KPL Antibodies and Conjugates Catalog

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YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYSERACARE KPL ANTIBODIES AND CONJUGATESAntibody PurificationWhen choosing a polyclonal antibody, either as aprimary or secondary antibody in an immunoassay,researchers are often inundated with an array of antibodypurification terms that they may not know muchabout. Understanding polyclonal antibody purificationsteps gives researchers the opportunity to pick the mostappropriate antibody purification technique for theirparticular immuno-application.Today, most polyclonal antibodies are referred to asbeing affinity purified. There are three major types ofaffinity purification: immunoglobulin-specific purification,antigen-specific purification, and serum adsorption.It should be noted that the different types ofpurification schemes are often used in combination toproduce a better antibody. But first, it is important tounderstand the early steps, serum purification and saltprecipitation.Serum PurificationPolyclonal secondary antibodies may be provided as aserum fraction. Serum is the amber colored supernatantobtained after blood is allowed to clot and is the simplestpurification technique. Serum contains different types ofantibodies as well as antibodies with a variety of affinities,some of which will bind to the antigen of interest andsome of which will bind to non-specific antigens. Serumis inexpensive and provides sensitivity, but not reliablespecificity. In fact, serum will likely contain less than10% specific antibody. The lack of specificity is due to amultitude of antibody clones and other proteins, includingalbumin, which may bind non-specifically in an immunoassay.Serum will often be found in individual labsprovided by researchers developing their own antibodies.Salt PrecipitationOne common technique for purifying proteins is saltprecipitation. The solubility of proteins is related to thesalt concentration of the solution. Increasing the amountof salt in a solution essentially removes water moleculesfrom the protein, causing the protein to precipitate. Thisis commonly called “salting out.” The differences in compositionand shape of proteins mean that proteins precipitateat varying concentrations of salt. Often proteinsare precipitated in a two-step or fractionated process. Inthe first step, salt is added so that the antibody of interestremains soluble, and unwanted proteins are removed.In the second step, more salt is added so that the antibodyof interest precipitates, and the unwanted solubleproteins can be removed. The precipitate is then dialyzedto remove the salt, and the antibody dissolved backinto solution. Serum can be precipitated this way usingammonium sulfate or caprylic acid. Salt precipitationis an inexpensive way to purify antibody from serum;however, the process does not remove proteins with aprecipitation pattern similar to antibodies.Step 1Serum with antibodies (Y)Extra proteins ( )Step 2Serum with antibodies (Y)Extra proteins ( )Low Salt PrecipitationYYYYYYYYYYYYYYYYYYYLow [salt]High [salt]CentrifugationSalt is added, forcing unwanted proteins to precipitate.Y YY YYHigh Salt PrecipitationYYYYYYYYYYYYYCentrifugationY YY YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYIncreasing the salt concentration eventually leads to antibody precipitation.BloodYYYYYYCentrifugationYYYYYYYYSerum with antibodiesYYYYBlood cellsAfter clotting, whole blood is centrifuged to separate serum fromother components.8 To order, call +1.301.948.7755 or toll free 1.800.638.3167 or visit www.kpl.com
SERACARE KPL ANTIBODIES AND CONJUGATESAffinity PurificationThere are three types of affinity purification. Many times,the three are used in combination when the purest antibodyis needed.Immunoglobulin-Specific Affinity PurificationIn certain bacteria, immunoglobulin-binding proteins areused to evade the host immune response. These proteinsbind immunoglobulins in an orientation that preventsnormal antibody function. Repurposed for purification,immunoglobulin-binding proteins are used to increasethe purity or to concentrate an antibody solution.There are three common immunoglobulin-bindingproteins used to purify antibodies, Protein A, Protein G,and Protein L. Protein A (from Staphylococcus aureus) andProtein G (from Streptococcus spp.) bind to the Fc regionof antibodies while Protein L (from Peptostreptococcusmagnus) binds to the antibodies through the light chain.Often these proteins are immobilized to a solid support(e.g., agarose beads) to form affinity matrices. Protein A,Protein G, and Protein L all have unique immunoglobulin-bindingcharacteristics according to species, antibodytype, and antibody isotype. Care must be taken when selectingan immunoglobulin-specific purification to makesure the antibody of interest will actually bind to theimmunoglobulin-binding protein. Because the techniquedoes not isolate antigen-specific antibodies, immunoassaysusing immunoglobulin-purified antibodies may stillhave background or specificity issues.Antigen Affinity Purification (Positive)Antigen affinity purification is another specific type ofaffinity purification and results in the purest antibodieswith the least amount of cross-reactivity. Affinity purifiedantibodies exhibit the highest specificity and sensitivitythat can be obtained from serum. Because these antigenspecificantibodies are polyclonal, they can be usedas both the capture and detection antibody in capture(sandwich) immunoassays.Despite all the care used to purify antibodies against aspecific antigen there are some epitopes that are sharedbetween proteins. These shared epitopes can causecross-reactivity. In order to remove the cross-reactivity,negative affinity selection is utilized. Negative affinitypurification selection is similar to antigen affinity purificationexcept that unwanted proteins are used to removecross-reacting antibodies. Often the unwanted proteinsare attached to a solid support, and an affinity column isconstructed using the cross-reacting antigen. During thepurification process a negative affinity column is utilizedto remove the unwanted antibody. Negative selection canbe performed one or more times depending on the levelof purity needed. The caveat to negative affinity columnsis that the total number of epitopes being recognizedby the polyclonal antibody is being reduced, and thus,the sensitivity will begin to decrease. A tradeoff must bemade between sensitivity and specificity.It should be noted that immunoglobulin-specific purificationis a specific type of affinity purification; however,manufacturers often do not distinguish between ProteinA, Protein G, or Protein L purification versus antigen affinitypurification.YAnti-Mouse IgGNonspecific Proteinsand ImmunoglobulinsColumn BoundMouse IgG EpitopesYYYY YYYYYYYAntibodyAgaroseBeadProtein A or GAntigen-specific antibodies bind to column matrix.To order, call +1.301.948.7755 or toll free 1.800.638.3167 or visit www.kpl.com9
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