• Attenuated whole-agent vaccines: uses live, but weakened microbes. Closely mimic actual infection. Lifelong immunity with 95% effective. Danger if backmutation to live
  • MMR
  • Sabin Polio
  • TB
  • Typhoid
  • Chickenpox
• Innactivated whole agent vaccines: use microbes that have been killed by formalin or phenol
  • Rabies
  • Salk Polio
  • Pneumococcal pneumonia
  • Cholera
  • Influenza [adults]
  • Hepatitis A
• Toxoids: inactivated toxins produced by a pathogen
  • Tetanus toxoid
  • Diptheria toxoid
  • Botulism toxoid
• Subunit vaccine: only use antigenic fragments of a microorganism that will best stimulate an immune response. Produced by genetic engineering techniques. Also called recombinant vaccines. Safer vaccines, since the microorganism cannont replicate in the recipient. Also contain little or no extraneous material that minimize adverse side effects. Two types of antigentic fragments used are capsular polysaccharides and outer surface lipoprotiens
  • Hepatitis B
  • Whooping cough [pertussis]
• Conjugated Vaccines: polysaccharides of capsule are combined with proteins
  • Influenza [children]
• Nucleic acid vaccines: DNA (plasmids) and RNA vaccines in trial studies

Vaccine choices: Safety, efficacy, prevents targeted diseases, ease of administration (oral, nasal, SQ, IM, air-gun)

Development of New Vaccines: Plants-> edible plant derived vaccines are undergoing clinical trials

Substances called adjuvants increase the efficacy of a vaccine or toxoid by increasing the availability of the antigen in the lymhatic system by slow release and ease of digestion by phagocytes. Examples include aluminum sulfate and aluminum hydroxide, mineral oil or peanut oil. The adjuvunct linked to antigen are taken up by the macrophages and presented to lymphocytes more effectively.

Diagnostic Immunology

Precipitation Reactions

Reactions of soluble antigens with IgG or IgM antibodies to form large, interlocking molecular aggregates called lattices. Reactions occur in two stages: First is the Ag|Ab complexes that take place in minutes. The second is a slower reaction over several hours, in which the reaction forms lattices that precipitate from solution. Forms a precipitation ring which is cloudy and white to cream between the antibody in the bottom of the tube and the soluble antigen added to the upper part of the tube.

Immunodiffusion Tests are precipitation reactions that are carried out in gel agar, as a line of precipitate develops in the wells as the antigen and antibody react.

Electrophoresis is combined with various immunology tests in gel to help speed up the process, known as immunoelectrophoresis. This process forms the basis of many immune tests.

Agglutination Reactions

Reactions involve particulate antigen or soluble antigens attached to particles.Antigens can be linked together by antibodies to form visible aggregrates called agglutination. Reactions are either classifed as direct agglutination or indirect agglutination tests.

Direct Agglutination: detect antibodies against large cellular antigens, such as those on RBCs, fungi, or bacteria. Test is done on microtiter plates with shallow wells. The amount of particulate antigen is the same, but the antibody serum is diluted by half [1:20, 1:40, 1:80, etc]. A positive reaction happens if sufficient antibodies are present in the serum to link the antigen together forming a Ag|Ab complex in the bottom of the well. A negative reaction occurs if not enough anibodies are present to cuase the linking of the antigens. Because of the serial dilutions, this is reported as a measure of titer. For diagnostic purpose, paired serum titers need to be performed to indicated response or immediate infection. Titer is defined as the most dilute concentration of serum antibody that yields a detectable reaction.

Indirect (Passive) Agglutination Tests: Soluble antigens are attached to latex beads and allowed to react with serum antibodies. Also known as a latex agglutination test. Also, the reverse can be set up as antibody is attached to the latex beads and the antigen added. A positive in either case, is agglutination. Therefore, the reactions can either test for antigens or test for antibodies. Used for rapid Streptococcal test. Urine drug testing, an agglutination is negative, since the drug coated latex particles are allowed to react with known antibody. If drugs are present in the urine, they bind to the antibody and prevent agglutination.

Hemagglutination: Agglutination reaction involves the clumping of red blood cell surface antigen and their complimentary antibodies. Routinely used in blood typing and in diagnosis of Infectious mononucleosis. Also known as the Coombs test. Other uses to help in diagnosis of measles, mumps, mononucleosis

 

Neutralization Reactions

Neutralization: Ag|Ab reaction that blocks harmful bacterial exotoxins or virus from affecting cells.

Bacterial toxin neutralization: Specific antibody, called an antitoxin, binds to bacterial exotoxin to neutralize it and prevents damage to cells.

Viral neutralization: Antiviral antibodies bind to viruses to inactivate them and prevent RBC agglutination. Used to diagnose influenza, measles, mumps

Complement Fixation Reactions

Used to detect very small amounts of antibody. Used in certain diagnosis of fungal, viral, and rickettsial disesaes. Antigen is combined with complement and added to serum. If serum has antibody against antigen, a Ag|Ab complex will form and complement will be activated. Sheep RBCs and antibody are added to see if complement has been previously fixed [activated]. A positive complement fixation = no hemolysis, that is, complement was activated by the original antigen and antibody exposure. A negative complement fixation reaction = hemolysis of sheep RBC since the second antibodies bind to RBCs and activate complement that wasn't activated by the first Ag|Ab reaction.

 

Fluorescent Antibody (FA) Techniques

FA techniques can identify microorganisms in clinical specimens and can detect the presenced of a specific antibody in serum. Test set up involves combining fluorescent dyes called fluorochromes with antibodies to make them emit visible light when under exposure to UV light. Two types of tests: Direct and Indirect

Direct FA tests: used to identify a microorganism in a specimen. Microorganism specimen containing the antigen is fixed to a slide. Then fluorescein labeled antibodies are then added. After incubation, the slide is washed and then examined under UV light for yellow-green fluorescence.

Indirect FA tests: detect presence of a antibody in serum following exposure to a microorganism. Known antigen is fixed to a slide and the test serum is added. Antigen | Antibody complexes form and another solution containing antihuman immune serum globulin is added to make the Ag|Ab complex visible

Flourescence-Activated Cell Sorter: Detect specific cell receptors, such as those for CD4 and CD8 T cells and sorting sperm cells that contain either X or Y chromosome. Certain cells that have antigens of interest with fluorescent-antibody markers. Laser beam is used to charge the flourescent cells and a fluorescence detector is used to identify the light emitted by the cell. The droplets are then passed by electrically charged plates that further separate the flourescent cells from the nonfluorescent cells.

Enzyme Linked Immunosorbant Assay [ELISA]

Most widely used tests. Two types: Direct ELISA tests for the presence of antigens and the Indirect ELISA tests for the presenced of antibodies.

Direct ELISA: antibody is absorbe into the well. Patient sample is added and Ag|Ab reaction occurs if complementary. Enzyme linked antibody is added to specifically test for presence of antigen. Enzyme substrate is then added to and reaction produces a color change if positive and no color change if negative.

Indirect ELISA: Antigen is absorbed to well and patient antiserum is added. Ag|Ab reaction occurs if complementary. Enzyme linked antihuman immune serum globulin is added and binds to bound antibody . Enzyme's substrate is added and reaction produces a color change if positive and no color change if negative.

Use of ELISA tests: Home pregancy tests, HIV, drug tests

Radioimmunassay (RIA)

Known amount of radioactive labeled antigens and unkown amount of unlabeled antigens is allowed to react with a known amount of specific antibody. Percentage of bound verse free radioactive antigen is measured and the concentration of the unkown antigen is then determined by a reference curve.

Radioallergosorbent Test [RAST}: detect antibodies or antigens involved with allergies.Antigen to suspected antibody is bound to a plastic plate. The test solution containing the antibodies is added.and if present and complementary, will bind to antigen. Radioactive antiglobulin antibody is added and will bind to the antibody and radioactivity will be detected.

Monoclonal Antibodies

Plasma cells are fused with Myeloma cancerous clone cellls to create a hybridoma that will secrete antibodies, known as monoclonal antibodies, which are pure and uniform and are used for research and laboratory tests.

 

Serotyping

Serological procedures used to differentiate strains [serovars, serotypes] of microorganisms that differ in the antigenic composition of a structure or product. Importance in identifying a pathogen out of the group, primarily using cell wall antigens.

Examples: Lancefield system to identify streptococci. Serotypes are identified by a letter A-O and is based on specific antibody agglutination reactions with cell wall carbohydrate [Polysaccharide O}. Further subdividing of Group A Strep based on specific M protein antigens can also be performed. Other examples of serotyping include identifying specific antigen-antibody reactions involving Flagella [H] antigens, capusular [K] antigens, and cell wall [O] antigens.