Antisera, production

As is the case with all other pharmaceutical substances, all aspects of antisera production must be undertaken by means conducive to the principles of GMP. Most regulatory authorities publish guidelines which outline acceptable standards/procedures for the production of such blood-derived products. Donor animals must be healthy and screened for the presence of (particularly blood-borne) pathogens. They must be housed in appropriate animal facilities, and withdrawal of blood must be undertaken by aseptic technique. Subsequent downstream processing must be undertaken according to the principles of GMP, as laid down in Chapter 3. 

Fig. 1 shows the polypeptide patterns obtained from PSl preparations of Anacvstis and soybean. In each preparation, two prominent bands appeared between 62-65 kDa, as the psaA and psaB gene products. The soybean preparation (Fig. IB) had a mimimal contamination of LHC2 polypeptides, observed also on a dodecyl-maltoside 2-dimensional gel which permits concommitant examination of PSl and PS2 proteins (7). Three LHCl proteins were observed (Fig. IB), and have been isolated and injected into rabbits for antisera production. 

Antisera Production and Immunoblot Analysis of Native Latrodectin... 

Antiserum Production The immunogen, carboxymethylmorphine-bovine-serum-albumin, is emulsified with equal volume of complete Freund s adjuvant. Initial immunization doses are injected into the New Zealand albino rabbits and later on this followed up with booster injections after a period of 6 weeks. The antiserum titer is determined with each booster dose injection and is duly harvested when the titre value is maximum. This is diluted suitably and employed in the radioimmunoassay. ... 

New Zealand rabbits are the preferred laboratory strain used for polyclonal antiserum production (see Note 2). 

Characteristic features Immunoaffinity chromatography (including antiserum production) MIP-based chromatography (including polymer preparation)... 

As a general rule, it is best to perform initial immunizations in mice for several reasons. First, mice can be maintained inexpensively and usually produce a readily detectable response within 6 weeks, thereby reducing the cost of initial antiserum production. Test bleeds of immunized mice typically produce 100-200 ll of serum, which at a titer of... 

Immunization and Antibody Production The lypphilized immunogen obtained above is dissolved in normal saline and emulsified with equal volumes of complete Freund s adjuvant into a thick paste. Three New Zealand albino rabbits are immunized with the immunogen-paste through intradermal injections. The process is repeated twice at 2-weeks intervals followed by booster doses at monthly intervals. The antiserum is harvested when the plasma titer value is attained maximum. 

The discovery of Lp(a) by Berg in 1962 (B6) relied on the production of rabbit antisera against beta-lipoprotein and on the selective absorption of these antisera with individual human sera. When certain human sera were used for absorption, the antisera retained precipitation capacity in radial immunodiffusion with 30-35% of individual human sera, which obviously contained a previous unknown antigen. The particle carrying the new antigen shared antigenic properties with beta-lipoprotein, but had an additional antigenic structure. This was evidenced from the only partial fusion of the precipitin bands formed between a positive human serum, the antibeta lipoprotein antiserum and the new absorbed antiserum. 

A strong link between the phosphorylation of nucleolin, its proteolysis and the production of ribosomal RNA has been observed (Bouche et al, 1984 Bourbon et al, 1983 Warrener and Petryshyn, 1991). The inhibition of proteolysis using leupeptin leads to a lower rRNA transcription in an in vitro transcription system (Bouche et al, 1984). In another series of experiments, the injection of nucleolin antiserum leads to 2-3.5 fold stimulation of pre-rRNA synthesis in Chironomus tentans salivary glands (Egyhazi et al, 1988), although it was not clearly demonstrated that these antibodies blocked specifically the homolog of nucleolin in this species. A model was proposed based on these observations where nucleolin was... 

The onset of botulism occurs generally between 18 and 36 hours after consumption of food products containing botulin toxin. However, it may affect patients earlier or later, even on the tenth day after food consumption. The first symptoms include stomach ache, nausea, vomiting, and diarrhea, followed by neurological disorders. Other symptoms include, skin, mouth and throat dryness, diplopia, blurred vision, dysphonia, dysarthria, dysphagia, and peripheral weakness. In lethal cases of botulism, respiratory muscles are involved. This leads to respiratory failure and death. Because all the symptoms are connected with toxemia, the first step of medical treatment is to provide a patient with antiserum. 

Production of antiserum with high titer and specility is done by trial and error, especially because each immunized animal gives antisera with different characteristics therefore, several groups of animals should be immunized with different antigen preparations. 

Fig. 1. Double label immunohistochemistry on rai liver. An acetone-fixed rat liver section was incubated with a polyclonal antiserum raised in rabbit to a hepa-tocyte cell surface protein (courtesy of Dr. S. Stamatoglou) and a mouse monoclonal antibody against a bile duct specific cytokeratin (courtesy of Dr. E. B. Lane). The hepatocyte protein was localized by use of a secondary peroxidase-conjugated antibody resulting in a red/brown product (thin arrow). The bile duct cytokeratin was identified by using an alkaline phosphatase-conjugated secondary antibody giving a blue color (thick arrow).

If antiserum to the foreign gene product is available, this can be used in the ELISA with an appropriate conjugate as the revealing reagent instead of antimouse X chain antibody as described below. 

---