Ammonium sulfate ascites precipitation

To increase the purity or to concentrate an antibody solution, it may be purified. Purification is done with a range of techniques applied to whole serum, supernatant, or ascites fluid. At the first level, the purified Ig will be separated from other serum proteins and will select all IgGs including the IgG of interest and other IgG molecules. These purification steps can be done by using ammonium sulfate to precipitate the Ig molecules or it can be done by binding antibodies to a Protein A and/or Protein G columns. Proteins A and G are produced by the bacteria. Staphylococcus aureus, and bind to different species and subclasses of antibodies by the Fc receptor. After the antibodies have attached, they are washed out by changing the buffer. 

Solubility curves for different types of Hp have been presented (H5, H7). The higher solubility of type 1-1 than that of the others is in conformity with its lower molecular weight. The irregularity of the solubility curves for Hp of 2-2 and 2-1 type reflects the molecular heterogeneity of these two proteins. It is known from fractionation experiments with ammonium sulfate as well as with ethanol that the slower Hp bands are enriched in those Hp fractions that are precipitated first. So far, we have not been able to separate any of the Hp bands completely from the others, except the 1-1 band in ascitic fluid of type 2-1. 

As with serum IgG (see Chapter 10), enrichment of MAbs by precipitation is a useful starting point for further purification. The use of ammonium sulfate, sodium sulfate, PEG, and caprylic acid, as described in Chapter 10, is applicable to hybridoma culture supernatants and ascitic fluid. 

Purification of Monoclonal Antibody. Immunoglobulins were precipitated from the pooled ascites by addition of an equal volume of saturated ammonium sulfate [50% (NH4)2S04]. The precipitate was collected by centrifugation (20 min 10,240 X g), dissolved in 0.01 M sodium phosphate (pH 6.8), and reprecipitated. After the second ammonium sulfate precipitation, the pellet was dissolved in a minimum volume of 0.01 M sodium phosphate (pH 6.8) and centrifuged for 10 min at 10,600 X g). The resulting supemate was applied to a P6G, gel filtration polyacrylamid, column (Bio-gel Biorad, Rockville Center, NY 1.5 X 40 cm). Fractions containing protein were pooled and applied to a hydroxyapatite column that had been equilibrated with 0.01 M sodium phosphate (pH 6.8). Proteins were eluted with a linear gradient of 0.01 to 0.3 M sodium phosphate. 

In cases where the cell line is a heterohybrid, that is, human/mouse hybrid, it may be possible to grow the ceil line as a tumor in suble-thally irradiated mice and produce ascitic fluid. If this proves successful, then the ascitic fluid can be treated with saturated ammonium sulfate and the precipitate redissolved and used in conventional lEC or... 

Serum, ascites, or hybridoma culture supernatant can, after filtration, be applied to the column directly. The crude antibody solution should then be diluted with 1/10 volume of IMTris-HCl, pH 8.0, and chromatography should be performed with 100 xaM Tris-HCl, pH 8.0. It is, however, advisable first to precipitate the immunoglobulins with ammonium sulfate see Chapter 2). This not only reduces the sample volume, but more importantly removes lipids, particularly from serum and ascitic fluid, extending the life of the column. 

Immunization and Fusion Protocol. Groups of five BALB/c female mice (4-6 weeks old), were given series of three injections every two weeks, with KLH-conjugated atrazine or hydroxyatrazine (50 ug/injection) mixed with Freund s adjuvant. After a rest period of two months, the mice were boosted with 500 ug of the conjugate. Three to four days later, the mice were sacrificed and the spleen cells were fused with the murine myeloma cell line Sp 2/0.Agl4 (12,13). The positive hybridomas were cloned and expanded in mice and the MAbs were purified from the ascitic fluid by ammonium sulfate precipitation, and DEAE-cellulose anion-exchange chromatography (14). 

Fractional precipitation methods can have damaging effects on certain mAbs. Pilot studies can examine this possibihty. Methods classically involve the use of ammonium sulfate, sodium sulfate, caprylic acid, Rivanol, and polyethylene glycol. These separate mAh from the majority of other proteins in ascites. Following dialysis, this procedure may well be sufficient for use in ELISA. 

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