Antibodies, fractionation

Purified C5-1 has been obtained from alfalfa leaf extracts by affinity chromatography on either a human IgG-Sepharose column or a Streamline rProtein A-Sepharose column. Interestingly, the purified product obtained with these two methods differed significantly. As shown in Fig. 1.5 a, the antibody fraction obtained from the human IgG column contained a mixture of different intermediate assembly forms of the heavy (H) and light (L) chains, ranging from H2 to the fully assembled H2L2 form. 

It is well known that a very important feature of many biological systems is specific recognition at the molecular level. Antibodies as a group are widely used for molecular recognition, e.g., affinity assays. This feature can be used by labeling an anti-human growth hormone antibody fraction with a fluorescent tag... 

Beh, K. J. 1973. Distribution of brucella antibody among immunoglobulin classes and a low molecular weight antibody fraction in serum and whey of cattle. Res. Vet. Sci. 14, 381-384. 

Dialyze the antibody fractions (detected by ELISA) against PBS or HBS to remove imidazole and NaCl (typical yields are 0.2-20 mg/L of culture). Some antibodies tend to precipitate during dialysis The precipitation is associated with the presence of the his-tag. Addition of 20 mM EDTA to the antibody sample before dialysis often solves this problem. 

M5. Miyai, K., Endo, Y., and Hata, N., Fluorescence immunoassay of thyroxine with clonotype antibody fractionated by ehromatofoeusing. Proc. Int. Fed. Clin. Chem., 12th, in press (1985). 

Antibody fraction from an HCIC column can be then adjusted at pH 8 and loaded into a column of hydroxyapatitte, previously equilibrated with 10 mM phosphate buffer, pH about 7. Impurities are found in the flow through and pure antibodies are desorbed by increasing the ionic strength by a phosphate-buffered solution of potassium chloride. 

Human normal immunoglobulin (HNIG) is prepared from screened, human plasma. The antibody fraction is extensively purified and contains immunoglobulins in glycine. Some products may contain sorbitol (refer to product information). HNIG is also used to treat possible infections as it contains antibodies to infectious pathogens against which the donors have been immunised. 

After the animal has been bled, the blood is allowed to dot by standing at room temperature for 1-2 h. The blood is centrifuged carefully at 5,000 g, avoiding lysis of the erythrocytes, to separate the serum and dotted fibrin factions. This antibody fraction can be stored at -80 °C for years without loss of activity. However, it is recommended that before aliquoting and freezing, the complement system should be inactivated, because this can interfere with many immunochemical reactions. Inactivation is carried out by simply heating to 56 °C for 10-20 min. 

Locate the antibody fractions by absorbance at 280 nm and by determining binding to antigen. 

PI. Peltre, G., and Brogren, C. H., Immuno-isoelectric focusing analysis of antibodies fractionated by isotachophoresis. In Electrofocusing and Isotachophoresis (B. ]. Radola and D. Craesslin, eds.), pp. 577-585. Walter de Gruyter, Berlin, 1977. Poehling, H. M., and Neuhoff, V., One- and two-dimensional electrophoresis in micro-slab gels. Electrophoresis 1, 90-102 (1980). 

Alternatively, combined antibody fractions can be desalted using size exclusion chromatography columns such as PD-10 from GE Healthcare BioSciences. 

The cross-reactions of Types III and VIII Pneumococcus in horse and rabbit antisera were studied quantitatively by Heidelberger and his colleagues. - At least three distinct kinds of anticarbohydrate (after removal of antibodies to the somatic C-substance) were evoked in horses in response to the stimulus of the type-specific pneumococcal antigen. Two of these, usually from one-quarter to one-third of the total, were completely precipitable by SIII or SVIII. However, the principal antibody fraction... 

The nature and preparation of antibody fractions and their relevance in disease and assay should also be examined (i.e., whole molecule, Fc, Fab, F[ab ]2, IgM, IgA). 

Antibodies specific for the active site of concanavalin A have been isolated from rabbit sera. Two antibody fractions, which were isolated after affinity chromatographic separation on immobilized concanavalin A, showed distinct differences in biological activity. One population of antibodies was directed to the active site of the lectin, and binding of the antibody in this case is inhibited by methyl cv-D-mannopyranoside, whereas the other antibodies were directed to the antigenic determinants on the concanavalin A molecule. 

This represents the amount and reactivity of the antibody fraction that was displaced from the immunoadsorbent by 5 M guanidine hydrochloride. After dialysis against 0.01 M phosphate buffer at pH 7.4 containing 0.15 M NaCl, its quantitative precipitin reactions with BSA and fragment 377-571 were determined. 

It seems not unlikely that certain processes auxiliary to antibody formation occur. The reported increase in globulin (aside from the antibody fraction) after immunization suggests the operation of a mechanism whereby the presence of antigen molecules accderates the synthesis of the globulin polypeptide chains. There is little... 

Here, we describe the details of an improved cDNA subtraction method based on a cDNA subtraction approach previously applied to single cells (6), which in turn is based on a global cDNA amplification protocol (PolyAPCR) used to produce amplified cDNAs, representing all the mRNAs present in the samples as small as a single cell (7,8). PolyAPCR has been applied successfully to a wide range of samples, including single micromanipulated cells (6,7,9,10), antibody fractionated populations (11), and fixed tissues (12). 

The affinity matrices were (0.5 ml bed volume) equilibrated with PBS-BT buffer and 0.5 ml of antiserum fractionated on each column. The unbound material was washed with 20.0 ml loading buffer (PBS-BT) followed by a 0.5 M NaCl to remove nonspecific binding. Specific antibody fraction was eluted in the same buffer adjusted to pH 2.0 and the fraction (2.0 ml each) were immediately neutralized. Assayed 100 tds of each fraction with either [3H]NAD (33,400 cpm = 0.90 ng) or 3H-5 AMP (33, 610 cpm = 0.83 ng) in PBS-BT buffer. 

Fig. 8.18 Principle of Fluorescence Polarization Immunoassay (FPIA). The rotational correlation time (0) is different for the free [(Re-L) -HSA and bound [(Re-L) -HSA]-anti-HSA-antibody fraction of the tracer.

Pipette 10 xl samples from each of the eluate fractions into coimting tubes and count them in a gamma counter in order to identify the tubes containing the labelled antibody fractions (typically tubes 3-5). Use or store the contents as required. 

Measure the OD 280 nm of each fraction on a UV spectrophotometer. Pool all antibody fractions with a concentration > 1 mg/ml. (Absorbance of 1 mg/ml solution dF IgG = 1.4/cm light path.) Measure the concentration of the pooled antibody. Cap the tube and keep on ice. Divide into suitable size aliquots (typically 250-500 pg) and freeze at the lowest temperature available. 

---