polyclonal antibody production modules 

Immunization

The immunization of a SPF rabbit (NZW strain) includes:

• Immunization standard protocol (4 injections), 2 control as well as final bleed on day 70
• Serum of pre-bleed (before first injection) can be sent on request for additional costs.
• Immunization is carried out by subcutaneous injections in combination with adjuvants.
• Delivery of serum (approximately 80 mL total: 1. and 2. bleed 3-5 mL each, final bleed approximately 70 mL)

*Various protocols for small and large animals also available.

Antibody Titer Determination

• Titer determination by ELISA using 7 dilution steps of the serum sample plus 2 controls (Example: 1 ELISA arresting the peptide on plate, approximately 100 μg peptide is necessary)

Purification of Serum Antibodies

For the purification process of serum antibodies, two alternative methods can be employed:

• Isolation of total immunoglobulin fraction- ammonium sulfate precipitation, protein A or G affinity chromatography, concentration of eluate, dialysis, determination of protein amount

• Isolation of antigen-specific (monospecific) antibodies- ammonium sulfate precipitation, dialysis against PBS, immunoaffinity purification by HYDRA^® purification column (please also see immobilization), elution of up to 3 affinity fractions, concentration of eluates, dialysis, purity analysis by gel filtration, determination of protein amount, documentation

Labeling of Polyclonal Antibodies

• Fluorescence labeling: antibody conjugation with FITC, Rhodamine, TRIIC, and others; removal of unconjugated fluorescence dye; determination of labeling efficiency; documentation
• Phycobiliprotein labeling (e.g., PE): conjugation of R-phycoerythrin to 1 mg antibody (1:1), FPLC chromatographic purification, determination of labeling efficiency, documentation
• Peroxidase (POD) labeling: thioether linkage by heterobifunctional crosslinker, FPLC chromatographic purification, determination of labeling efficiency, documentation (please inquire for other enzymes)
• Alkaline phosphatase (AP) labeling: thioether linkage by heterobifunctional crosslinker, AP > 7000 U/mg, FPLC chromatographic purification, determination of labeling efficiency, documentation (please inquire for other enzymes)
• Biotin or LC-biotin labeling: conjugation of biotin or LC-biotin to antibody, removal of unconjugated biotin, documentation

Fragmentation of Antibodies

• Production of mouse Fab antibody fragments*: papain cleavage of up to 3 mg purified mouse IgG, preparative separation of the fragments and isolation of Fab fragments, concentration and rebuffering, OD₂₈₀ determination, FPLC-based purification control, determination of total yield
  *mouse IgG1 upon request
  
• SDS-PAGE gel electrophoresis (optional): analysis of antibody fragments by SDS PAGE gel electrophoresis, includes 2 mini gels with 1 standard each and up to 8 samples per gel; documentation
• Fc, Fab, F(ab)2 fragmentation of IgG: papain, pepsin, or ficin cleavage of monoclonal IgG, preparative separation of the fragments and isolation of Fc, Fab or F(ab)₂fragments, concentration and rebuffering, OD₂₈₀ determination, FPLC-based purification control, determination of total yield, documentation.

Immobilization

• Preparation of a HYDRA® antigen affinity column for isolation of antigen-specific antibodies from serum or cell culture supernatant: coupling of protein, peptide, or low-molecular compound to HYDRA® gel matrix (1-2 mg peptide, low-molecular compound, or 5 mg protein are needed per gel, respectively).