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 γglobulin. 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 Rituximab Trastuzumab Gemtuzumab ozogamicin* Alemtuzumab Ibritumomab tiuxetan* Tositumomab* Cetuximab Bevacizumab Panitumumab Trade Name Rituxan Herceptin Mylotarg Campath Zevalin Bexxar Erbitux Avastin Vectibix Used to Treat: Non-Hodgkin lymphoma Breast cancer Acute (AML) Chronic (CLL) myelogenous lymphocytic leukemia leukemia Approved in: 1997 1998 2000 2001 2002 2003 cancer 2004 2006 2004 2006 2004

Non-Hodgkin lymphoma Non-Hodgkin lymphoma Colorectal Head & neck cancers

Colorectal cancer Non-small cell lung cancer Colorectal cancer

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. 2. 3. 4. Lane 1, HIV+ serum (positive control) Lane 2, HIV- serum (negative control) Lane A, Patient A Lane B, Patient B viral envelope precursor (env) gp160 gp120 p24 p31 viral envelope protein (env) binds to CD4 viral core protein (gag) reverse transcriptase (pol) 5. Lane C, Patient C

HIV,

the

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 following types of proteins: