1. These protocols are broadly applicable to the generation of polyclonal antisera in hosts other than rabbits, however if it is intended to generate mouse monoclonal antibodies (Mabs) the following should be noted: mouse responses are often best in F1 crosses (e.g., BALB/c x C57Bl/6) rather than pure strains. Anticarrier antibodies often comprise a significant proportion of Mabs generated using conventional carriers and it is, therefore, worth considering the use of an alternative carrier, such as the purified protein derivative (PPD) of tuberculin. If PPD conjugates are used for peptide immunizations, the animals must first be primed with live attenuated bacteria (BCG strain), which express PPD on their surface. This priming step elicits a strong T-cell helper response against subsequent PPD-linked immunogens. Freund's complete adjuvant appears to interfere with this priming process and so should be avoided if using this method. Immunizations should be performed using FIA. Immunizations immediately prior to hybridoma fusions should not be done with persistent adjuvants such as Freund's, because in this case the aim is to induce a rapid and transient immune response whose early (lymphoproliferative) stage coincides with fusion to myeloma cells. Therefore, acetone precipitates are particularly useful for immunizations prior to spleen fusions for the development of Mabs.

2. A single-step affinity purification of anti-phosphopeptide antibodies on phosphopeptide matrix is normally sufficient to purify the antiserum to a significant degree. However, a second purification step on nonphosphopeptide matrix, to deplete residual non-phosphopeptide immunoreactivity, may be necessary.

3. Affinity purification may be performed using gravity flow through a disposable column (e.g., a Bio-Rad Poly-Prep chromatography column). In this case, flow reversal is not possible and recovery of high affinity antibodies may be compromised. Nevertheless, acceptable results are often achieved.

4. Not all peptides will be soluble at 1 mg/mL in PBS. Addition of SDS with gentle heating and shaking can aid solubilization.

5. NHS-activated Sepharose 4 Fast Flow incorporates a spacer arm and is a good alternative to CNBr-Sepharose.

6. 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.

7. Avoid contamination of substrate solutions by skin contact as this can sometimes increase background activity. Read HRP reactions immediately as atmospheric oxidation will gradually react with substrate in all wells.

8. P-Galactosidase conjugates are suitable alternatives to HRP conjugates as second layer reagents. Substrate solution for P-galactosi-dase: 4 mg/mL o-nitrophenyl-P-D-galactopyranoside dissolved in TBS containing 0.7% 2-mercaptoethanol and 1 ml MgCl2. Stop solution for P-galactosidase is 1 M Na2CO3.

9. If the ELISA color development takes more than a few minutes, continue the incubation in the dark. P-galactosidase reactions tend to have a lower spontaneous background than peroxidase reactions but the yellow o-nitrophenyl-P-D-galactopyranoside reaction product can take longer to develop. P-galactosidase reactions can be speeded up by incubation at 37°C.

10. Ammonium sulfate precipitation of IgG inevitably leads to residual ammonium sulfate in the pellet. Dialysis will remove this, but may also lead to a significant loss of antibody. For most purposes, addition of 10XTBS to the resuspended pellet is an acceptable alternative.

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