragments. The sample is then
electrophoresed to identify its size and specificity. Thus, it allows amplification of minute amounts
of DNA. Since this technique involves amplification of DNA, the most obvious application of the
method is in the detection of minuscule amounts of specific DNAs. This important in the detection
of low level bacterial infections or rapid changes in transcription at the single cell level, as well as
the detection of a specific individual's DNA in forensic science . It can also be used in DNA
sequencing, screening for genetic disorders, site specific mutation of DNA, or cloning or
subcloning of cDNAs.

PCR requires a template molecule of the DNA or RNA you want to copy and two primer
molecules to get the copying process started. The primers are short chains of the four different
base pairs that make up any strand of genetic material.

There are three basic steps in PCR. First, the target genetic material must be denatured. That is,
the strands of its helix must be unwound and separated by heating to 90-96C. The second step is
hybridization or annealing, in which the primers bind to their complementary bases on the now
single-stranded DNA. The third is DNA synthesis by a polymerase. Starting from the primer, the
polymerase can read a template strand and match it with complementary nucleotides very
quickly. The result is two new helixes in place of the first, each composed of one of the original
strands plus its newly assembled complementary strand.

To get more of the DNA, repeat the process, beginning by denaturing the DNA already made. The
amount will double every time. With the cycle of rapid heating and cooling controlled
automatically, nature-aided by scientist-supplied primers, polymerase, nucleotides, and chemical
reagents-does the rest. Each cycle takes only 1-3 minutes, so repeating the process for just 45
minutes can generate millions of copies of a specific DNA strand.

10.ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) is one of the most sensitive methods of
detecting specific antigens and antibodies. Antigen is attached to a plate , and antibody is added. If
present in the serum, the antibody will bind to the antigen. Unbound antibody is washed away, and an
enzyme is added to react tot possible antigen-antibody complexes. The enzyme causes a color change of
the plate/substrate, and by spectrophotometry, the amount of antibody is measured.

11. WESTERN BLOT is a modified ELISA procedure wherein the antigens to be assayed are separated
first by immunoelectrophoresis. Then they are transfer to a nitrocellulose medium (blot step).The
nitrocellulose with the antigen is now flooded with serum containing antibodies.The excess antibodies are
washed, and anti-antibodies are added. Instead of enzymes, radioactive isotopes are used and the
position of the antibody is determined by autoradiography. This is often used in detection of HIV.

In 1987 the Centers for Disease Control along with several other organizations established criteria for
serologic interpretation of HIV Western blot tests. The criteria are listed below.

No bands present Negative

Bands at either p31 OR p24 AND

bands present at either gp160 OR Positive
gp120

Bands present, but pattern does not

Indeterminate
meet criteria for positivity
Band pattern Interpretation

1. Lane 1, HIV+ serum (positive control)

2. Lane 2, HIV- serum (negative control)
3. Lane A, Patient A
4. Lane B, Patient B
5. Lane C, Patient C
viral envelope precursor (env)
gp160

gp120 viral envelope protein (env) binds to CD4

p24 viral core protein (gag)

p31 reverse transcriptase (pol)

HIV, like any other virus, is composed of a number of different

proteins. The Western blot positive control lane contains
proteins from patient sera as well as HIV proteins. HIV
positivity can therefore only be confirmed by the presence of
the following types of proteins: