Immune sera preparation

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),...

Polyclonal antibodies are produced by injecting an antigen into an animal in the presence of an adjuvant containing bacterial lipopolysaccharides that stimulate the immune system. Serum prepared from the blood contains several different classes of antibodies that interact with different domains in the antigen molecule, each of... 

A simple and fast radioimmunoassay for saxitoxin has been developed using an anti-saxitoxinol antibody. The immune serum required for the immunoassay was raised in rabbits using an immunogen conjugate prepared from saxitoxinol and bovine serum albumin... 

Although the serum of animals immunized with small amounts of toxins has been used for many years to help people recover from diphtheria or to provide protection against snake or scorpion venom, there have been other situations where a specific antibody was needed in a concentrated form. Unfortunately, there were limits to what animal antibodies could do. First, the animal serum preparations, even with repeated injections, were mixtures of antibodies—not collections of many of the same antibody. That is just the way the immune system works in animals, including humans. In addition, there was always the danger of taking the serum from a diseased animal remember the case of Jim, the horse with tetanus ... 

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. 

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. 

Prepare three different pools of modified MB-Tosyl magnetic beads (i) with the anti-sulfonamide class-specific antibodies (ii) with the non-specific antibody (with the non-immunized serum) and, (iii) without any antibody, only treated with the corresponding coupling buffer [2],... 

A further quantity of the same substance was prepared by these workers and purified by repeated precipitation with lead acetate and ammonia. The product had [q ]d + 67.7° which changed to —10° after hydrolysis with dilute sulfuric acid for four hours. The amount of reducing sugar present was 75.6%. Mannose, D-arabinose and an unidentified sugar acid were obtained from the hydrolyzate. The substance did not produce a skin reaction, nor did it stimulate antibody formation. It reacted with immune serum in high dilution (1 2,000,000) in the precipitin test. It was claimed that this product was closely related to the polysaccharide isolated by Laidlaw and Dudley. ... 

The classical method for the prevention of non-specific adsorption of antibodies to the sample, particularly in the PAP method, is the pretreatment of the tissue preparation with normal serum from the species which produced the antiserum (3% normal serum in the pretreatment, 1% normal serum included in the next steps Stern-berger and Joseph, 1979). Grube and Weber (1980), however, reported that IgG or F(ab )2 from non-immune serum was bound non-specifically to gastrin cells (to unidentified anionic constituents) and... 

Figure 2. Immunolocalization of carbonic anhydrase. Sections of the anterior region of Chlamydomonas. prepared as described previously, show the same pattern of gold particle distribution. Significant staining of the cell wall (cw) even in the region of flagella (fl) attachment is apparent. Some non-specific staining of the starch grains was also found to occur with either pre-immune serum or with the polyclonal antibody directed against carbonic anhydrase.

Hyperimmune serum A preparation of immune serum globulins having high titers of specific kinds of antibodies. 

Fig. 6. Immunoprecipitation of wound- and auxin-induced ACC synthases from winter squash and tomato. Enzyme preparations were mixed with an antibody against winter squash wound-induced ACC synthase open symbol) or non-immune serum closed symbol), and the antigen-antibody complex was precipitated by protein A-Sepharose. ACC synthase activity remaining in the supernatant was assayed. Panel 1 the mesocarp wound-induced enzyme A, a), and the hypocotyl lAA-induced enzyme D/d)ol winter squash. The wound- and lAA-induced enzymes were mixed in 2 1 B, b) and 1 2 (C, c) activity ratios. A-D, treated with the antibody a-d, with non-immune serum. Panel 2 The wound-induced enzyme of tomato pericarp panel 3 the wound-induced enzyme of winter squash hypocotyls panel 4 the lAA-induced enzyme of winter squash hypocotyls panel 5 the lAA-induced enzyme of tomato hypocotyls and panel 6 the lAA-induced enzyme of mung bean hypocotyls [From 10]...

The purified protein was used to raise polyclonal antibodies in a rabbit. The purified IgG showed significantly enhanced immunoreactivity towards a purified cytokinin oxidase preparation, when compared to non-immune control serum, by dot blotting using a gold labelled goat anti-rabbit second antibody procedure with silver enhancement. Most significantly, the antibody preparation was able to precipitate 80% of the cytokinin oxidase activity of a highly active, partially purified preparation from Zea mays kernels in the presence of fixed Staphylococcus aureus cells. No precipitation occurred with non-immune serum. 

---