rev: November 11, 1999
HOME (index page)
Return (alphabetical antibody page)
Return (anti-Rat/Mouse Immunoglobulin..index)
anti-RAT
and MOUSE
ANTIBODIES
Mouse Immunoglobulin Purification Information
RDI Division of Fitzgerald Industries Intl offers a wide line of antibodies. Since no one antibody works best for all applications (neutralization, blotting, ELISA, etc), we offer many different types of antibodies to help solve this problem. Please inquire for other applications or types of antibodies not listed below.
TECHNIQUE OF PURIFICATION OF MOUSE Ig BY IMMUNOAFFINITY CHROMATOGRAPHY
"x" ml of serum or ascitic fluid from BALB/c mice bearing or not a hybridoma are applied at a rate of about 2 ml/min at room temperature to a column of Sepharose-4B (Pharmacia, Belgium) on which "y" mg of rat monoAb anti-mouse Ig has been immobilized. The column is washed with 100-120 ml of phosphate buffered saline (PBS), then 100 ml of PBS containing 2.5 M NaCl, and then again at normal salinity with 100 ml of PBS. Mouse immunoglobulins are eluted by decreasing the pH with glycin HCL 0.1 M + 0.15 M NaCl buffer at pH 2.8*. The eluted fractions are neutralized as rapidly as possible after the elution with glycin NaOH buffer (0.1 M, pH 8.6) (Bazin et al., 1986; Cormont et al., 1986).
Examples of capacity of monoAb immunoaffinity column can be found in Bazin and Malache (1986).
For example, with the present experimental conditions, they were of 0.2 mg of mouse IgM for 1 mg of LO-MM-9 coupled to Sepharose-4B and 1 mg of mouse IgG1 for 1 mg of LO-MG1-2 coupled to Sepharose-4B.
*Depending on the rat monoclonal antibodies, the pH of the eluting buffer could be from pH 2.8 to pH 4.5.
REFERENCES
BAZIN H., CORMONT F., DE CLERCQ L. Purification of rat monoclonal antibodies.
Methods in Enzymology 1986,
121, 638-652.
CORMONT F., MANOUVRIEZ P., DE CLERCQ L., BAZIN H. The use of rat monoclonal
antibodies to characterize,
quantify and purify polyclonal or monoclonal mouse IgM. Methods
in Enzymology 1986, 121, 622-631.
BAZIN H., MALACHE J.M. Rat (and mouse) monoclonal antibodies.V.A simple automated
technique of antigen
purification by immunoaffinity chromatography. J. Immunol.
Methods 1986, 88, 19-24.
IMMUNOAFFINITY PURIFICATION OF MOUSE IMMUNOGLOBULINS BY RAT MAb: INDICATIVE VALUES OF ELUTION (pH or ionic strength)
| MAB
Sepharose 4B |
Elution buffer
NaCl 2.5M pH 7.2 |
Elution buffer
NaCl 0.9M pH 4.5 |
Elution Buffer
NaCl 0.9M pH 3.8 |
Elution Buffer
NaCl 0.9M pH 2.8 |
| LO-MK-2 | 0 | 0 | X | XX |
| LO-MM-9 | 0 | 0 | XX | XX |
| LO-MG1-2 | 0 | 0 | XX | XX |
| LO-MG2a-3 | 0 | 0 | X | XX |
| LO-MG2b-1 | 0 | 0 | XX | XX |
| LO-MG2b-2 | 0 | 0 | XX | XX |
0: no elution; X: partial elution; XX:complete elution
Examples of Binding Capacities of mouse Immunoglobulin by Rat MAB
| MAB-Sepharose | Number of runs already done with the column | Capacity (in mg) of binding per mg of MAB |
| LO-MK-2 | 2 | 0.45 |
| LO-MM-9 | 38 | 0.2 |
| LO-MG!-2 | 26 | 1.0 |
| LO-MG2a-3 | 28 | 0.55 |
| LO-MG2b-1 | 7 | 0.16 |
| LO-MG2b-2 | 28 | 0.41 |
| Immunoglobulin to be purified | column | 1st peak (fall through) | 2nd peak (eluted with acidic buffer) |
| 5ml of PB-1 BALB/c IgM secreting plasmacytoma mouse ascitic fluid | 30mg of LO-MM-9 (rat MaB anti-mouse IgM) coupled t 1.5g of Sepharose | Mouse ascitic fluid without IgM | 7.8mg of mouse PB-1 and polyclonal IgM |
| 10ml MARD-3 BALB/c IgG1 secreting Mab mouse ascitic fluid | 25 mg of LO-MG1-2 (rat Mab anti-mouse IgG1) coupled to 1.0g of Sepharose-4B | mouse ascitic fluid without IgG1 | 20mg of mouse MARD-3 and polyclonal IgG1 |
| 5ml of MOPC-173 Balb/c IgG2a secreting plasmacytoma mouse ascitic fluid | 25 mg of LO-MG2a-3 (rat Mab anti-mouse IgG2a) coupled to 1.0g of Sepharose | mouse ascitic fluid without IgG2a | 17.2mg of mouse MOPC-173 and polyclonal IgG2a |
| 7ml of C1907 B3* Balb/c IgG2a secreting plasmacytoma mouse ascitic fluid | 50mg of LO-MG2b-2 (rat Mab anti-mouse IgG2b) coupled to 2.0g of Sepharose-4B | mouse ascitic fluid without IgG2b | 21.9 mg of mouse C1907 B3 and polyclonal IgG2b |
| 7.5ml of MOPC-173 BALB/c IgG2a kappa secreting plasmacytoma mouse ascites | 35 mg of LO-MK-2 (rat MAB anti-mouse Ig kappa) coupled to 2.0g of Sepharose-4B | mouse ascitic fluid without kappa light chain Ig | 12.4mg of mouse MOPC-173 and polyclonal Ig kappa |
| 45ml of normal mouse ascitic fluid | 30mg of LO-MM-9 (rat Mab anti-mouse IgM) coupled to 1.5g of Sepharose-4B | normal mouse ascitic fluid without IgM | 3.7mg of mouse polyclonal IgM |
| 30ml of normal mouse ascitic fluid | 25 mg of LO-MG1-2 (rat Mab anti-mouse IgG1) coupled to 1.0g of Sepharose-4B | normal mouse ascitic fluid without IgG1 | 16.0mg of mouse polyclonal IgG1 |
| 25ml of normal mouse ascitic fluid | 25 mg of LO-MG2a-3 (rat Mab anti-mouse IgG2a) coupled to 2.0g of Sepharose-4B | normal mouse ascitic fluid with IgG2a | 9.1mg of mouse polyclonal IgG2a |
| 25 ml of normal mouse ascitic fluid | 50mg of LO-MG2b-2 (rat MAB anti-mouse IgG2b) coupled to 2.0g of Sepharose-4B | normal mouse ascitic fluid without IgG2b | 7.5 mg of mouse polyclonal IgG2b |
| 15ml of normal mouse ascitic fluid | 35 mg of LO-MK-2 (rat Mab anti-mouse Ig kappa) coupled to 2.0g of Sepharose-4B | normal mouse ascitic fluid without kappa-l-chain Ig | 12.0mg of mouse polyclonal Ig kappa |
*Kindly provided by Dr J. Van Snick (UCL)
RDI Division of Fitzgerald Industries Intl
34 Junction Square Drive
Concord MA 01742-3049
USA
phone (978) 371-6446 or (800) 370-2222
fax (978) 371-2266
EMAIL:antibodies@fitzgerald-fii.com
Return (anti-Rat/Mouse Immunoglobulin..index)
Ordering terms