Immunization antiserum

Figure 9. Anti-PbTx antiserum inhibition of [ H]PbTx-3 binding to its receptor site in rat brain membrane preparations. Labeled toxin (0.5 nM in 1 ml PBS) was incubated with rat brain membranes (125 fig total protein) and increasing amounts of anti-PbTx antiserum (- -) or pre-immune serum (- -) for 1 hr at 4 C. Membrane-bound radioactivity was then measured in a centrifugation assay as previously described (8),...

Antiserum (plural antisera) Blood serum containing one or more polyclonal antibodies that are specific for one or more antigens. The antibodies in an antiserum result from previous immunization or exposure to an agent of disease. 

Sensitivity defines the degree to which an assay can distinguish one compound from another of the same nature and an immunoassay is a function of the particular antibody molecules contained in the antiserum. Specificity of the antiserum is a function of the particular antigen used to immunize the animal. Affinity usually measures how strongly bound is the antigen to the antibody. Titer refers to the concentration level of, in the context of the usage, antibody contained in the obtained serum. 

Antiserum Production The immunogen, carboxymethylmorphine-bovine-serum-albumin, is emulsified with equal volume of complete Freund s adjuvant. Initial immunization doses are injected into the New Zealand albino rabbits and later on this followed up with booster injections after a period of 6 weeks. The antiserum titer is determined with each booster dose injection and is duly harvested when the titre value is maximum. This is diluted suitably and employed in the radioimmunoassay. ... 

Immunization and Antibody Production The lypphilized immunogen obtained above is dissolved in normal saline and emulsified with equal volumes of complete Freund s adjuvant into a thick paste. Three New Zealand albino rabbits are immunized with the immunogen-paste through intradermal injections. The process is repeated twice at 2-weeks intervals followed by booster doses at monthly intervals. The antiserum is harvested when the plasma titer value is attained maximum. 

The PAP method was pioneered by Sternberger in 1979 (1). The method uses an immunological sandwich amplification and the enzyme peroxidase to effect a signal. The unique feature of this procedure is the enzyme/antibody solution, the PAP immune complex. The horseradish peroxidase enzyme, itself an immunogenic protein, is used to inoculate a given species, and a polyclonal immune response is generated against the enzyme. This antiserum is harvested and placed in solution with the enzyme so that immune complexes form that... 

Production of antiserum with high titer and specility is done by trial and error, especially because each immunized animal gives antisera with different characteristics therefore, several groups of animals should be immunized with different antigen preparations. 

To reduce unspecific binding, mix a 200-pl aliquot of the clear supernatant with 2 pi pre-immune serum or unspecific antibody and a further 200 pi aliquot with 50 pi precipitation aid. Rock at 0 °C for 1 h and spin at 1000 x g. Transfer the supernatant into a fresh container and fill it up to 1000 pi with Soln. A. Add 0.5 - 5 pi of the specific antiserum and monoclonal antibody, respectively, and incubate on ice for 1 h. Prepare a second sample containing pre-immune serum instead of antiserum. 

Three basic approaches are used to control viral diseases vaccination, antiviral chemotherapy, and stimulation of host resistance mechanisms. Vaccination has been used successfully to prevent measles, rubella, mumps, poliomyelitis, yellow fever, smallpox, chickenpox, and hepatitis B. Unfortunately, the usefulness of vaccines appears to be limited when many stereotypes are involved (e.g., rhinoviruses, HIV). Furthermore, vaccines have little or no use once the infection has been established because they cannot prevent the spread of active infections within the host. Passive immunization with human immune globulin, equine antiserum, or antiserum from vaccinated humans can be used to assist the body s own defense mechanisms. Intramuscular preparations of immune globulin may be used to prevent infection following viral exposure and as replacement therapy in individuals with antibody deficiencies. Peak plasma concentrations of intramuscular immune globulins occur in about 2 days. In contrast, intravenously administered immune globulin provides immediate passive immunity. 

Antiserum can be raised againsf lymphocyfes or fhymo-cyfes by fhe repealed injection of human cells into an appropriate recipient, usually a horse. The use of such antiserum or the immune globulin fraction derived from it has been used to produce immunosuppression. Although antilymphocytic serum can suppress cellular and often humoral immunity against a variety of tissue graft systems, the responses are variable, particularly from one batch of serum to another. 

Antisera directed against lymphocytes have been prepared sporadically for over 100 years. With the advent of human organ transplantation as a therapeutic option, heterologous antilymphocyte globulin (ALG) took on new importance. ALG and antithymocyte globulin (ATG) are now in clinical use in many medical centers, especially in transplantation programs. The antiserum is usually obtained by immunization of large animals such as horses or sheep with human lymphoid cells. 

Injected forms of antibodies which have been generated in another body or animal can be isolated, purified, and administered as standard human immune serum globulin (ISG), and ISG plus preparation, or as an animal antiserum or antitoxin. Some serums which are available are those for rabies, snake and insect bites, botulism, and tetanus. Temporary immunity of up to six months to hepatitis can be imparted by one "gamma globulin" shot. More permanent active immunity is available to health care workers. 

The neutral FDPase and SDPase activities, which were present in the crude spinach extracts, were precipitated at lower ammonium sulfate concentration and could thus be separated from the specific alkaline FDPase. These activities appeared to be associated with the chloroplast fraction and did not require the presence of a divalent cation for activity. In crude extracts only the alkaline FDPase activity was inhibited by antiserum prepared by immunizing rabbits with the purified alkaline FDPase. The neutral FDPase was also active with ribulose diphosphate (RuDP) (98). 

The disaccharide was synthesized as its -nltrophenyl derivative and subsequently, as described above, covalently linked to BSA to yield the glycoconjugate AR-BSA (18). Immunized rabbits responded with antibody production with specificity for the saccharide hapten, as demonstrated in ELISA studies (19). When tested in immunofluorescence, the AR-BSA antiserum proved to be of the same excellent specificity for the identification of Salmonella serogroup C2-C3 bacteria as had been shown previously for the other dldeoxyhexose-contalnlng dlsaccharlde haptens (Figure 1) (19). 

Saunders et al. (1997) have raised a polyclonal antiserum using a peptide specific for ER 3. The peptide (CLSKAKRNGGHAPRVLEL) corresponding to amino acids 196-213 of rat ER(3 was conjugated to keyhole limpet hemocyanin and used to immunize rabbits according to standard procedures. Polyclonal IgGs were purified from serum on a Hitrap protein A Sepharose column based on the manufacturer s instruction (Pharmacia). 

We also investigated the specificities of purified antibodies against G i and Gp, from the serum of a rabbit immunized with bovine brain gangliosides. As shown in Figure 6, the whole antiserum had substantial activity both against the two ganglio-... 

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