Lecture Guide MLAB 1335 Immunology/Serology 2. … · Lecture Guide MLAB 1335 Immunology/Serology ... 1335 Immunology/Serology 2. Basic Immunologic Procedures 27 2) ... decrease in

Lecture Guide MLAB 1335 Immunology/Serology

2. Basic Immunologic Procedures 26

2. Basic Immunologic Procedures

I. Introduction

A. General characteristics of antigen/antibody reactions.

1. Sensitization is the basic reaction of an antigen and antibody binding.

2. Factors that affect antigen/antibody reactions include:

a. Concentration of the reactantsb. Temperaturec. Length of incubationd. pH of the test system

3. Classification of Antigen/Antibody reactions:

a. Primary phenomenon (sensitization)

1) Combination of single antibody to single antigen site.

2) Tests to detect this reaction are technically difficult, complex, expensive,may require special equipment and are time consuming.

3) Techniques utilized to detect this type of reaction include:immunofluorescence, radioimmunoassay and enzyme immunoassay.

b. Secondary phenomenon

1) Basic antigen/antibody reactions are taken a further step, forming crosslinks or lattice formation to create large molecules that are easilydetectable.

2) Methods used to detect these reactions are quick and easy to perform, lessexpensive, less time consuming and usually do not require specialequipment.

3) These methods are less specific, less sensitive and have moreinterferences.

4) Techniques utilized to detect this type of reaction include precipitation,agglutination and complement fixation.

A. Tertiary phenomenon

1) Antigen/antibody reaction is not visible but is detected by the affect thereaction has on tissues or cells.



2) These types of reactions include: inflammation, phagocytosis, depositionof immune complexes, immune adherence, deposition of immunecomplexes, and chemotaxis.

3. The secondary phenomena is a method of choice for many serological tests to detect in-vitro presence of antigen or antibody.

a. Precipitation involves combination of soluble antibody with soluble antigen toproduce insoluble complexes.

b. Agglutination is the process by which particulate antigen such as cells areaggregated to form large, visible aggregates if the specific antibody is present.

c. Complement fixation is the triggering of the classical complement pathway due tocombination of antigen with specific antibody.

B. Antigen-Antibody Binding

1. Primary union of antigen and antibody are depends on two characteristics, affinity andavidity.

2. Affinity is the initial force of attraction that an antibody for a specific antigenic epitope ordeterminant.

a. As they come close together a chemical bond forms which is rather weak and caneasily dissociate.

b. How well the antibody fits to the shape of the antigen will determine the stabilityof the bond.

c. Antibodies may react with antigens that are structurally similar to the originalantigen and results in cross reactivity, the greater the similarity of the originalantigen, the stronger the reaction.

d. Antigens and antibodies with the perfect lock and key fit will have the strongestaffinity.

3. Avidity is the sum of all attractive forces between an antigen and an antibody.

a. It is the force that stabilizes the antigen-antibody reaction, keeping the moleculestogether.

b. The stronger the chemical bonds which form between the antigen and antibody,the less likely that the reaction will reverse.

4. Law of Mass Action governs the reversibility of the antigen-antibody reaction.a. Free reactants are in equilibrium with bound reactants.

b. The equilibrium constant represents the rates of antigen/antibody binding anddissociating.



c. Measures the “goodness of fit” of the reaction, as the avidity increases the rate ofdissociation decreases.

C. Precipitation Curve

1. Precipitation reactions are also dependent on the amount of antigen and antibody presentin the test system

2. Prozone phenomenon occurs when excess antibody is present.a. So much antibody is present that all antigen sites are coated and lattice formation

cannot occur.

b. Results in false negative reaction.

3. Postzone phenomenon occurs when excess antigen is present.a. There is so much antigen present that the antibody molecules bind in such a way

that lattice formation cannot occur.

b. The antibody may bind to antigen sites on two separate molecules, sensitizationmay occur, but the binding is not on enough adjacent molecules to form lattice.

c. Results in a false negative reaction.

4. Zone of equivalence is when antigen and antibody are present in optimal proportions,sufficient antibodies can bind to antigens on adjacent molecules resulting in latticeformation.



II. Measurement of Precipitation By Light Scattering

A. Turbidimetry

1. Measures the turbidity or cloudiness of a solution by detection of light passing thru.

2. Turbidity is caused by formation of antigen-antibody complexes resulting in theformation of a precipitate, the more antigen or antibody present, the greater the turbidity.

3. The tube is placed in the direct path of a light source, the more turbid the solution, theless light will pass through.

4. The amount of substance being quantitated (antigen or antibody) is calculated based onresults obtained on standards and controls.

5. Very simple method but not very sensitive.

B. Nephelometry

1. Instead of measuring the decrease in light in turbidimetry, nephelometry measures theamount of light scattered by the antigen-antibody complexes.

2. The amount of light scattered is dependent on the number and the size of particles in thelight beam.

3. Nephelometers measure light scattered at an angle, laser beams are the most accurate asthe detect light deflected on a few degrees from the original light path.

4. Nephelometry is more sensitive as immunocomplexes tend to scatter light forward, whichinterferes with absorbance values.

5. In endpoint nephelometry the reaction is allowed to run to completion, but particles tendto fall out of solution and decrease light scatter.

6. Kinetic nephelometry measures the rate of increase in scattering immediately after thereagent is added, this rate is directly proportional to antigen or antibody concentration.

7. This technique is used to quantitate immunoglobulins, complement components, C-reactive protein, haptoglobin and other acute phase proteins.

III. Passive Immunodiffusion Techniques

A. Introduction

1. Antigen and antibody reactions occur in a gel, migrating towards each other and forminga detectable precipitate in the gel.



2. The rate of diffusion is affected by:

a. Size of the particlesb. Temperaturec. Gel viscosity and hydrationd. Interaction of the reactants with the gel

3. Classified into 4 types:

a. Single diffusion (one reactant moving) single dimension (up or down)b. Single diffusion, double dimension (moving our radially from a well)c. Double diffusion (both reactants moving) single dimensiond. Double diffusion, double dimension

B. Oudin - (single diffusion/single dimension)

1. Oudin first one to employ gels.

2. Antibody is added to agarose gel and placed in a tube, antigen is layered on top of the geland will diffuse down into the gel.

3. If the antibody present reacts with the added antigen a precipitin band will form in thegel.

C. Radial Immunodiffusion-RID (single diffusion/double dimension)

1. Antibody is added to the gel and poured into a plate, wells are cut into the plate.

2. Antigen is added to the well and will diffuse out radially from the well.

3. If the antibody present is specific for the antigen added a ring of precipitate will form, thesize of the ring is directly proportional to the concentration of the antigen.

4. Standards are run at the same time and a standard curve is created.

5. Two methods:

a. Endpoint method allows the reaction to go to completion.

b. Kinetic method employs measurements taken before the zone of equivalence isreached.

6. Technical sources of error:

a. Overfilling or under filling the well.b. Spilling sample outside of the well.c. Nicking the well.d. Improper incubation time or temperature.



D. Ouchterlony Gel Diffusion (double diffusion/double dimension)

1. Ouchterlony Immunodiffusion is a method used for comparison of antigens.

2. Holes are cut in the agar, one central hole surrounded by other wells.

3. Antibody is added to the central well, antigens are added to the outer wells, the positionof the bands formed between the antigens allows for comparison of the antigens to eachother.

4. Three possibilities:

a. Identity-the bands form an arc.b. Partial identity-fusion of 2 lines with a spur.c. Non-identity- pattern of lines which cross each other.

IV. Electrophoretic Techniques

A. Introduction

1. Immunodiffusion can be combined with electrical current to speed things up.

2. Electrophoresis is a technique which separates molecules according to differences in theirelectrical charge when they are exposed to an electric current.

3. A direct current is applied to the gel, the antigen and antibody migrate through the gel, asdiffusion takes place precipitin bands are formed.

4. Can be applied both as a single or double diffusion method.

B. Rocket Immunolectrophoresis

1. Description

a. One-dimensional electroimmunodiffusion,it is an adaptation of radialimmunodiffusion (RID).

b. A quatitative method for measuring serum proteins.

c. Involves electrophoresis of antigen into a gel contains antibody.

d. The technique is restricted to detection of antigens that move to the positive poleduring electrophoresis.

2. Procedure

a. Agar mixed with antibody and poured into a plate, wells (holes) cut into agar.

b. Antigen is placed in the wells, an electrical current is applied and precipitationbegins.



c. As the concentration of antigen changes, there is dissolution and reformation ofthe precipitate at ever increasing intervals.

d. The end result is a precipitin line with a conical shape resembling a rocket.

e. The height is measured and is directly proportional to the concentration ofantigen.

f. If standards are run a standard curve is constructed and concentration determined.

3. Advantage over RID is the results are obtained in a few hours.

4. Primarily used to quantitate immunoglobulins and to assay proteins whose concentrationsare too low for nephelometry but too high for RID

C. Immunoelectrophoresis (IEP)

1. Description.

a. A two-step double-diffusion technique which first involves the electrophoreticseparation of proteins.

b. Followed by the linear diffusion of antibodies into the electrophoretic gel from atrough which extends through the length of the gel adjacent to the electrophoreticpath.

2. Procedure

a. Proteins are first electrophoresed to separate them.

b. A trough is cut in the gel parallel to the line of separation, antiserum is added tothe trough and the gel is incubated overnight.

c. Double diffusion occurs as the antibody and separated proteins diffuse towardsone another in the gel and form precipitin lines in the gel.

d. The lines can be compared in shape, intensity and location to that of normal serumcontrol to detect abnormalities and is semiquantitative.

3. Excellent screening test to differentiate serum proteins and detect abnormalities such as inmyelomas, Waldenstrom’s macroglobulinemia, malignant lymphomas and otherlymphoproliferative disorders.

4. Can also identify immunodeficiencies.



D. Immunofixation Electrophoresis (IFE)

1. Description

a. Similar to IEP except that after electrophoresis is performed the antiserum isapplied directly to the surface of the gel.

b. The most sensitive method used to detect, confirm and characterize monoclonalgammopathies such as those found in plasma cell dyscrasias such as multiplemyeloma Waldenstrom’s macroglobulinemia, monoclonal gammopathy ofunknown significance, light or heavy chain disease.

c. Particularly useful in identifying minor monoclonal proteins, the light chaincomponent of monoclonal proteins, and unknown bands seen in serum proteinelectrophoresis.

2. Procedure

a. Gel with antibody is electrophoresed

b. The antibody is impregnated into a cellulose acetate strip which is placed over thegel.

c. The antibody from the strip diffuses down into the gel and a precipitate will formif the specific antigen is present.

d. After washing the strip to remove extraneous proteins a stain is applied.

3. The best adaptation of this test is the Wester Blot test to detect antibodies to the HumanImmunodeficiency virus 1 (HIV-1).

a. A mixture of HIV antigens is placed on a gel and electrophoresed to separate thecomponents of the HIV antigen.

b. The components are transferred to nitrocellulose paper.

c. Patient serum is added to the paper and allowed to react.

d. The strip is washed and stained to detect precipitin bands indicating the presenceof antibodies to the HIV antigen.

e. Must have several bands present to be positive.

E. Sources of Error in Electrophoresis

1. Applying current in wrong direction.2. Incorrect buffer pH.3. Incorrect timing.4. Amount of current applied.



V. Agglutination

A. Overview

1. Definition - the clumping together in suspension of antigen-bearing cells,microorganisms, or particles int the presence of specific antibodies (agglutinins).

2. Two step process involving sensitization and lattice formation.

3. Particles used in the test can be red blood cells (hemagglutination), bacterial cells, or inertparticles such as latex or charcoal.

B. Steps in Agglutination

1. Sensitization

a. First step involves attachment of antibody to it’s specific antigen.

b. Rapid and reversible reaction.

c. This step affected by antibody affinity and avidity which will determine howmuch antibody remains attached.

d. IgM much more efficient than IgG in agglutination reactions.

e. Antigen bearing surfaces must have sufficient quantities of the epitope present, ifsparse or obscured less likely to interact with antibody.

2. Lattices Formation

a. Second stage represents sum of interaction between antigen and antibody.

b. Dependent upon environmental conditions and concentrations of antigen andantibody.

c. Physicochemical factors such as ionic strength, pH and temperature affectreaction.

d. Antibody must be able to bridge gap between two particles to form lattice.

e. Electrical charge of particles may hinder lattice formation, especially important ifreaction involves IgG, IgM usually able to overcome.

3. Enhancement of Agglutination

a. Surface charge must be controlled, can be accomplished by additve whichneutralizes surface hcarge.

b. Increasing viscosity can also neutralize charge.



c. Treatment of antigen with certain enzymes can remove substances on cell surfaceto reduce interference of electrical charge.

d. Agitation and centrifugation provides physical means to increase cell-to-cellcontact and enhance strength of reaction.

e. Temperature is critical and depends on antibody class involved: IgM prefers roomtemperature or lower, IgG reacts best at body temperature 37C.

f. Optimal pH of 6.7 to 7.2.

B. Types of Agglutination Reactions

1. Introduction

a. Agglutination reactions have many advantages: easy to carry out, no complicatedequipment and can be performed as needed.

b. Most agglutination reactions are available as prepackaged kits with all reagentsand controls needed present.

c. Can detect either antigen or antibody, critical to know principle of procedure.

d. Most reactions are qualitative, either positive or negative, which indicatespresence or absence, although titers can be performed to give semi-quantitativeresults.

2. Direct Agglutination

a. Agglutination of an antigen found naturally on a particle.

b. Can be used to identify bacteria as when serotyping Salmonella species.

c. Test patient serum against known bacteria in suspensions when cultivation ofbacteria is difficult: Tularemia, rickettsial diseases, and typhoid fever.

d. Hemagglutination utilized to determine ABO blood grouping.

e. Hemagglutination kits available for detection of antibodies to hepatitis B, C andHIV I and II

3. Passive Agglutination

a. The agglutination of inert particles by antibody directed against antigen bound totheir surface.

b. Employs particles that are coated with antigens such as RBCs, polystyrene latex,bentonite or charcoal.

c. Artificial particles have advantage of consistency, uniformity and stability.



d. Reactions are generally very easy to read macroscopically.

e. Many antigens adsorb to RBCs spontaneously, tanned sheep RBCs frequentlyused..

f. IgG naturally adsorbs to surface of latex particles.

g. Passive agglutination routinely used for the following tests: antinuclearantibodies, group A strep, Rheumatoid factor, and antibodies to viruses such ascytomegalovirus (CMV), rubella and varicella-zoster.

4. Reverse Passive Agglutination

a. Antibody attached to carrier particle instead of antigen.

b. Antibody must be active and be oriented such that the active site is facingoutward.

c. Often used for detection of microbial antigens such as: Group A and BStreptococcus, Staphylococcus aureus, Neisseria Meningitidis, Haemophilusinfluenzae, Cryptococcus neoformans, Mycoplasma pneumoniae and Candidaalbicans.

d. Some of these organisms very difficult to grow and/or quick diagnosis needed socorrect treatment can be started.

e. Widest application is in detecting soluble antigens in urine, spinal fluid andserum.

f. Antigens present in these fluids will attach to antibodies on particles.

g. Extraction step may be necessary.

5. Agglutination Inhibition

a. Based on competition between particulate and soluble antigens for limitedantibody combining sites.

b. Patient sample added to reagent antibody specific for antigen being tested, ifantigen is present it binds to reagent antibody.

c. Reagent particles (latex or RBCs) coated with the same antigen are added, ifantigen was present in the sample all reagent antibody binds to it so no antibody ispresent to react with antigens coating the particles.

d. Used for detecting antibodies to certain viruses such as: rubella, mumps, measlesand influenza for which RBCs have neaturally occurring viral receptors.

e. Test: incubate patient serum with viral preparation, add RBCs which the virus isknown to agglutinate, if patient antibody present it will bind to free viurs and



cannot attach to RBCs, no agglutination is a positive result.

f. Used as a turbidimetric procedure for therapeutic drug monitoring

6. Coagglutination

a. Name given to systems using bacteria as the inert particles to which antibody isattached.

b. Directly detects soluble bacterial antigens.

c. Staphylococcus aureus most frequently used.

d. Bacteria are not colored and reactions are difficult to read.

e. Not as sensitive for detecting small quantities of antigen.

f. Used to identify Streptococci, Neisseria meningitidis, Neisseria gonorrhea andHaemophilus influenzae.

g. These may provide true positive results when culture and gram stain results arenegative and for patients who have already received antimicrobial therapy.

h. Antigen detection methods should never be substituted for culture and gram stain.VI. Labeled Immunoassays

A. Introduction

1. Need rapid, specific, sensitive assays.

2. Labeled immunoassays

a. Some antigen/antibody reactions not detected by precipitation or agglutination.

b. Measured indirectly using a labeled reactant.

c. Referred to as receptor-ligand assays.

d. Ligand is the substance to be measured and is defined as a molecule that binds toanother molecule of a complementary configuration, usually it binds to the substance the test is trying to detect.

e. The receptor is what binds the specific target molecule.

3. Sometimes referred to as a sandwich technique.

a. Have a receptor of some kind that will bind to the antigen or antibody the systemis trying to detect.



b. If the substance is present it will bind and a washing process eliminatesextraneous substances present.

c. A labeled ligand is added which also has specificity for the substance beingdetected and results in a labeled product.

d. This results in a sandwich, receptor, substance to be detected and ligand.

e. Depending upon the ligand label a visible or detectable reaction will occur.

B. Constituents of Labeled Assays

1. Labels which may be used include: fluorescent, radioactive, chemiluminescent andenzymes.

2. The assay includes the use of:

a. Labeled and non-labeled ligandsb. Specific antibodyc. Standards and calibratorsd. Means of separating bound from free componentse. Means of label detection.

3. Competitive binding

a. A labeled ligand is mixed with the patient serum.

b. It is added to the receptor.

c. Competitive binding occurs in which patient sample and labeled ligand “compete”for antibody sites.

d. If little or no antigen is present in the patient sera a strong positive occurs.

e. If there is a lot of antigen in the patient sera, it will successfully bind in largequantities, causing a decrease in the binding of the labeled ligand, causing adecrease in color, radioactivity, etc.

4. Antibodies used in labeled immunoassay reactions must have high affinity.

5. Standards or calibrators are substances of known concentration.

a. Standards are run at the same time as patient sample.

b. Usually three standards are run, the results are graphed, and a standard curve isdrawn.

c. The concentration of the unknown patient sample can be determined from thestandard curve.



6. Once a reaction has occurred there must be a way to remove unbound analytes from thereaction, this is done through a separation method.

a. Can measure either bound ligand or free ligand remaining after separationmethod.

b. Unreacted ligand can be removed by adsorption onto inert particles added to thesystem.

c. Precipitation of the antigen-antibody complexes.

d. Use a second antibody to precipitate out the antigen-antibody complexes.

e. Solid phase is the most popular method, antigen or antibody is physically attachedto a tube, plate, etc, the substance being detected will bind, the excess is washedaway.

f. In all procedures, the separation method is a limiting factor.

g. Separation procedure must be precise and reproducible.

7. Detection of the labeled analyte.

a. There must be an accurate system for detecting and measuring the labeled productproduced.

b. For radioimmunoassay, radioactivity is measured.

c. For labels such as enzymes, fluorescence or chemiluminescence, changes inabsorbancy on a spectrophotometer are used.

8. Quality control procedures must always be performed to ensure the accuracy of the resultsobtained.

a. “Blanks” are tubes filled with a clear solution or, if the solutions added havecolor, with the solution only, to determine the “background”, any substancespresent in the original solution must be calculated out.

b. Controls are substances with a known range of values and generally three levelsare run: normal, high and low.

c. If controls do not give the expected values the results cannot be reported out.

C. Radioimmunoassay (RIA) Techniques

1. Competitive Binding Assays

a. Uses radioactive substance as a label, usually I .125



b. Antibody is bound to a tube or other solid matrix.

c. A measured amount of patient sample is added to a measured amount ofradiolabeled analyte or ligand.

d. The antigen in the patient sample and the radiolabeled antigen compete for thebinding to the antibody.

e. If there is no antigen in the sample then there will be a high level of radiationsince the radiolabeled antigen can bind to all antibody sites.

f. If antigen is present in the patient sample then radioactivity will be decreasedproportionally to the amount of antigen present.

g. Standards are run to create a standard curve.

h. Controls are run to ensure proper technique and reagent reactivity.

2. Immunoradiometric Assay (IRMA)

a. Excess labeled antibody is added to a tube with patient antigen.

b. All patient antigen is bound by the antibody.

c. Solid phase antigen is added which will then bind up all excess labeled antibody.

d. The tube is spun and the solid phase antigen will go to the bottom, all antibodybound to patient antigen remains in solution.

e. The radioactivity of the supernatant solution is determined.

f. The count obtained is directly proportional to the amount of patient antigenpresent in the specimen.

g. Advantages: faster, reaction, increased sensitivity and specificity.

h. Disadvantage: pure antigen and antibody are needed.

3. Procedures which utilize RIA are: human chorionic gonadotropin (HCG), follicle-stimulating hormone (FSH), gastrin, insulin, carcinoembryonic antigen (CEA), thyroxine,estrogens, androgens, IgE and erythropoietin.

4. Disadvantages of using RIA:

a. Health hazardb. Disposal of radioactive wastec. Short shelf lifed. Expensive equipment



D. Enzyme Immunoassay (Perform ELISA Virutal Lab - Serology Online Activities)

1. Introduction

a. Advantages of enzyme immunoassay:

1) labels cheap and plentiful.2) labels have a long shelf life3) easily adapted to automation4) reaction measured using inexpensive equipment5) very sensitive6) no health hazards associated with reagents7) can be used for qualitative OR quantitative procedures.

b. Enzymes chosen for us as labels according to the following:

1) number of substrate molecules converted per molecule of enzyme2) purity3) sensitivity4) ease and speed of detection5) stability6) absence of interfering substances7) availability8) cost

c. Typical enzymes used include:

1) horseradish peroxidase - cheap, very popular, reacts with a number ofchromogens

2) glucose oxidase3) glucose-6-phosphate dehydrogenase - uses fluorimetric means4) alkaline phosphatase - expensive5) β-D-galactosidase

d. The enzyme label is linked to antibody or ligand.

e. Two classification of enzyme assays:

1) Heterogenous requires a step to physically separate bound ligand from free.

2) Homogeneous assays require no separation step.

3. Heterogenous Enzyme Immunoassays

a. Competitive Enzyme Linked Immunosorbent Assays (ELISA)

1) Enzyme labeled ligand competes with unlabeled patient ligand for bindingsites on antibody molecules attached to solid phase.

2) After reacting, a washing step is performed.



3) Enzyme activity is determined.

4) Enzyme activity is inversely proportional the concentration of the testligand.

5) Sensitivity in nanograms (10 g/mL) can be achieved.-9

b. Noncompetitive ELISA

1) Referred to as indirect ELISA test.

2) Antigen is bound to solid phase, unlabeled patient antibody is added.

3) After incubation a wash step is performed and an enzyme labeled substrateis added.

4) The amount of enzyme label detected is directly proportional to theamount of antibody in the specimen.

5) Indirect ELISAs are very sensitive.

6) Disadvantage is there is more manipulation of the test.

c. Immunoenzymometric Assay

1) Non competitive ELISA

2) Detects unknown antigen by means of excess labeled antibody.

3) Antibody and patient sample allowed to react.

4) Antigen attached to solid phase, such as glass beads, added to test system.

5) Solid-phase antigen combines with unreacted labeled antibody.

6) Centrifuge tube and measure supernatant which contains antigen-antibodycomplexes.

7) This method requires less time, eliminates nonspecific reactivity and hasgreater sensitivity than other competitive ELISAs.

d. Sandwich or Capture Assays

1) Used with antigens having multiple epitopes.

2) Excess antibody attached to solid phase, allowed to combine with testsample.

3) After incubation, enzyme labeled antibody is added.



4) Second antibody may recognize same or different epitope than solid phaseantibody.

5) Enzyme activity directly related to concentration of antigen.

6) Detects antigens present in low concentrations.

7) Suited to antigens with multiple epitopes.

8) Epitope must be unique to the organism and present in all strains.

9) Major use of this technique is for measurement of immunoglobulins, theimmunoglobulin is the antigen and an anti-antibody is added to the testsystem.

10) Disadvantage: linear relationship does not exist, must prepare standardcurve, test conditions must be carefully controlled.

4. Homogeneous Enzyme Immunoassay

a. Reagent antigen is labeled with enzyme tag, patient sample is added.

b. When antibody binds to antigen, stearic hindrance to the enzyme occurs whichinactivates the enzyme.

c. Antigen competes with enzyme labeled analyte for limited number of antibodybinding sites, this is a competitive assay.

d. Enzyme activity is directly proportional to concentration of patient antigen.

e. No separation step necessary.

f. Sensitivity of the procedure is determined by:

1) detectability of enzymatic activity2) change in activity when antibody binds to antigen3) strength of antibody bond4) susceptibility of the assay to interferences such as cross reactivity.

g. Sensitivity far less than heterogenous enzyme assays.

5. Advantages of Enzyme Immunoassay

a. Sensitivity similar to RIA without health hazards and reagent disposal problems.b. Expensive equipment not necessary, spectrophotometers or notation of color change.c. Most are fairly simple



6. Disadvantages

a. Some samples may have natural inhibitors.b. Size of enzyme label limiting factor in designing some assays.c. Nonspecific protein binding may occur.d. Enzyme reactions very sensitive to temperature.

E. Fluorescent Immunoassay

1. Introduction

a. Use fluorescent compounds called fluorophores or fluorochromes as markers.

b. Markers have the ability to absorb energy and convert it into light.

c. Fluorescent probe must exhibit high intensity to distinguish from background.

d. Two compounds most frequently used are fluorescein and rhodamine.

1) Fluorescein emits green color, it has high intensity and goodphotostablility.

2) Tetramethylrhodamine emits red light.

3) Newer compounds include algae, porphyrins and chlorophylls.

e. Fluorescent tags first used for localization of antigen in tissues.

1) Immunofluorescent assay (IFA) is qualitative observation of fluorescentreaction using fluorescent scope for visualization.

2) Can detect antigens in fixed cells or live cell suspensions.

3. Fluorescent Microscopy

a. Uses light source that emits light of the appropriate wavelength necessary toexcite fluorochrome used.

b. The presence of a specific antigen is determined by the appearance of localizedcolor against a black background.

c. Fluorescent staining

1) Direct immunofluorescent assay the antibody has a fluorescent tags,added directly to unknown antigen fixed to slide, after incubation andwashing observe for fluorescence.



2) Indirect immunofluorescent assay react patient serum with knownantigen attached to solid phase, wash, add anti-human antibody attached toflourescent tag, wash, observe for fluorescence.

3. Heterogeneous Fluorescent Immunoassays

a. Fluorescent immunoassays (FIA) are classified as heterogeneous or homogeneous.

b. Heterogeneous assays require a separation step and include:

1) indirect2) competitive3) sandwich4) capture

c. Same principle as EIA except fluorescent label is used.

d. Can detect either antigen or antibody.

e. Solid phase uses antigen or antibody attached to beads which may be magnetized.

f. When beads are utilized the reaction occurs, the tube centrifuged, supernatantdiscarded, and the beads observed for fluorescence.

g. Magnetized beads are placed on a magnetic surface to attract beads to the bottomof the tube.

h. Solid phase can also employ a dipstick coated with antigen or antibody, react withpatient sample then add a labeled antibody.

5. Homogenous Fluorescent Immunoassays

a. Just like EIA, no separation necessary, one incubations step and no wash step.

b. Usually involves competitive binding.

c. Direct relationship between amount of fluorescence and quantity of antigen inpatient sample.

d. This technique suffers from lack of sensitivity.

6. Fluorescence Polarization Immunoassay (FPIA)

a. Based on change in polarization of fluorescent light emitted from labeledmolecule bound by antibody.

b. When fluorophores are excited, emit partially polarized fluorescence.

c. Antibody bound labeled antigens will emit more polarized light.



d. Competitive binding.e. Degree of fluorescence inversely related to concentration of analyte.f. Technique limited to small molecules.g. Used most frequently for therapeutic drugs and hormones.h. Requires sophisticated instrumentation.

7. Advantages of fluorescent techniques:

a. Methods fairly simple.b. No hazardous reagents to use or discard.c. Increased sensitivity over radiolabeled and enzyme reactions.

8. Disadvantages include:

a. Fluorescent compounds are sensitive to environmental changes.b. Stability of labelsc. Nonspecific binding can diminish signal.d. Bilirubin or hemoglobin can absorb the excitation or emission energy.e. Requires expensive dedicated instrumentation.

9. Chemiluminescent Immunoassays.

a. Chemiluminescence is the production of light energy due to a chemical reaction.

b. Most common substances used are: luminol, acridium esters and dioxetanephosphate.

c. The substance is oxidized, usually with hydrogen peroxide and an enzyme for acatalyst.

d. Energy given off in the form of light.

e. Heterogeneous and homogenous assays available.

f. Disadvantages:

1) False results may be obtained if lack of precision in addition of hydrogenperoxide.

2) Some biologic materials cause quenching.3) Need dedicated instrumentation.

g. Advantages:

1) reactive stability of label2) speed of detection3) sensitivity comparable to RIA and EIA4) low cost



G. Summary1. Labeled assays are used to measure antigen OR antibody.2. Different labels can be used.3. Two classification of enzyme assays:

a. Heterogenous requires a step to physically separate bound ligand from free.b. Homogeneous assays require no separation step.

4. Immunoassay labels must be very specific and have high affinity.5. Radioactive labels

a. RIA is COMPETITIVE - known antibody added in specific amount to unknownsample, competes with patient antibody to attach. Indirect measurement.

b. IRMA - all unknown antigen reacts due to excess antibody being added.6. Enzymes can be used as labels, results in a color reaction.

a. Most assays today are non-competitive.b. Sandwich technique very popularc. Homogeneous enzyme assays - no separation necessary, antibody binding causes

antigen labeled with enzyme to lose activity.7. Fluorescence

a. Direct immunofluorescence detects antigen directly by using labeled antibody.b. Indirect immunofluorescence utilizes sandwich technique.



VII. Molecular Biology Techniques

A. Characteristics of Nucleic Acids1. Molecular biological techniques exceptional new and powerful tools.2. Based on detection of specific nucleic acid sequences in microorganisms and particular

cells.3. Procedures include: enzymatic cleavage of nucleic acids, gel electrophoresis, enzymatic

amplification of target sequences and nucleic acid probes.4. Two types of nucleic acid:

a. DNA and RNA have same purine bases, adenine and guanine, differ in thepyrimidine bases.

b. Deoxyribonucleic acid (DNA)1) sugar deoxyribose present2) DNA is organized as two complementary strands, head-to-toe, with bonds

between them that can be "unzipped" like a zipper, separating the strands;3) DNA is encoded with four interchangeable "building blocks", called

"bases", Adenine, Thymine, Cytosine, and Guanine, with U rarelyreplacing T, each base "pairs up" with only one other base: A+TorU,TorU+A, C+G and G+C ; that is, an "A" on one strand of double-strandedDNA will "mate" properly only with a "T" or "U" on the other,complementary strand;

4) Carries primary genetic information5) Human genome more than 3 billion DNA bases long

c. Ribonucleic acid (RNA)1) Ribose primary sugar2) RNA has five different bases: adenine, guanine, cytosine, uracil, and more

rarely thymine. 3) The first three are the same as those found in DNA, but uracil usually

replaces thymine as the base complementary to adenine.



4) Exceptions include transfer RNA, which always has thymine on one of itsloops. This may be because uracil is energetically less expensive toproduce.

5) In DNA, however, uracil is readily produced by chemical degradation ofcytosine, so having thymine as the normal base makes detection and repairof such incipient mutations more efficient.

6) Thus, uracil is appropriate for RNA, where quantity is important butlifespan is not, whereas thymine is appropriate for DNA wheremaintaining sequence with high fidelity is more critical.

7) Structurally, RNA is indistinguishable from DNA except for the criticalpresence of a hydroxyl group attached to the pentose ring in the 2' position(DNA has a hydrogen atom rather than a hydroxyl group). This hydroxylgroup makes RNA less stable than DNA because it makes hydrolysis ofthe phosphosugar backbone easier

5. Replication of DNAa. DNA very stable, but loses conformational structure under extreme conditionsb. Replication straightforward, semi-conservative process uses one strand as

template to create two daughter molecules, each an exact copy.c. in lab double helix separated or denatured by heat (melting) or alkaline solution.d. As DNA strands returned to normal physiological conditions, complementary

strands spontaneously rejoin or anneal. e. Replication is performed by splitting (unzipping) the double strand down the

middle via relatively trivial chemical reactions, and recreating the "other half" ofeach new single strand by drowning each half in a "soup" made of the four bases.

f. Since each of the "bases" can only combine with one other base, the base on theold strand dictates which base will be on the new strand.

g. This way, each split half of the strand plus the bases it collects from the soup willideally end up as a complete replica of the original, unless a mutation occurs.



B. Nucleic Acid Probes

1. Spontaneous pairing of complementary DNA strands forms basis for techniques used todetect and characterize genes.

2. Probe technology used to identify individual genes or DNA sequences.3. Nucleic acid probe short strand of DNA or RNA of known sequence used to identify

presence of complementary single strand of DNA in patient sample.4. Binding of 2 strands (probe and patient) known as hybridization.5. Two DNA strands must share at least 16 to 20 consecutive bases of perfect

complementarity to form stable hybrid.6. Match occurring as a result of chance less than 1 in a billion.7. Probes labeled with marker: radioisotope, fluorochrome, enzyme or chemiluminescent

substrate.8. Hybridization can take place in solid support medium or liquid.

C. Hybridization Techniques

1. Hybridization is the process of forming a double-stranded DNA molecule between asingle-stranded DNA probe and a single-stranded target patient DNA.

2. Dot-blot and sandwich hybridizations are simplest solid support hybridization assays.

3. Dot-blota. Dot-blot clinical sample applied to membrane, heated to denature DNA.b. Labeled probes added, c. Wash to remove unhybridized probe and measure reactants.d. Qualitative test only.e. May be difficult to interpret.

4. Sandwich hybridizationa. Modification of dot-blot.b. Uses two probes, one bound to membrane and serves as capture target for sample

DNA, second probe anneals to different site on target DNA and has label fordetection., sample nucleic acid sandwiched between the two.

c. Two hybridization events occur, increases specificity.d. Can be adapted to microtiter plates.e. Restriction endonucleases cleave both strands of double stranded DNA at specific

sites, approximately 4 to 6 base pairs long.f. Further separated on the basis of size and charge by gel electrophoresis.g. Digested cellular DNA from patient/tissue added to wells in agarose gel and

electric field applied, molecules move.h. Gel stained with ethidium bromid and vieuwed under UV light.i. Differences in restriction patterns referred to as restriction fragment length

polymorphisms (RFLPs)j. Caused by variations in nucleotides within genes that change where the restriction

enzymes cleave the DNA.k. When such a mutation occurs different size pieces of DNA are obtained.



5. Southern Blota. DNA fragments separated by electrophoresis.b. Pieces denatured and transferred to membrane for hybridization reaction.c. Place membrane on top of gel and allow buffer plus DNA to wick up into it.d. Once DNA is on membrane heat or use UV ligth to crosslink strands onto

membrane to immobilize.e. Add labeled probe for hybridization to take place.

6. Northern Blota. Similar, but RNA is extracted and separated.b. Used to determine level of expreseeion of particlar messenger RNA species to see

if a gene is actually being expressed.c. Because RNA is a short, single-stranded molecule it does not have to be digested

before electrophoresed.d. Must be denatured to ensure it is unfolded and linear.e. probes are used to identify presence of specific bands.

7. Solution Hybridizationa. Both target nucleic acid and probe free to interact in solution.b. Hybridization of probe to target in solution is more sensitive than hybridization on

solid support c. Requires less sample and is more sensitive.d. Probe must be single-stranded and incapable of self-annealing.e. Fairly adaptable to automation, especially tose using chemiluminescent labels.f. Assays performed in a few hours.

8. In Situ Hybridizationa. Target nucleic acid found in intact cells.b. Provides information about presence of specific DNA targets and distribution in

tissues, the word "in situ" means that the hybridization occurs "in place", in thiscase, within the nucleus of specimen cells that have been fixed to a microscopeslide.

c. Fluorescence in situ hybridization (FISH) is a type of hybridization in which aDNA "probe" is labeled with fluorescent molecules so that it can be seen with amicroscope.

d. To conduct a FISH analysis, one warms fixed cells mounted on a microscope slideto unwind their chromosomal DNA and allow access of the DNA probe.

e. After adding the probe, the specimen cells are then cooled to allow the DNAprobe to hybridize with its complementary target DNA.

f. Once hybridized, the fluorescent molecules on the probe will show preciselywhere their target DNA lies along a chromosome.

g. Depending upon the design of the probe DNA, one can detect many types ofgenetic changes.

h. Probes must be small enough to reach nucleic acid.i. Radioactive or fluorescent tags used and requires experience.

9. DNA Chip Technologya. A DNA chip (DNA microarray) is a biosensor which analyzes gene information

from humans and bacteria. b. This utilizes the complementation of the four bases labeled A (adenine), T

(thymine), G (guanine) and C (cytosine) in which A pairs with T and G pairs withC through hydrogen bonding.



c. A solution of DNA sequences containing known genes called a DNA probe isplaced on glass plates in microspots several microm in diameter arranged inmultiple rows.

d. Genes are extracted from samples such as blood, amplified and then reflected inthe DNA chip, enabling characteristics such as the presence and mutation of genesin the test subject to be determined.

e. As gene analysis advances, the field is gaining attention particularly in the clinicaldiagnosis of infectious disease, cancer and other maladies.

f. Used to examine DNA, RNA and other substances.g. Allow thousands of hybridization reactions to be performed at once.h. Florescent tags used, excited and emission measured with optical scanner.i. Example is determination of genes associated with drug resistance in HIV testing.

10. Drawbacks

a. Stringency, or correct pairing, is affected by:1) salt concentration2) temperature3) concentration of destabilizing agent such as formamide or urea.

b. If conditions not carefully controlled mismatches can occur.c. Patient nucleic acid may be present in small amounts, below threshold for probe

detection.d. Sensitivity can be increased by amplification: target, probe and signal

D. Target Amplification1. Introduction

a. In-vitro systems for enzymatic replication of target molecule to detectable levels.b. Allows target to be identified and further characterized.c. Examples: Polymerase chain reaction, transcription mediated amplification,

strand displacement amplification and nucleic acid sequence-based amplification.2. Polymerase Chain Reaction (PCR)

a. Capable of amplifying tiny quantities of nucleic acid.b. Cells separated and lysed.c. Double stranded DNA separated into single strands.d. Primers, small segments of DNA no more than 20-30 nucleotides long added.e. Primers are complementary to segments of opposite strands of that flank the target

sequence.f. Only the segments of target DNA between the primers will be replicated.g. Each cycle of PCR consists of three cycles:

1) denaturation of target DNA to separate 2 strands.2) annealing step in which the reaction mix is cooled to allow primers to

anneal to target sequence3) Extension reaction in which primers initiate DNA synthesis using a DNA

polymerase.4) These three steps constitute a thermal cycle

h. Each PCR cycle results in a doubling of target sequences and typically allowed torun through 30 cycles, one cycle takes approximately 60-90 seconds.



i. Taq polymerase ("Taq pol") is a thermostable polymerase isolated from thermusaquaticus, a bacterium that lives in hot springs and hydrothermal vents. "Taqpolymerase" is an abbreviation of Thermus Aquaticus Polymerase. It is often usedin polymerase chain reaction, since it is reasonably cheap and it can survive PCRconditions. Its halflife at 95 degrees Celsius is 1.6 hours. Unfortunately, it doesn'thave a very high fidelity since it lacks the 3' --> 5' exonuclease proofreadingcapacity to replace an accidental mismatch in the newly synthesized DNA strand.

j. With PCR, it is routinely possible to amplify enough DNA from a single hairfollicle or single sperm cell for DNA typing.

k. The PCR technique has even made it possible to analyze DNA from microscopeslides of tissue preserved years before.

l. The great sensitivity of PCR makes contamination by extraneous DNA a constantproblem

3. Transcription Mediated Amplificationa. TMA is the next generation of nucleic acid amplification technology.b. TMA is an RNA transcription amplification system using two enzymes to drive

the reaction: RNA polymerase and reverse transcriptase. c. TMA is isothermal; the entire reaction is performed at the same temperature in a

water bath or heat block. This is in contrast to other amplification reactions suchas PCR or LCR that require a thermal cycler instrument to rapidly change thetemperature to drive the reaction.

d. TMA can amplify either DNA or RNA, and produces RNA amplicon, in contrastto most other nucleic acid amplification methods that only produce DNA.

e. TMA has very rapid kinetics resulting in a billion fold amplification within 15-30minutes.

E. Probe Amplification1. Amplify detection of molecule or probe instead of target molecule.2. QB Replicase

a. uses an RNA directed RNA polymerase that replicates the genomic RNA of abacteriophage named QB.

b. The RNA genome of QB is essentially the only substrate recognized by thepolymerase.

c. Because a short probe can be inserted into the QB RNA this becomes the systemfor amplification.

d. After the probe has annealed to the target, unbound probe is treated with RNaseand washed away.

e. The hybridized probe is RNase resistant.f. When QB replicase is added the probe is enzymatically replicated to detectable

levels.3. Ligase Chain Reaction LCR

a. The LCR test employs four synthetic oligonucleotide probes (two per DNAstrand) anneal at specific target sites on the cryptic plasmid.

b. Each pair of probes hybridize close together on the target DNA template, with 1-to 2- nucleotide gap between.

c. Once the probes are annealed, the gap is filled by DNA polimerase and close bythe ligase enzyme.

d. This two-step process of closing the gap between annealed probes makes theLCR, in theory, more specific than PCR technology.



e. The ligated probe pairs anneal to each other and, upon denaturation, from thetemplate for successive reaction cycles, thus producing a logarithmicamplification of the target sequence.

f. Like PCR, LCR is made in a thermocycler. g. The LCR product is detected in an automated instrument that uses an

immunocolorimetric bead capture system. h. At the end of the LCR assay, amplified products are inactivated by the automatic

addition of a chelated metal complex and a oxidizing agent. F. Signal Amplification

1. Replicates signal rather than either the target or the probe.2. Based on the reporter group (the labeled tag) being attached in greater numbers to the

probe molecule or increasing the intensity generated by each labeled tag.3. Patient nucleic acid not replicated or amplified technique is less prone to contamination.4. Sensitivity is lower.5. Branched chain signal amplification employs several simultaneous hybridization steps.6. Author states similar to decorating a Christmas tree and involves several sandwich

hybridizations.a. first, target specific oligonucleotide probes captures target sequence to solid

support.b. Second set of target specific probes called extenders hybridize to adjoining

sequences and act as binding site for large piece called branched amplificationmultimer.

c. Each branch has multiple side branches capable of binding numerousoligonucleotides.

d. Branched chains are well suited to detection of nucleic acid targets with sequenceheterogeneity such as hepatitis C and HIV.

G. Drawbacks of Amplification Systems1. Potential for false-positive results due to contaminating nucleic acids.2. PCR and LCR, DNA products main source of contamination.3. QB replicase and TMA, RNA products are possible contaminants.4. Must have product inactivation as part of QC program.5. Separate preparation areas from amplification areas and use of inactivation systems such

as UV light help alleviate contamination.6. Very expensive.7. Closed system, automation will also decrease number of problems.

H. Future of Molecular Diagnostic Techniques1. Despite expense may be times that rapid diagnosis will result in decreased cost.2. Example: Mycobacteria - quick diagnosis no need for expensive respiratory isolation.3. Detection of multi-drug resistant M. Tuberculosis will lead to more timely public health

measures.4. Incredibly useful in serology and microbiology.5. Increased specificity and sensitivity of molecular testing will become the standard of

practice in immunology and microbiology.6. Testing will continue to become more rapid as assays are automated which will also bring

down the costs.7. Author states will not replace culture for routine organisms, but it already is, and as DNA

chip technology improves, the ability to test for multiple organisms will become easier.

Lecture Guide MLAB 1335 Immunology/Serology 2. … · Lecture Guide MLAB 1335 Immunology/Serology ... 1335 Immunology/Serology 2. Basic Immunologic Procedures 27 2) ... decrease in

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