Immunization rabbit

The adjuvant effect of different doses of lipid A in lipospheres was also examined by immunizing rabbits with lipospheres containing R32NS1 and prepared at different final concentrations of lipid A. The ELISA titers of the individual rabbit groups immunized, as determined by dilution of serum obtained at 6 weeks after primary immunization, have shown a gradual increase in IgG antibody titer with increasing... 

It has been observed that while normal, rabbit serum failed to bind labelled phenobarbital, the serum from immunized rabbits bound 75 to 80% of the added pentobarbital and there exists a linear relationship between 14C-phenobarbital and the concentration of added antibody. Besides, when variable quantities of 14C-pentobarbital are added to a constant quantity of antibody, there exists a linear relationship between added and bound 14C-phenobarbital as depicted in Figure 32.4. 

Preparation of Aqueous Extract of Cotton Bract. Field-dried cotton bract, 10 g, picked directly from plants in a field near Lake Providence, Louisiana, was ground with mortar and pestle, extracted twice with 100 ml of deionized water at pH 7 for 30 min each time at 250c with stirring. The extracts were combined and the solution was filtered the filtrate was freeze-dried. This bract extract (bAg) was also used to immunize rabbits. Additional bAg was further purified by treatment with 85% methanol in a similar manner as with cotton dust. 

These reactions differ from the pseudoimmune reactions reported by Edwards and Jones (30). because the cotton dust anti gen(s) does not react nonspecifically with both normal and immunized rabbit sera. Precipitin arcs in a reaction of identity between antigens in bracts and dust in Figure 2 confirm that antigens present in dust are derived at least partly from cotton bract. 

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). 

Historically, antibodies have been obtained from the serum of animals. The serum contains a mixture of polyclonal antibodies. In 1890, Emil Behring immunized rabbits and mice against tetanus and diphtheria and reported that the antitoxin serum could protect against a lethal dose of the toxin. Since then, antisera have been used to protect from pathogens and toxins, but serum sickness was a major drawback for their clinical use. Antisera may produce immune responses, which could cause severe allergic reactions, and may even lead to anaphylactic shock and death. 

Mangold, B.L. and Dean, D.A. (1 992) The role of IgG antibodies from irradiated cercaria-immunized rabbits in the passive transfer of immunity to Schistosoma mansoni-infected mice. American journal of Tropical Medicine and Hygiene 47, 821-829. 

Miller, H.M. and Kerr, K.B. (1932) Attempts to immunize rabbits against a larval cestode, Cysticercus pisi-formis. Proceedings of the Society for Experimental Biology and Medicine 29, 670-671. 

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). 

To localize ERp on paraffin sections, 65-kDa antirat ER antibodies are used (Upstate Biotechnology, Lake Placid, NY). This antibody is obtained by immunizing rabbits with synthetic peptides representing the N-terminal amino acid residues 46-63 of human ER(3. The deparaffinized sections on slides are placed in the Target Retrieval Solution (pH 6.1)... 

To localize ER[3 in frozen sections, antigen retrieval with heating is not required. 210-180-C050 antibodies (Alexis Corporation, Nottingham, UK) are obtained by immunizing rabbits with synthetic peptides representing the C-terminal amino acid residues 467-485 of human estrogen. 

Numerous studies of specificities of anti-glycolipid antibodies produced by immunizing rabbits have been reported (8,9), and some discrepancies have been noted between laboratories (9). The widespread occurrence of natural antibodies might cause confusion in analyzing specificities produced by experimental immunization. Because of this we have purified both immune and natural anti-glycolipid antibodies from rabbit sera, and we have compared their specifities against the same, and different, glycolipids. 

Figure 7.2 A. Immunostaining of caspase 14 in exponentially growing NHEKs (400x). B. NHEKs treated with 50 pM EGCG for 24 h (400x). C. Confluent OSC2 cells (400x). Pre-immune rabbit serum was used as negative control (data not shown), and cell nuclei were counterstained with Mayer s hematoxylin.

Immune rabbit serum, 5 yl is mixed with varying amounts of inhibitor in buffer in a final volume of 300 pi. After incubation for 2 h at 25°, 6.7 pinole of [ 3H ]mannotetraose (103 cpm) in 100 pi of buffer is added and incubation is continued for 2 h at 25°. Binding of tritiated sugar is determined as described in Figure 2. 

Guar is a polysaccharide of galacto-mannan type [59], Guar was used to immunize rabbits to produce anti-carbohydrate antibodies. Inhibition experiments showed that galactose was an inhibitor but the mannose was not. Guar was subjected to periodate oxidation [37] and to oxidation by galactose oxidase [51], and in both cases the oxidized guar did not react with the... 

The glycoconjugate a-L-fucose-BSA was used to immunize rabbits. The immunization was performed interdermally at multisites on the back of the neck and repeated weekly for 10 weeks. Blood samples were collected weekly and immune sera samples were prepared. It was found in later experiments that on immunization by this glycoconjugate two types of antibodies were produced. One type was specific only for a-L-fucose and the other for BSA and the conjugate with a BSA moiety, Fig (40). 

Rabbit serum (normal or pre-immune rabbit serum and anti-/3-galactosidase serum) ... 

Normal or pre-immune rabbit serum—Each group will require 180 /xl. Store at —20°C. We have also purchased normal rabbit serum from Sigma (catalog R-4505). 

Serum from immunized rabbit added to well. A=desired antibodies Q =other serum proteins... 

Figure 7.6. Diagrammatic representation of the use of ELISA to assess antibody titer in the serum of an immunized rabbit.

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