Sera & Immunoglobulins

Normal Sera
Mouse Myeloma Proteins
Purified Immunoglobulins

Normal Sera
Normal serum is useful for reducing background or as a negative control in immunoassays. Pre-incubation of immunoblots or tissue sections in buffer with 1-10% normal serum of the same species as the conjugated antibody host can reduce background due to nonspecific binding of the conjugate, and is sometimes more effective than albumin alone. In indirect immunoassays up to 1% normal serum of the same species as the blotted protein or tissue section can be added to the secondary antibody conjugates to reduce background due to cross-reactivity. Normal serum can be used in assays as a negative control to measure background staining due to nonspecific binding or cross-reactivity, substituting for pre-immune serum if that is not available.

Normal sera from animal species commonly used in antibody production are tested for protein concentration by Biuret and assayed for a normal protein profile by immunoelectrophoresis. They contain 0.01% thimerosal as a preservative.

Store normal sera at -20 °C in working aliquots to avoid freeze-thaw cycles.

See the table of normal serum immunoglobulin ranges.

Mouse Myeloma Proteins

Myeloma is a cancer of the antibody-producing plasma cells. It occurs spontaneously in humans and mice, and can be induced by injecting some substances such as mineral oil, pristane, or viruses into the peritoneum. Myeloma cells continue to secrete immunoglobulins in a normal fashion, but exhibit uncontrolled growth. As the number of myeloma cells increases, the level of myeloma immunoglobulins in plasma also rises, eventually inhibiting the production of immunoglobulin by normal plasma cells.

Plasmacytomas have been cloned from myelomas, yielding cell lines that produce a completely homogeneous population of myeloma immunoglobulin bearing a single heavy chain isotype and a single light chain type. This myeloma protein retains the antigen specificity of the Ig produced by the plasma cell from which the myeloma originated, although that specificity is not usually known. Plasmacytomas may be propagated as tumors in the peritoneum of animals, secreting myeloma Ig into the surrounding ascites fluid, or in cell culture, secreting myeloma Ig into the culture supernatant. Mouse plasmacytomas bear designations consisting of an alphabetic prefix, an accession number, and sometimes a letter to indicate a subclone. The prefix is usually derived from the tumorigenic agent or the last initial of the investigator who induced the tumor, sometimes combined with PC for plasmacytoma.¹ The table below lists some prefixes used for the myelomas offered by Sigma, along with their meaning:

MO = Mineral oil (such as MOPC)

HO = 7-n-Hexyloctadecane (such as HOPC)

TE = Tetramethylpentadecane (pristane) (such as TEPC)

Y = Yancy

AB = Abelson virus (such as ABPC)

Mouse myeloma proteins are produced by plasmacytoma lines carried intraperitoneally in BALB/c mice. Ascites fluid produced by the mice contains elevated levels of the myeloma protein produced by the plasmacytoma, as well as normal immunoglobulins and other serum proteins. Sigma offers mouse myeloma proteins as clarified ascites or purified immunoglobulins. Each purified myeloma immunoglobulin represents a single subclass and light chain type.

Note: The light chain designation following the subclass refers to the type of light chain carried on the immunoglobulin molecule.

Mouse myeloma proteins have many applications:

1. Due to the homogeneity of these immunoglobulins, they are useful for amino acid sequencing studies.²
2. They may serve as positive isotype controls in the characterization of monoclonal antibodies and as negative controls for monoclonal antibodies in immunoassays.³ When using mouse myeloma proteins as negative controls, it is important to match not only the immunoglobulin isotype but also the level of purity and the immunoglobulin concentration as closely as possible. For instance, if the test monoclonal is in ascites, then a clarified ascites product should be used for the negative control. If the monoclonal has been purified, then the purified myeloma immunoglobulin is a better choice. In either case, the concentration of the myeloma protein should be adjusted to approximate the concentration of the test monoclonal antibody. When testing monoclonal antibodies in cell culture supernatant, use the purified myeloma protein diluted in cell culture medium to the approximate concentration of the test monoclonal antibody.
3. Where the antigen specificity is known, mouse myeloma proteins may be used as capture antibodies in capture ELISA or as primary antibodies in immunohistochemistry or immunocytochemistry for targets bearing that antigen (i.e., in the case of M 2046, dinitrophenyl-labeled proteins).
4. Myeloma IgG and IgM may be used as molecular weight markers.

References

1. Potter, M., Immunoglobulin-producing tumors and myeloma proteins of mice. Physiol. Rev., 52, 631-719 (1972).
2. Kehry, M. et al, Amino acid sequence of a mouse immunoglobulin µ chain., Proc. Nat. Acad. Sci. USA, 76, 2932-2936 (1979).
3. Kramer, K. et al., Prognostic value of TrkA protein detection by monoclonal antibody 5C3 in neuroblastoma. Clin. Cancer Res., 2, 1361-1367 (1996).

Clarified Ascites Fluid
Ascites fluid is clarified by centrifugation and filtration. Immunoelectrophoresis and Ouchterlony double diffusion confirm the myeloma identity. Electrophoresis and quantitative densitometry determine myeloma content. Each vial contains at least 5 mg of myeloma protein. Control ascites NS-1 (Product Code M 8273), is produced by a non-secreting plasmacytoma. It has been tested as a negative control for indirect ELISA, indirect immunofluorescence staining of cell suspensions, and immunoperoxidase methods.

Clarified ascites are supplied lyophilized from 0.01 M phosphate buffered saline, pH 7.2, except for NS-1, which is supplied as liquid ascites fluid containing 15 mM sodium azide as preservative. They may be stored at 2-8 °C prior to reconstitution. Reconstitute with 1 ml deionized water per vial. Reconstituted and liquid ascites should be stored frozen in working aliquots to avoid repeated freeze-thaw cycles. Storage in a frost-free freezer is not recommended.

Purified Mouse Immunoglobulins
Purified myeloma immunoglobulins are isolated from ascites fluid by a combination of fractionation, ion-exchange chromatography, and/or affinity chromatography. The identity of the immunoglobulins is confirmed by immunoelectrophoresis against anti-whole serum and anti-subclass specific antisera. Ouchterlony double diffusion against subclass specific antisera is used to confirm the absence of contaminating subclasses. Purity of the immunoglobulin is determined by SDS-PAGE. Protein content is determined by spectrophotometry.

IgG subclasses and IgA are supplied in 0.02 M Tris buffered saline, pH 8.0. IgM is supplied in 0.05 M Tris, 0.5 M sodium chloride, pH 8.0. Sodium azide (0.02%) is added as preservative. For applications that may be affected by the presence of azide, the azide may be removed by dialyzing the protein against the desired buffer. Sigma offers a disposable dialyzer (Product Code Z36,839-3) with a capacity of 2 ml and a molecular weight cutoff of 10,000 that is suitable for dialysis of the purified myeloma immunoglobulins. Liquid proteins should be stored frozen in working aliquots to avoid repeated freeze-thaw cycles. Storage in a frost-free freezer is not recommended.

Purified Immunoglobulins
A wide range of human and animal immunoglobulins (Igs) for use in immunological and biochemical applications have been isolated from normal or myeloma sources. Purified Igs may be used in various immunoassays and biochemical applications as positive or negative controls, standards, coating antigens, or blocking agents. They may also serve as starting material for the preparation of immunogens, tolerogens, or solid-phase immunosorbents. Our high standards and rigorous testing procedures ensure a high level of product quality, purity, and consistency. Normal Igs are offered in two grades, Reagent Grade and Technical Grade, to meet a wide range of customer needs.

Reagent Grade
Reagent Grade Igs are prepared from normal sources, such as the serum of animals that have not been immunized with a specific protein. A combination of fractionation, ion-exchange chromatography and/or gel filtration chromatography is used to isolate the Igs. IgG and IgM are prepared from pooled normal serum. IgA is prepared from normal colostrum and, therefore, is in the secretory form.

Reagent grade Igs have been prepared to meet the highest purity standards. They are determined to be > 95% pure by non-reducing SDS-PAGE.

The high purity of Reagent Grade Igs makes them excellent reagents for sensitive immunoassays. They may be used as:

• Standards
• Blocking agents
• Negative controls

They are also suitable starting materials for:

• Immunogens for the production of antiserum
• Tolerogens to induce tolerance to a specific class of immunoglobulin
• Conjugation to solid supports to prepare immunosorbents for the purification or removal of antibodies and other proteins with a specificity for the individual Igs.

Reagent Grade Igs (except for chicken IgG and mouse IgG) are supplied as liquids or as lyophilized powders which are greater than 90% protein by weight. The lyophilized powders (except for chicken and mouse IgG) are essentially salt-free (less than 1% sodium by weight as determined by atomic emission) and contain less than 10% water by weight as determined by the Karl-Fischer method. Mouse and chicken IgG are lyophilized from phosphate buffer and are approximately 80% protein by weight. The protein content is determined by spectrophotometry using = 14. Liquid preparations are supplied in buffered solutions with 15 mM sodium azide as preservative.

Technical Grade
Technical Grade Igs are isolated from pooled normal serum by fractionation. Their purity is determined to be > 80% by non-reducing SDS-PAGE.

Technical Grade Igs are an economical alternative for applications that do not require the high level of purity of the Reagent Grade Igs. They may be used as negative control antibodies when the experimental antibody is in the form of an IgG fraction (i.e. not as pure as an affinity isolated antibody). They may also serve as blocking agents in immunoassays or as starting material for procedures involving downstream purification such as papain or pepsin digestions to produce Fc, Fab, or F(ab)₂ fragments of IgG.

Technical grade Igs are supplied as solutions in 10 mM phosphate buffered saline, pH 7.2, with 15 mM sodium azide as a preservative.

Human IgG Subclasses
Sigma offers human IgG subclasses as purified immunoglobulins. Each purified immunoglobulin represents a single subclass and light chain type. Human IgG subclasses have been isolated from myeloma plasma by fractionation, ion-exchange chromatography, and/or affinity chromatography. The identity and purity of the immunoglobulins are determined by immunoelectrophoresis, indirect ELISA, and SDS-PAGE. Protein content is determined by spectrophotometry. The antigen specificity of these myelomas has not been determined. The source material for human IgG subclasses has been tested and found negative for antibody to HIV and HCV, and for HbsAG.

Human IgG subclasses are supplied as solutions and should be stored frozen in working aliquots to avoid repeated freeze-thaw cycles. Storage in a frost-free freezer is not recommended.

Note: The light chain designation following the subclass refers to the type of light chain carried on the immunoglobulin molecule.