Specificity immunized rabbit serum

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). 

The nature of the specificity of antihapten antibodies can be demonstrated with a few examples of experimental results. Tables 2.1 and 2.2 present data on the specificity of rabbit antibody directed to the p,p -azophenylazobenzoate hapten group (2a). The immunizing antigen consisted of bovine serum proteins to which several hapten groups, per molecule of protein, had been conjugated by diazotization. (See Fig. 2.3.)... 

These specific substances or blood group factors are detected by their property of inhibiting agglutinin reactions between human erythrocytes and heterologous human sera. This is the so-called isoagglutinin test which can detect the factors in dilutions of more than 1 part in 10 million. An even more sensitive but less specific test is their power to inhibit hemolysis of sheep s erythrocytes by the serum of rabbits immunized with certain human corpuscles. 

Antibody Preparation and Characterization. Our best [STX0L]n-BSA immunogen (n=9) was used to hyperimmunize a total of 6 rabbits. The formation of anti-STXOL antibodies was monitored by determining the quantity of specific binding protein (presumably IgG) in the serum samples. Table I shows that 5 of the 6 rabbits produced anti-STXOL antibodies within 100 days after initial immunization. 

Especially if antisera are used, an incubation of a parallel blotting strip with a non-specific control serum from the same species (better pre-immune serum from the same animal) with the same dilution ratio should be done to exclude false-positive results. These false-positive results for proteins with higher molar mass are often found if rabbit sera are used. 

Figure 12. Protective efficiency in mice of passively transferred rabbit antisera against intraperitoneal challenge with S. typhimurium bacteria (U 106 bacteria = 25 LD 0 dose). Percentage ofsurvivors plotted against ELISA titers with S. typhimurium LPS as antigen. Key U, anti whole heat-killed bacterial serum (04, 12 specific) a, anti[GM(A )R],-ITC-BSA serum (04,12) . anti[GM(A )R]2-ITC-BSA serum (04,12) and o, anti AM-ITC-BSA serum (04). (Reproduced with permission from Ref. 65. Copyright 1981, Infect. Immun.j...

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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