KPL Antibodies and Conjugates Catalog

More documents

Recommendations

Info

SERACARE KPL ANTIBODIES AND CONJUGATESSerum Adsorption (Negative Affinity Purification)Polyclonal antibodies are most commonly used asanti-immunoglobulin secondary antibodies in variousimmunoassays. Even though polyclonal antibodies oftendisplay higher sensitivity than monoclonal antibodies,they can suffer from a lack of specificity. In certainapplications even antigen affinity purified antibodiesmay suffer from a lack of specificity. Despite beingaffinity purified as species-specific, anti-immunoglobulinscan bind other species to varying degrees due toshared epitopes across species. Depending on the immunoassays,researchers may need this cross-reactivityremoved. Traditionally, the cross-reactivity was removedby adding serum of the unwanted species. Thecross-reacting antibodies bound to the serum and wereremoved from the total antibody pool. Cross adsorptionis easy to perform but introduces a complex proteinmixture of serum from a different species back intoa relative pure antibody solution. The antibody mustthen be repurified.YYCross-ReactingAnti-Mouse IgGAnti-Mouse IgGColumn BoundCross-Reacting AntigenYYYYYYYYY YYCross-reacting antibodies bind to column matrix.YYYYYYY YYY YY YYYWhile the above technique is historically valid, thesedays most manufactures of secondary antibodies attachserum to a solid support and make negative affinitycolumns from serum. Thus serum adsorption is bettercategorized as a specific type of negative affinity, ratherthan a purification technique. Despite the wide use ofnegative affinity for purification purposes, manufacturershave traditionally only listed which antibodies havebeen serum adsorbed because knowledge of serumadsorption is critical for researchers performing multiplexassays.10 To order, call +1.301.948.7755 or toll free 1.800.638.3167 or visit www.kpl.com
SERACARE KPL ANTIBODIES AND CONJUGATESHow KPL Antibodies Are PurifiedThe proprietary purification technology used results inhigher affinity antibodies, which increase sensitivity andreduce background. Selected antibodies may be purifiedusing negative affinity columns to minimize cross-reactivitybetween animal species or to reduce shared reactivity withother immunoglobulin classes. These processes yieldpure antibodies with defined specificities and result inconsistent lot-to-lot performance.Considerable effort is spent developing and purifyingan antigen formulation to generate high titer, specificantiserum, since pure antigen results in a more potentand specific antibody.ImmunizationPrior to immunization, animals are carefully screenedfor reactivity toward the antigen. This allows thescreening out of animals that will not produce aneffective antibody. Next, multiple animals are involvedin the production of one antibody, thus allowing forthe collection the most potent antibodies possible. Inaddition, pooling antiserum from multiple animalsminimizes natural serum variability, resulting in uniformlarge-scale antibody lots.Production bleeds are obtained from the animals andare screened for potency. Production bleeds that do notmeet quality requirements are rejected. The immunoglobulinfraction of antisera is then isolated and testedfor purity as well as potency against the antigen.PurificationEven with the purity testing, the antiserum will containmany lipids and serum proteins. The most prevalentamong them is albumin, which accounts for 50% of theprotein in serum. The first step in the affinity purificationprocess is accomplished by a series of salt precipitationsteps in which 99% of these substances are removed.The resultant immunoglobulin fraction is likelyto contain only 5-10% specific antibody. At this point,affinity purification using both positive and negativeselection is utilized to purify the specific antibody fromthe nonspecific antibody. Then, the antibody is testedagain for potency and cross-reactivity.Species Cross-ReactivityImmunoglobulins of related animal species often sharesimilar epitopes derived from homologous proteinstructure and sequence. For example, affinity purifiedantibodies against mouse IgG may recognize epitopeson human IgG. To minimize cross-reactivity betweenmouse and human species, antibodies to mouse immunoglobulinare adsorbed against immobilized humanserum on a negative affinity column. The resultingantibody, anti-mouse IgG, human serum adsorbed(HSA), is highly specific to mouse IgG with minimalreactivity to human IgG or to any other human serumcomponents. Select antibody preparations are furtheradsorbed against multiple animal species to reducereactivity to shared regions among these species.Heavy and Light Chain Cross-ReactivityPolyclonal antibodies recognize different regions on IgGheavy and light chains. Some of these regions are similarto IgA and IgM heavy and light chains. Therefore,these antibodies consist of subpopulations that also recognizeIgM and IgA. To make an antibody specific forthe IgG heavy chain, these subpopulations need to beremoved or reduced. To do this, the purified antibodiesare passed over a negative affinity column with immobilizedwhole IgM and IgA antibodies containing bothheavy and light chains. Antibodies that recognized IgMand IgA bind to the column while antibodies specificfor the IgG heavy chain pass through and are collected.The Finished ProductThe final purified antibody is characterized for potencyand specificity by two distinct ELISA formats and forpurity by SDS-PAGE. After the testing is complete, thepurified antibody is lyophilized and undergoes onemore round of testing for potency and solubility.To order, call +1.301.948.7755 or toll free 1.800.638.3167 or visit www.kpl.com11
Page 1 and 2: See moresignalwith less noiseSeraCa
Page 3 and 4: SERACARE KPL ANTIBODIES AND CONJUGA
Page 9: SERACARE KPL ANTIBODIES AND CONJUGA
Page 34 and 35: SeraCareCustom SolutionsSeraCare ha
Page 40: BacTrace is a registered trademark

KPL Antibodies and Conjugates Catalog

Create successful ePaper yourself

Delete template?

Save as template?