Antibodies rabbit preparation

Primary antibody incubation. Prepare primary antibody cocktail in the blocking buffer (5% normal horse serum, buffer + azide), and incubate 1 h at room temperature. We typically use a mix of mouse, rabbit, goat, and/or chicken primary antibodies together see Table 5.2 for appropriate dilutions of the recommended antibodies. At this point (or during secondary antibody incubation), it is usually safe to incubate the samples overnight at 4°. 

Dilute the various rabbit serum antibody solutions prepared on Day 1 in hlocking buffer as follows ... 

Nonviable cells of S. faecalis strain N were used for the immunization of rabbits to activate the immune system to synthesize antibodies. To prepare the vaccine, the cells from 500 mL of freshly grown culture were collected by centrifugation at 10,000 rpm and then shaken in 100 mL of 0.2% formaldehyde in saline for 48 h. After removal of the formaldehyde by washing the cells with 0.01 M phosphate buffer of pH 7.2 in saline, the cells were suspended in 80 mL of sterile saline. Viability tests showed that the cells were made nonviable by this treatment. This suspension exhibited high absorbance at 600 nm and was used for immunizing rabbits. 

Indirect Two-Site Immunoradiometric Assay of Human Proinsulin. The method used is that described by Rainbow et al. Plastic tubes coated with purified guinea pig anti-insulin antibodies are prepared as described above 200-jul samples containing human proinsulin are added to these coated tubes and incubated at 4° for 24 hr. After removal of the sample, tubes are washed twice with 400 /tl of NIGP buffer. Rabbit antibody to human C-peptide is diluted to 1/1000 in 50 mM sodium phosphate buffer, pH 7.4, containing 150 mM sodium chloride, 10 g of bovine serum albumin per liter, and 100 mg of guinea pig IgG per liter 200 /til are added to each tube. After a further 24 hr of incubation at 4 the tubes are washed twice as previously and 200 /u,l of I-labeled sheep anti-rabbit IgG (10,000 cpm) are added in the same buffer as that used for diluting the C-peptide antiserum. After a final 24 hr of incubation and two further washes as above, the tubes are counted. 

An antibody is prepared by injecting a small amount of a foreign compound, antigen, into an animal, such as a horse, rabbit, mouse, chicken, donkey, goat, or sheep. The animal usually will form antibodies to combat the invasion. If these antibodies then are separated from the animal s body fluids, usually blood, they will react selectively with the antigen in another system. Although research chemists and biochemists can make their own for a specific use, dozens of antibodies are commercially available. There are monoclonal (one compound) and polyclonal (many compounds) antibodies. The first is used to isolate a specific compound, whereas the latter is used to isolate a class of compounds. 

In an immunochemical approach to learning more about the receptor site, an antibody was prepared against choline phenyl ether 12.86) in the rabbit. Unfortunately, when this antibody was used as a model for nicotinic receptors, it was found unable to distinguish between muscarinic and nicotinic agents, nor between agonists and antagonists (Marlow, Metcalf and Burgen, 1969). 

Hybrid antibodies have found practical application in electron microscopy, where they have been used, for example, to localize surface antigens on cells. For such an experiment (96), mouse lymphocytes may first be coated with mouse antibodies to H-2 antigens present on the cell surface. Hybrid rabbit antibody is prepared with one combining site spe-... 

Rodkey (115) immunized rabbits with protein conjugated to p-azophenyl-N-trimethylammonium groups, collected and purified the antibodies, and prepared F(ab )2 fragments. The latter were polymerized and injected into the same rabbit after a rest period of 14 months. Antiidiotypic antibodies were synthesized as a consequence of this procedure. 

Fig. 4 b. Arrangement same as in fig. 4 a, except rabbit antibodies were prepared against human lipoproteins of density <1.063 ( anti-LDL ). Here both patients with Tangier disease demonstrated LDL, which was further shown by immunoelectrophoresis. Plates prepared by Dr. R. I. Levy. Source Hoffman and Fredrickson (1965). (Reproduced with kind permission of The American Journal of... 

Peptides formed during tryptic digest of Salmonella flagellin were immobilized on the WPG-PG to prepare immunoadsorbents for the isolation of monoreceptor antibodies from rabbit serum against H-antigens of Salmonella spp. [129]. The... 

Miyake et al reported an ELISA method for the determination of pesticide residues in the aquatic environment. The polyclonal antibody and three monoclonal antibodies of acifluorfen were prepared by immunization of rabbits and mice with acifluorfen-bovine serum albumin conjugates. The polyclonal antibody reacted with acifluorfen at concentrations of 1.5-800 mg L , while the monoclonal antibodies reacted with acifluorfen at concentrations of 1.5-144 mg L . Among three monoclonal antibodies, AF 75-144 reacted with chlornitrofen, which did not react with the other two antibodies. It seems that the ELISA method is effective for the determination of herbicide residues in the aquatic environment. 

K. Matsumoto, A. Torimaru, S. Ishitobi, T. Sakai, H. Ishikawa, K. Toko, N. Miura, and T. Imato, Preparation and characterization of a polyclonal antibody from rabbit for detection of trinitrotoluene by a surface plasmon resonance biosensor. Talanta 68, 305-311 (2005). 

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... 

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