Immunized rabbit serum

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. 

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). 

Rabbit HCP-mix antiserum. The polyclonal rabbit anti-HCP serum was obtained by a cascade immunization of rabbits with a HCP mix from Vero cells. Three days before immunization blood was obtained from the rabbits. This pooled serum serves as pre-immune rabbit serum. The HCP mix, a blank culture of Vero cells is three times frozen and thawed to induce cell lysis. The culture supernatant was pooled and filtered. The pore size of the filters was comparable with those used during the IPV Vero production process. The HCP mix was divided in small aliquots, rapid frozen using dry ice and stored at -70 °C until use. 

FIGURE 8. Plot of integral CL versus time. Each curve depicts the luminol-amplified CL response from a suspension of human PMN in barbital buffer, pH 7.2, following addition of P. aeruginosa. Curve a did not receive serum curves b through e received the quantities of pre-imaune and immune rabbit serum indicated. 

Post-DEAE fraction (105 fxg total protein, containing approx. 5 /ig receptor) was incubated for 15 h at 4° C in phosphate-buffered saline at pH 7.0 with 0-40 fig of IgG purified from immune or pre-immune rabbit serum (by ammonium sulfate fractionation and anion exchange chromatography). Binding of [ C]-NAA (saturable by 0.1 mM unlabelled NAA) was then measured either directly or in the supernatant obtained after removal of immunoprecipitate by centrifugation (100 000 g, 5 min), using the ammonium sulfate precipitation method [20]. The immuno-precipitates were analyzed by SDS-PAGE. 

The data in Fig. 1 illustrate that the thyroid-stimulating activity of the immunized rabbit serum is not inhibited by a dose of antithyrotropin antiserum which inhibited the concentration of rabbit thyrotropin giving a similar 2 hr response in the bioassay. Further, the activity was apparently bound to thyroid microsomes when the extract of rabbit serum was incubated with a human thyroid microsome preparation and the microsome pellet resedimented by ultracentrifugation, the supernatant contained no thyroid stimulating activity [distinct from results (not shown) found with a human liver microsome preparation which did not bind the thyroid stimulator]. The importance of the latter data is marginal since. 

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. 

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. 

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). 

Figure 4. Binding of sH-lacto-Fl-tetraitol by rabbit serum (R31) in response to immunization with edestin-etNH-lacto-N-tetraose. Antigen in complete Freunds adjuvant was administered on Days 1, 38, and 58 (arrows). The binding assay is performed as described in the legend to Figure 2.

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.

Four rabbits were used for immunizations with vaccine of equal parts of nonviable cells of S. faecalis suspension and Freund s complete adjuvant. Each week the rabbits are injected intravenously with 0.2 mL of the vaccine for 3 consecutive days, followed by a rest period of 4 days. This regime is performed for 5 weeks and is followed by a 1-week rest period. The immunization is performed in the same manner as before for 6 more weeks. Blood samples are drawn weekly after the fourth week of initial immunization. The serum is separated and tested by agar diffusion for the presence of antibodies. 

Dissolve agar (Agar Noble, Difco Laboratories) in 50 mM PB, pH 7.3, containing 150 mM NaCI in an electric oven. Spread the gel on a slide glass. Make a 3-mm diameter well in the center of the gel, and 6 wells around the center well at a distance of 7.5 mm from center to center of the wells. Add 10 [il of antigen (100. ig/ml) to the central well and 10 [il of 2-fold serially diluted serum to the outer wells. Incubate the gel at 4 °C for 1-2 days in a humid atmosphere in a Petri dish. Read the maximum dilution that results in an immunoprecipitin line. Usually, the antibody titer (the dilution factor) of rabbit serum immunized with component II is higher than that for component I. 

Reading (1982) adapted this to obtain concentrates of NSF for in vitro immunization. For this purpose, thymocytes are prepared (Section 5.4.2.4) from both BALB/c and C57BL/6 mice and equal numbers are mixed to a final concentration of 5x 10 cells/ml and cultured in plastic culture flasks in DMEM supplemented with 4.5 g dextrose/1, MEM non-essential amino acids, sodium pyruvate, 2-ME, HAT, HEPES, and 2% rabbit serum. After 48 h, the cells and other debris are pelleted and the supernatant (thymocyte-conditioned medium, TCM) is filtered (200 nm) and stored at — 70°C in aliquots of 10 ml. 

Solomon, D. H., and Beall, G. N., Thyroid-stimulating activity in the serum of immunized rabbits. II. Nature of thyroid-stimulating material. J. Clin. Endocrinol. Metab. 28, 1496-1502 (1968). 

For this, a subcutaneous or intradermic injection is made, at different points to five or six mice, rats or rabbits of the hapten-bound protein (immunogen) with certain additives. This multi-point primary injection corresponds to around 100 gig of pesticide. Animals having the same genetic background do not necessarily react in the same way. Two weeks later the same process is repeated with one fifth of the immunogen concentration. Two weeks after that, blood samples are taken from the animals to verify if the antibodies have appeared. The injections are repeated two or three times in order to create hyper-immunization. The serum of the animals is now a source of the desired antibody, which is of the polyclonal type. By contrast, monoclonal antibodies are derived from a single cell line. 

---