Pre-immun serum

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),...

Figure 3. Mortality of mice 24 hr after i.p. injection of 1 x 10" mg/kg palytoxin in the presence of pre-immune serum from Rabbit 633 undiluted immune serum, 10% immune serum, and 1% immune serum. Numbers above bars represent the number of animals dead and the number tested. (Reproduced with permission from Ref. 8. Copyright 1987 Pergamon Press.)...

Appropriate controls should always be run with any immunocytochemical procedure. Controls may include omitting the primary antibody, substituting pre-immune serum, normal serum, or normal IgG for the primary antibody, adsorbing the primary antibody against the antigen, or immunostaining with an unrelated antibody. 

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. 

An immunization scheme for rabbits is given in Table 4.5. Do not forget to take some milliliters of blood for pre-immun serum immediately before or after the first immunization. 

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. 

A Pre-immunization serum sample from the test animal serves as an excellent negative control serum. This can be taken at the time of the first immunization. 

It will be noted in pattern A of Figure 4, that only serum protein was present in the pre-immune serum and antibodies were not eluted by galactose or lactose. Pattern B of the Figure shows that, in addition to the serum proteins, two 280 nm absorbing components were obtained from the anti-S. faecalis serum. One component eluted with galactose and the other eluted with lactose. 

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.

Fig. 1. (left) Identification of the immunoprecipitated enzyme after gel electrophoresis and autoradiography. This experiment was designed to test die specificity of the immuno-assay using either the free or immobilized antibody. Lane 1 shows total P S]-labelled enzyme obtained using the immobilized antibody lane 2, unmodified enzyme obtained after boronate chromatography lane 3, identical to lane 1 except that free antibody was used and lane 4, pre-immune serum control for lane 3. 

Fig. 3. Enzyme-bound poly(ADP-ribose) content. The autoradiography (A) and corresponding densitometric scan (B) were carried out with enzyme samples from [3B[1 adenosine labelled cells which were treated with MNNG (lane 6) 3AB and MNNG (lane 4) and without treatment (lane 2). The pre-immune serum controls for lanes 6, 4 and 2 are represented by lanes 5, 3, and 1, respectively.

Immune, but not pre-immune, serum is able to inhibit auxin binding (Fig. 3b) and remove binding activity from solution (Fig. 3a). This removal coincides with the appearance of a 22 kDa polypeptide in the immunoprecipitate when analyzed by SDS-PAGE (not shown). 

The anti-idiotypic serum 455 2 inhibits GA4-induction of a-amylase in aleurone protoplasts (Table 5). This inhibition is of high titre and can be reversed by MAC 182, but not by pre-immune serum. Another anti-idiotypic serum, 455 1, behaves similarly but is of lower titre (data not presented). Both these sera will agglutinate aleurone protoplasts (Fig. 5), indicating that they probably recognize determinants at the protoplast surface which are involved in GA-perception. 

Immunogold-labelled sections of cells from germinating rapeseed cotyledons 5 days after germination A. Control section treated with pre-immune serum. No gold labels are visible. Many full or depleted oil-bodies and micro-bodies can be seen. 

Fig.2 Immunogold labelled sections of cells from maturing cotyledons of (A) rapeseed (B) mustard (C) radish. Sections were treated with IgG raised against the rapeseed 19kDa "olein . Note specific labelling of the oil-body membranes of all three oilseed species. No gold labelling was observed in control sections treated with pre-immune serum.

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