UNIT-2 Antigen-Antibody Reactions Vaccines PDF

UNIT – 4 Antigen – Antibody Reactions (Antigen-Antibody Interactions In Vitro) Antigens and antibodies combine with each other specifically and in observable manner. Antigen-Antibody reactions serve several purposes, which are as follows— In the body, they fo...

UNIT – 4 Antigen – Antibody Reactions (Antigen-Antibody Interactions In Vitro) Antigens and antibodies combine with each other specifically and in observable manner. Antigen-Antibody reactions serve several purposes, which are as follows— In the body, they form the basis of antibody-mediated immunity in infectious diseases, or of tissue injury in some types of hypersensitivity and autoimmune diseases. In the laboratory, they help in the diagnosis of infections, in epidemiological surveys, in the identification of infectious agents and of non-infectious antigens such as enzymes. In general, these reactions can be used for the detection and quantitation of either antigens or antibodies. Antigen-Antibody reactions in vitro are known as ‘Serological reactions’. Serology: The branch of medical immunology concerned with antigen-antibody reactions in vitro is serology [serum and -ology]. The reactions between antigens and antibodies occur in 3 stages— 1. Primary stage 2. Secondary stage 3. Tertiary stage 1. Primary stage: - The primary stage is the initial interaction between antigen and antibody without any visible effects. This reaction is rapid, occurs even at low temperatures and obeys the general laws of physical chemistry and thermodynamics. The reaction is reversible. 2. Secondary stage: - In this stage demonstrable events occur such as— 1  Precipitation Agglutination Lysis of cells Killing of live antigens Neutralization of toxins and other biologically active antigens Fixation of complement Immobilization of motile organisms Enhancement of phagocytosis 3. Tertiary stage Some antigen-antibody reactions occurring in vivo initiate chain reactions that lead to neutralization or destruction of injurious antigens, or to tissue damage. These are tertiary reactions and include humoral immunity against infectious diseases, clinical allergy and other immunological diseases. =======================================================  General features of antigen-antibody reactions 1. The reaction is specific, an antigen combining only with its homologous antibody and vice versa. The specificity, however, is not absolute and ‗cross reactions‘ may occur due to antigenic similarity or relatedness. 2. Entire molecules react and not fragment. 3. There is no denaturation of the antigen or the antibody during the reaction. 4. The combination occurs at the surface and is firm but reversible. The firmness of the binding is influenced by the affinity and avidity of the reaction. Affinity refers to the intensity of attraction between the antigen and antibody molecules. Avidity is the strength of the bond after formation of antigen-antibody complexes. 5. Both antigens and antibodies participate in the formation of precipitates or agglutinates. 6. Antigens and antibodies can combine in varying proportions, unlike chemicals with fixed valencies. Both antigens and antibodies are multivalent. Antibodies are generally bivalent, though some molecules may have 5 or 10 combining sites (IgA, IgM). Antigens may have valencies up to hundreds. 2 * Measurement of antigen and antibody Many methods are available for the measurement of antigens and antibodies. Measurement may be in terms of mass (e.g. mg Nitrogen) or more commonly as units or titre. # Antibody titre: - It is the highest dilution of the serum which shows an observable reaction with the antigen in the particular test. # Sensitivity: - It is the ability of the test to detect even very minute quantities of antigen or antibody. # Specificity: - It is the ability of the test to detect reactions between homologous antigens and antibodies only, and with no other. # Types of antigen-antibody reactions 1. Precipitation, Flocculation 2. Agglutination 3. Complement fixation 4. Neutralization 5. Opsonization 6. Imunofluorescence 7. Enzyme Linked Immunosorbent Assay (ELISA) 1. Precipitation, Flocculation: - When a soluble antigen combines with its antibody in the presence of electrolytes (NaCl) at a suitable temperature and pH, the antigen-antibody complex forms an insoluble precipitate. The reaction is called as Precipitation. Flocculation: - When, instead of sedimenting, the precipitate remains suspended as floccules, the reaction is known as flocculation. Precipitation can take place in liquid media or in gels such as agar, agarose or polyacrylamide. The amount of precipitate formed is greatly influenced by the relative proportions of antigens and antibodies. If increasing quantities of antigens are added to the same amount of antiserum in different tubes, precipitation will be found to occur most rapidly and abundantly in one of the middle tubes in which the antigen and antibody are present in optimal or equivalent proportions. 3  Mechanism of precipitation [ The Lattice hypothesis of Marrack ] According to this hypothesis, multivalent antigens combine with bivalent antibodies in varying proportions, depending on the antigen-antibody ratio in the reacting mixture. Precipitation results when a large lattice is formed consisting of alternating antigen and antibody molecules. This is possible only in the zone of equivalence. In the zones of antigen or antibody excess, the lattice does not enlarge, as the valencies of the antibody and the antigen, respectively, are fully satisfied. The lattice hypothesis holds good for agglutination also. * Applications of precipitation reaction 1. The precipitation test may be carried out as either a qualitative or quantitative test. It is very sensitive in the detection of antigens but relatively less sensitive for the detection of antibodies and as little as 1 g of Antigen can be detected by precipitation tests. 2. Forensic applications: - It is used in the identification of blood and seminal stains in murder case and rape case. Also it is used in food adulterations. 3. Ring test: - This is the simplest type of precipitation test, consists of layering the antigen solution over a column of antiserum in a narrow tube. E.g. Grouping of streptococci by the Lancefield technique. 4. Slide test (VDRL Test): - When a drop each of antigen and antiserum are placed on a slide and mixed by shaking, floccules appear. The VDRL test for syphilis is an example of flocculation. 4 5. Tube test: - A quantitative tube flocculation test is used for the standardization of toxins and toxoids. Serial dilutions of the toxin/toxoid are added to the tubes containing a fixed quantity of the antitoxin. The amount of toxin or toxoid that flocculates optimally with one unit of the antitoxin is defined as an Lf dose. 6. Immunodiffusion (Precipitation in gel): - There are several advantages of precipitation in gel rather than in liquid medium. The reaction is visible as a distinct band of precipitation, which is stable and can be stained for preservation. As each antigen-antibody reaction gives rise to a line of precipitation, the number of different antigens in the reacting mixture can be readily observed. Immunodiffusion also indicates identity, cross-reaction and non-identity between different antigens. Immunodiffusion is usually performed in a soft (1 %) agar gel. Different modifications of immunodiffusion are as follows— A) Single diffusion in one dimension (Oudin procedure): - The antibody is incorporated in agar gel in a test tube and the antigen solution is layered over it. The antigen diffuses downward through the agar gel, forming a line of precipitation. The number of bands indicates the number of different antigens present. 5........ Antigen ::::::: Precipitate line Agar gel with antibody B) Double diffusion in one dimension (Oakley-Fulthorpe procedure): Here, the antibody is incorporated in gel, above which is placed a column of plain agar. The antigen is layered on top of this. The antigen and antibody move towards each other through the intervening column of plain agar and form a band of precipitate where they meet at optimum proportion......... ::::::: Antigen Plain gel column Precipitate line Agar gel with antibody C) Single diffusion in two dimensions (Radial immunodiffusion) Here the antiserum is incorporated in agar gel poured on a flat surface (Slide or petridish). The antigen is added to the wells cut on the surface of the gel. It diffuses radilly from the well and forms ring shaped bands of precipitation (halos) concentrically around the well. The diameter of the halo gives an estimate of the concentration of the antigen. 6  This method is used for the estimation of the immunoglobulin classes in sera and for screening sera for antibodies to influenza viruses, among others. Agar gel with antiserum Antigen well Ring shaped band of precipitate D) Double diffusion in two dimensions (Ouchterlony procedure) This is the immunodiffusion method most widely used and helps to compare different antigens and antisera directly. Agar gel is poured on a slide and wells are cut using a template. The antiserum is placed in the central well and different antigens in the surrounding wells. If two adjacent antigens are identical, the lines of precipitate formed by them will fuse. If they are unrelated, the lines will cross each other. 7 7) Immunoelectrophoresis: - This involves the electrophoretic separation of a composite antigen (such as serum) into its constituent proteins, followed by immunodiffusion against its antiserum, resulting in separate precipitin lines, indicating reaction between each individual protein with its antibody. This enables identification and approximate quantitation of the various proteins present in the serum. The technique is performed on agar or agarose gel on a slide, with an antigen well and an antibody trough cut on it. The test serum is placed in the antigen well and electrophoresed for about an hour. Antibody against human serum is then placed in the trough and diffusion allowed to proceed for 18-24 hours. The resulting precipitin lines can be photographed and the slides dried, stained and preserved for record. Over 30 different proteins can be identified by this method in human serum. This is useful for testing for normal and abnormal proteins in serum and urine. 1 2 3 4 5 6 8 1. Semisolid agar layered on the glass slide. A well for antigen and trough for antiserum cut out of agar. 2. Antigen well filled with human serum. 3. Serum separated by electrophoresis. 4. Antiserum trough filled with antiserum to whole human serum. 5. Serum and antiserum allowed to diffuse into agar. 6. Precipitin lines form for individual serum proteins. 8) Electroimmunodiffusion: - The development of precipitin lines can be speeded up by electrically driving the antigen and antibody. Various methods have been described combining electrophoresis with diffusion. I) One-dimensional single electroimmunodiffusion (Rocket electrophoresis) The main application of this technique is for quantitative estimation of antigens. The antiserum to the antigen is incorporated in agarose and spread on the glass slide. The antigen is placed in wells punched in the gel in increasing concentrations. The antigen is then electrophoresed into the antibody containing agarose. The pattern of immunoprecipitation resembles a rocket and hence the name. + 1 2 3 4 _ 9  4 Rocket electrophoresis 1 -- Antibody in agarose gel 2 -- Precipitation arcs 3 -- Antigen wells 4 -- Increasing antigen concentration II) One dimensional double electroimmunodiffusion: - (Counterimmunoelectrophoresis, CIE) This involves simultaneous electrophoresis of the antigen and antibody in gel in opposite directions resulting in precipitation at a point between them. This method produces visible precipitation lines within 30 minutes. The clinical applications are for detecting various antigens such as specific antigens of cryptococcus and meningococcus in the cerebrospinal fluid. Electrophoretic current _ + Ag Ab Counterimmunoelectrophoresis (CIE) Antigen and antibody are driven together by an electric current and a precipitin line forms. ========================================================= 10 2. Agglutination reaction 1. When a particulate antigen is mixed with its antibody in the presence of electrolytes at a suitable temperature and pH, the particles are clumped or agglutinated. 2. Agglutination is more sensitive than precipitation for detection of antibodies. 3. The same principles govern agglutination and precipitation. Agglutination occurs optimally when antigens and antibodies react in equivalent proportions. The zone phenomenon may be seen when either an antibody or an antigen is in excess. 4. Applications of agglutination reaction: - I) Slide agglutination: - When a drop of the appropriate antiserum is added to a smooth, uniform suspension of a particulate antigen in a drop of saline on a slide or tile, agglutination takes place. A positive result is indicated by the clumping together of the particles. The reaction is facilitated by mixing the antigen and the antiserum with a loop or by gently rocking the slide. Depending upon the titre of the serum, agglutination may occur instantly or within seconds. Agglutination is usually visible to the naked eye but may sometimes require confirmation under the microscope. e.g. Blood group determination and cross matching. Identification of many bacterial isolates. Blood group determination Requirements: - i) Clean grease free slide ii) Sterile needle or lancet iii) Absolute alcohol iv) Cotton, pins v) Anti-A, Anti-B and Anti-D (Anti Rh) antiserum Procedure: - i) Sterile finger of left hand with alcohol. ii) Prick it with sterile lancet and wipe out 1 to 2 drops. iii) Take drops of blood at 3 places on a clean grease free slide. iv) Add Anti-A, Anti-B and Anti-D antiserum on first, second and third drop of blood respectively. v) Mix all drops with separate pins. 11 Observation: - Observe for agglutination in each drop. Result: - Sr. Agglutination in Agglutination Agglutination Blood group No Anti-A added in Anti-B in Anti-D drop added drop added drop 1 Present Absent Present A Positive 2 Present Absent Absent A Negative 3 Absent Present Present B Positive 4 Absent Present Absent B Negative 5 Present Present Present AB Positive 6 Present Present Absent AB Negative 7 Absent Absent Present O Positive 8 Absent Absent Absent O Negative II) Tube agglutination This is the standard quantitative method for the measurement of antibodies. When a fixed volume of a particulate antigen suspension is added to an equal volume of serial dilutions of an antiserum in test tubes, the agglutination titre of the serum can be estimated. E.g. Diagnosis of typhoid by Widal test 12 III) The antiglobulin test (Coombs test) (Haemolytic disease of the newborn) This test was devised by Coombs, Mourant and Race (1945) for the detection of anti-Rh antibodies that do not agglutinate Rh-positive RBCs in saline. When sera containing incomplete anti-Rh antibodies are mixed with Rh- positive RBCs, the antibody globulin coats the surface of the RBCs, but they are not agglutinated. When such RBCs coated with the antibody globulin treated with a rabbit antiserum against human gammaglobulin (antiglobulin or Coombs serum), the RBCs are agglutinated. E.g. Detection of haemolytic disease of the newborn due to Rh incompatibility. RBCs of newborn are washed to become free from unattached protein and then mixed with a drop of Coombs serum, agglutination results. 1. Rh positive RBCs are mixed with incomplete antibody 2. The antibody coats the RBCs 3. Antibodies being incomplete, cannot produce agglutination 4. On addition of antiglobulin serum, which is complete antibody to immunoglobulin, agglutination takes place. 2 3 1 + 5 4 13 IV) Passive agglutination test (RA factor determination test)/ Rose-Waller test: - The only difference between the requirements for the precipitation and agglutination tests is the physical nature of the antigen. By attaching soluble antigens to the surface of carrier particles, it is possible to convert precipitation test into agglutination test. Such tests are known as passive agglutination tests. The commonly used carrier particles are RBCs, Polystyrene Latex particles (0.8 – 1 mm in diameter) or Bentonite. E.g. Rose-Waller test (RA factor determination test) In Rheumatoid Arthritis, an auto-antibody (RA factor) appears in the serum, which acts as an antibody to gammaglobulin. The RA factor is able to agglutinate RBCs coated with globulins. The antigen used for this test is suspension of sheep RBCs sensitized with a dose of rabbit anti-sheep RBC antibody (Amboceptor). ======================================================== 3. Complement fixation test (CFT) / Wasserman test for syphilis 1. Complement takes part in many immunological reactions and is absorbed during the combination of antigens with their antibodies. 2. In the presence of the appropriate antibodies, complement lyses RBCs, Bacteria, immobilizes motile microorganisms, promotes phagocytosis and contributes to tissue damage in certain types of hypersensitivity. 3. The ability of antigen-antibody complex to ‘fix’ complement is made use of in the complement fixation test (CFT). 4. This is a very versatile and sensitive test, applicable with various types of antigens and antibodies and capable of detecting as little as 0.04 mg of antibody nitrogen and 0.1 mg of antigen. 5. Requirements: - i) Antigen (May be soluble or particulate) 0 ii) Antibody / Antiserum (Should be heated at 56 C to destroy complement activity. iii) Complement (Taken from guinea pig serum). iv) Sheep RBCs v) Amboceptor (Rabbit antibody to sheep RBCs) vi) Physiological saline with added calcium and magnesium ions. 14 vii) Each of these reagents has to be separately standardized. viii) Example Wasserman test for syphilis. Procedure: - Step – I The inactivated serum of the patient is incubated at 37 0 c for one hour with Wasserman antigen and a fixed amount (two units) of guinea pig complement. If the serum contains syphilitic antibody, the complement will be utilized during the antigen-antibody interaction and will be fixed. If the serum does not contain the antibody, no antigen-antibody reaction occurs and the complement will not be fixed. Step – II Whether the complement is fixed or not is detected in this step. It consists of addition of sheep RBCS and amboceptor and incubation at 37 0 C for 30 minutes. Result: - Positive test: - One drop of reaction mixture is taken on slide and observed under microscope. Absence of lysis of RBCs indicates that the complement was used up in the first step and, therefore, the serum contained the antibody. Negative test: - Lysis of RBCs indicates that complement was not fixed in the first step and, therefore, the serum did not have the antibody. I) ANTIGEN + TEST SERUM -- Complement Fixed (Contains Antibody) + COMPLEMENT + HEMOLYTIC SYSTEM (Sheep RBCs + Amboceptor) -- Result – No haemolysis CF TEST POSITIVE II) ANTIGEN + TEST SERUM -- Complement Not Fixed (Contains NO Antibody) + COMPLEMENT + HEMOLYTIC SYSTEM (Sheep RBCs + Amboceptor) -- Result – Haemolysis CF TEST NEGATIVE 15 Complement fixation test. In this example, two serum samples are being tested for antibodies to a certain infectious agent. In reading this test, one observes the cloudiness of the tube. If it is cloudy, the RBCs are not hemolyzed and the test is positive. If it is clear and pink, the RBCs are hemolyzed and the test is negative. ======================================================== 16 4. NEUTRALIZATION TESTS Specific antibodies are able to neutralize the biological effects of viruses, toxins and enzymes. Virus neutralization tests: Neutralization of viruses by their antibodies can be demonstrated in various systems, Neutralization of bacteriophages can be demonstrated by the plaque inhibition test. When bacteriophagcs are seeded in appropriate dilution on lawn cultures of susceptible bacteria, plaques of lysis are produced. Specific antiphage serum inhibits plaque formation. Neutralization of animal viruses can be demonstrated in three systems — animals, eggs and tissue culture. Toxin neutralization: Bacterial exotoxins are good antigens and induce the formation of neutralizing antibodies (antitoxins) which are important clinically, in protection against and recovery from diseases such as diphtheria and tetanus. The toxicity of endotoxins is not neutralized by anti-sera. Toxin neutralization can be tested in vivo or in vitro. Neutralization tests in animals consist of injecting into them toxin antitoxin mixtures and estimating the least amount of antitoxin that prevents death or disease in the animal. With the diphtheria toxin, which in small doses causes a cutaneous reaction, neutralization tests can be done on the human skin. The Schick test is based on the ability of circulating antitoxin to neutralize the diphtheria toxin given intradermally, and indicates the immunity or susceptibility to the disease. Toxin neutralization in vitro depends on the inhibition of some demonstrable toxic effect. An example is the antistreptolysin O test, in which antitoxin present in patient‘s sera neutralizes the haemolytic activity of the streptococcal O haemolysin. ========================================================= 5. IMMUNOFLUORESCENCE / Coons Test Fluorescence is the property of absorbing light rays of one particular wavelength and emitting rays with a different wavelength. Fluorescent dyes show up brightly under ultraviolet light as they convert ultraviolet into visible light. Coons and his colleagues (1942) showed that fluorescent dyes can be conjugated to antibodies and that such ‗labelled‘ antibodies can be used to locate and identify antigens in tissues. This 'fluorescent antibody' or immunofluorescence technique has several diagnostic and research applications. Direct immunofluorescence test: In its simplest form, it can be used for the identification of bacteria, viruses or other antigens, using the specific antiserum labelled with a fluorescent dye. For example, direct immunofluorescence is now 17 routinely used as a sensitive method of diagnosing rabies, by detection of the rabies virus antigens in brain smears. A disadvantage of this method is that separate fluorescent conjugates have to be prepared against each antigen to be tested. Indirect immunofluorescence test overcomes this difficulty by using an antiglobulin fluorescent conjugate. An example is the fluorescent treponemal antibody test for the diagnosis of syphilis. Here a drop of the test serum is placed on a smear of T. pallidum on a slide and after incubation, the slide is washed well to remove all free serum, leaving behind only antibody globulin, if present, coated on the surface of the treponemes. The smear is then treated with a fluorescent labelled antiserum to human gammaglobulin. The fluorescent conjugate reacts with antibody globulin bound to the treponemes. After washing away all the unbound fluorescent conjugate, when the slide is examined under ultraviolet illumination, the treponemes will be seen as bright objects against a dark background, if the test is positive. If the serum does not have anti treponemal antibody, there will be no globulin coating on' the treponemes and, therefore, they will not take on the fluorescent conjugate. A single antihuman globulin fluorescent conjugate can be employed for detecting human antibody to any antigen. Fluorescent dyes may also be conjugated with complement. Labelled complement is a versatile tool and can be employed for the detection of antigen or antibody. Antigens also take fluorescent labelling, but not as well as antibodies do. Sandwich technique For detection of antibodies by immunofluorescence, the 'sandwich' technique can be employed. The antibody is first allowed to react with unlabelled antigen, which is then treated with fluorescent labelled antibody, thus forming a sandwich, the antigen being in the middle, with labelled and unlabelled antibody on either side. Fluorescent dyes The fluorescent dyes commonly used are fluorescein isothiocyanate and lissamine-rhodamine, exhibiting blue-green and orange-red fluorescence, respectively. By combining the specificity of serology with the localizing capacity of histology, immunofluorescence helps in the visualization of antigen antibody reactions in situ. The major disadvantage of the technique is the frequent occurrence of nonspecific fluorescence in tissues and other materials. 18 Immunofluorescent testing. (a) Direct: Unidentified antigen (Ag) is directly tagged with fluorescent Ab. (b) Indirect: Ag of known identity is used to assay unknown Ab; a positive reaction occurs when the second Ab (with fl uorescent dye) affixes to the first Ab. 19  ====================================================== 6. Enzyme-linked immunosorbent assay (ELISA) This is a simple and versatile technique, which is as sensitive as radioimmunoassay and needs only microlitre quantities of test reagents. ELISA has found application for the detection of a variety of antibodies and antigens, such as hormones, toxins and viruses. The test may be done in polystyrene tubes (macro-ELISA) or polyvinyl microtitre plates (micro-ELISA). The principle of the test can be illustrated by outlining its application for the detection of the rotavirus antigen in faeces. The wells of a microtitre plate are coated with goat antirotavirus antibody. After thorough washing, the faecal samples to be tested are added and incubated overnight at 4°C or for 2 hours at 37°C. Suitable positive and negative controls are also put up. The wells are washed and guinea pig antirotavirus antiserum, labelled with alkaline phosphatase, added and incubated at 37°C for one hour. After washing, a suitable substrate (paranitrophenyl phosphate) is added and held at room temperature till the positive controls show the development of a yellow colour. The phosphatase enzyme splits the substrate to yield a yellow compound. If the test sample contains rotavirus, it is adsorbed to the antibody coating the wells. When the enzyme-labelled antibody is added subsequently, it is in turn adsorbed. The presence of residual enzyme activity, indicated by the development of yellow colour, therefore, denotes a positive test. If the sample is negative, there is no colour change. 20 21 ======================================================================== 22  General methods of prophylaxis. Many issues are involved contained by the prevention of microbial diseases. 1. Immunoprophylaxis against microbial illnesses includes the use of vaccines or antibody-containing preparations to provide a susceptible individual with immunologic protection against a specific disease. Immunization against microbial illnesses can be either active or passive. With active immunity, protection is achieved by stimulating the body's immune system to produce its own antibodies by immunization with a virus preparation. Passive immunity is conferred by administering antibodies formed in another host. For example, an antibody-containing gamma globulin preparation may protect a susceptible individual exposed to a microbial illness. 2. Avoidance of microbial exposure. This is an effective means of preventing the transmission of HIV-1, which is spread through sexual contact and exposure to blood of infected individuals. Blood bank testing, e.g., for hepatitis B surface antigen and for antibodies to HIV-1, HIV-2, HTLV-I, and hepatitis C, also avoids exposure by identifying and discarding blood units contaminated with these infectious agents. 3. Control of nonhuman microbial reservoirs: The most notable success was the control and elimination of rabies in some countries through removal of stray dogs, quarantine of incoming pets, and vaccination of domestic animals. 4. Vector control: Transmission of viral disease by the bite of an arthropod vector, yellow fever was transmitted by mosquitoes, malaria by mosquitoes, gastro by flies etc. These procedures include draining swamps, applying insecticide, screening homes, and using insect repellant or protective clothing. 5. Improvement in sanitation: The well-known link between the discharge of raw sewage into tidal waters, contamination of shellfish, and type A hepatitis is an example of a situation readily reversible by improved sanitary practices. 23  Toxoids A toxoid is a bacterial toxin (usually an exotoxin) whose toxicity has been inactivated or suppressed either by chemical (formalin) or heat treatment, while other properties, typically immunogenicity, are maintained. Thus, when used during vaccination, an immune response is mounted and immunological memory is formed against the molecular markers of the toxoid without resulting in toxin-induced illness. E. G. There are toxoids for prevention of diphtheria, tetanus and botulism. Toxoids are used as vaccines because they induce an immune response to the original toxin or increase the response to another antigen since the toxoid markers and toxin markers are preserved. For example, the tetanus toxoid is derived from the tetanospasmin produced by Clostridium tetani which causes tetanus. Botulin is produced by Clostridium botulinum and it causes the deadly disease botulism. While patients may sometimes complain of side effects after a vaccine, these are associated with the process of mounting an immune response and clearing the toxoid, not the direct effects of the toxoid. The toxoid does not have virulence, as did the toxin before inactivation. Preparation The Corynebacterium diphtheria and Clostridium tetani organisms are grown on modified Mueller's media 1, 2 which contains bovine extracts. Tetanus and diphtheria toxins produced during growth of the cultures are detoxified with formaldehyde. The detoxified materials are then separately purified by ammonium sulfate fractionation. The diphtheria toxoid is further purified by column chromatography. The tetanus and diphtheria toxoids are individually adsorbed onto aluminum phosphate. Each 0.5 ml dose is formulated to contain the following active ingredients: 2 Lf of tetanus toxoid and 2 Lf of diphtheria toxoid. Each 0.5 ml dose also contains aluminum adjuvant (not more than 0.53 mg aluminum by assay), < 100 mcg (0.02%) of residual formaldehyde, and a trace amount of thimerosal [mercury derivative, (