Coupling to Sepharose

Specific IgE Assay. Two radioimmunoassays are available in France using a quaternary ammonium compoimd coupled to Sepharose [30, 31]. The sensitivity of these tests was equivalent at 88%, the specificity reaches 90%. A morphine-based immunoassay has been proposed in Australia [14]. More recently, Ebo et al. [32] investigated a rocuronium ImmimoCAP and set the sensitivity at 85%, the specificity being absolute, provided an assay-specific decision threshold is applied. An ImmimoCAP (Phadia A) is available. 

Huot et al. [38] used affinity chromatography to identify and partially purify an amiloride-binding protein with characteristics of the renal brush border Na /H exchanger. The high-affinity amiloride analog A35 (5-A-(3-aminophenyl)amiloride) was coupled to Sepharose CL-4B through a triglycine spacer. Rabbit renal brush border membranes were solubilized with 0.6% Triton X-100, incubated with the... 

Zopf, D.A., Smith, D.F., Drzeniek, Z., Tsai, C.-M., and Ginsburg, V. (1978a) Affinity purification of antibodies using oligosaccharide-phenethylamine derivatives coupled to Sepharose. In Methods in Enzymology, (V. Ginsburg, ed.), Vol. 50, pp. 171-175. Academic Press, New York. 

Figure 6. Affinity chromatography of EGD from Clostridium thermocellum. Nucleic acid preparation, heat treatment and ammonium sulfate precipitation (0-70%, 70-100%) were carried out as described (10). The final precipitate ( 50 mg protein), dissolved in 50 mM sodium acetate, pH 5.0, was applied (after centrifugation) on the affinity column (2 x 25 cm) (4 -aminobenzyl l-thio-/ -cellobioside coupled to Sepharose 4B) (11). Protein was monitored at 280 nm and the activity of the fractions (2 ml) determined using 2 -chloro-4 -nitrophenyl / -cellobioside (pH 6.5, 25°C) as described in the text. Elution with 10 mM G2 was started as indicated.

Human albumin and polymixin B sulfate are each coupled to Sepharose according to the manufacturer s instructions. 

Cell-binding studies on human erythrocytes, several human, urinary-bladder, carcinoma cell-lines, and an osteogenic-sarcoma cellline have been conducted.189 After treatment with neuraminidase, 80% of human lymphocytes will bind the H. pomatia lectin.5728 Neuraminidase-treated lymphocytes can also be fractionated on Helix pomatia hemagglutinin coupled to Sepharose beads.572b... 

Human serum albumin coupled to Sepharose-entrapped iron oxide particles from a ferrofluid (108). 

To illustrate this principle, we have chosen a lectin-glycoprotein system (130). The glycoenzymes glucose oxidase and peroxidase are bound to immobilized Concanavalin A or lentil lectin coupled to Sepharose. The immobilized lectin is packed in a small column inside a simple flow calorimeter. A continuous buffer stream (flowrate 0.75 mL/min) is pumped through a small column, at the outlet of which is placed a thermistor. This unit is well insulated from the surroundings. 

Fig. 2. SDS-PAGE of affinity-purified MAbs specific for human IL-3. Track 5 shows standard mol-wt markers (from top to bottom myosin heavy chain, 200 kD p-galactosidase, 116 kD phosphorylase b, Mf 97.4 kD bovine albumin, 66 kD egg albumin, Mr 45 kD and carbonic anhydrase, M, 29 kD). Track 1 shows Cohn fractionated human IgG. The remaining three tracks show three different IgG antibodies purified using a column of human recombinant DNA-derived IL-3 coupled to Sepharose 4B. The m jor bands are kappa and gamma chains the faint higher Mr bands are caused by incomplete dissociation of heavy and li t chains.

Vol. L [16]. Affinity Purification of Antibodies Using Oligosaccharide-Phenethylamine Derivatives Coupled to Sepharose. D. A. Zopf, D. F. Smith, Z. Drzeniek, C.-M. Tsai, and V. Ginsbuig. 

Bi2-Sepharose has also been used in the preparation of human intrinsic factor from gastric juice. Affinity chromatography was the sole purification technique and yielded a protein 853-fold purified [91]. A series of derivatives of B12, lacking the nucleotide portion of the molecule, coupled to Sepharose were used in the resolution of two hog B12 binding proteins, termed intrinsic and non-intrinsic factor. The total B12 binding protein population, from which the two forms were resolved, was isolated by affinity chromatography on B -Sepharose [92]. 

Eldefrawi and Eldefrawi [98] reported the purification of the acetylcholine of Torpedo electroplax on an affinity column consisting of cobra (Naja naja siamensis) toxin coupled to Sepharose 4B. Desorption with 10 mM benzoquinonium produced a protein that bound [ I]a-bungarotoxin but not [ H]acetyl-choline. However, desorption with 1 mM carbamylcholine gave a receptor protein that bound pH]acetylcholine decamethonium, [ H]nicotine [ C]dimethyl-(-l-)-tubocuranrine, and [ I]a bungarotoxin. Schmidt and Raftery [99] also purified acetylcholine receptor, from Narcine, on a N-(e-aminohexanoyl)-3-aminopropyltrimethyl-ammonium bromide-HBr-agarose column. 

Protein A present in the cell wall of Staphylococcus aureus is a marker probe for the Fc portion of IgG-1, -2 and -4. Thus, a one step purification of protein A from extracts of 5. aureus was achieved using IgG coupled to Sepharose 4B affinity media [146]. The preparations obtained were pure as judged by electrophoresis. Conversely, affinity... 

In the second separation procedure, ConA coupled to Sepharose 2B was added to the vesicle mixture to preferentially bind the sensitized vesicles. After a 10-min incubation on ice, the mixture was filtered and... 

The 7-0-hemlsucclnate-7-desacetyl derivative of forskolin has been coupled to Sepharose to form an affinity resin. 2 Solubilized adenylate cyclase from rat brain and heart, but not pigeon erythrocytes, binds to this resin and can be eluted from the resin when forskolin is included in the buffer. Preparations of adenylate cyclase devoid of Ns activity are recovered from this column consistent with the proposal that forskolin binds to the catalytic subunit(s). Adenylate cyclase preactivated with guanosine-5, 3>Y i idodiphosphate (GppNHp) also binds to the forskolln-Sepharose column and elutes from the column as a complex of the catalytic subunlt(s) and the activated Ns protein. 

These results are consistent with the previous observation that RubisCO was covalently coupled to Sepharose by virtue of the epsilon amino on the large subunit(3). Thus, when part or all of the small subunits are present in the RubisCO-Sepharose, both the large and small subunit could be dissociated by 4 M urea. However, if all of the small subunits from the RubisCO-Sepharose are removed, 8 M urea but not 4 M urea was required to dissociate the large subunit core. This suggests that the binding between large subunits became tighter... 

Neuromuscular blocking drug (NMBD) or analog—choline for succinylcholine and decamethonium triethylcholine for gallamine Drug or analog covalently coupled to Sepharose Vecuronium produced 50 % inhibition at 0.2 nmol/tube... 

In addition to the conventional methods of peptide isolation, affinity chromatography may be used to isolate the labeled peptide. The method is based on the fact that the native antibody coupled to Sepharose can be used to bind specifically the labeled peptide by virtue of the ligand that is attached to the peptide. Thus peptides from affinity-labeled anti-DNP antibodies can be purified on anti-DNP Sepharose column. By this means, the positions of affinity labeled residues in the heavy and light chains of protein 315 were established. These results together with those obtained by other reagents are presented in Table... 

Both human (Vahlquist et al., 1971) and rat (Poole et al., 1975) RBP have been isolated by affrnity chromatography on human TTR coupled to Sepharose. Affinity chromatography with retinoic acid linked to Sepharose has also been used (Fex and Hansson, 1978) to isolate delipidated apo-RBP from serum and urine. Finally, RBP has been isolated by a number of investigators from the urine of patients with tubular proteinuria, using procedures similar to those employed for the isolation of RBP from serum (Peterson and Berggard, 1971 Vahlquist et al., 1971 Poulik et al., 1975 Fex and Hansson, 1979). 

The lectin from Ricinus communis coupled to Sepharose 4B has been used as an affinity-chromatography matrix in the purification of glycoproteins containing terminal D-galactosyl residues. ... 

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