Understanding Secondary Antibodies Note

Understanding secondary antibodies Fragment antigen binding antibodies and isotypes IgG D2 D2 F(ab’)2 after pepsin cleavage VH S S S CL S CH2 SS CH2 S S S S SS Hinge VL S S S S S CL S S S CH1 CH1 S S VL S S VH SS CH3 SS CH3 F(ab) antibodies The light chain (LH) folds into a variable domain (VL) and a constant domain (CL) whereas the heavy chain is composed of one variable domain (VH) and three (IgG and IgA) or four constant domains (IgE). Antigen binding sites Antibody structure Antibody structure and F(ab) antibodies F(ab) after papain cleavage F(ab’)2 after pepsin cleavage F(ab’) after pepsin and βmercaptoethanol The F(ab) fragment is an antibody structure that still binds to antigens but is monovalent with no Fc portion. An antibody digested by the enzyme papain yields two F(ab) fragments of about 50 kDa each and an Fc fragment. In contrast, F(ab')2 fragment antibodies are generated by pepsin digestion of whole IgG antibodies to remove most of the Fc region while leaving intact some of the hinge region. F(ab')2 fragments have two antigen-binding F(ab) portions linked together by disulfide bonds, and therefore are divalent with a molecular weight of about 110 kDa. Secondary antibodies F(ab) fragments Host Target Donkey Goat Goat Goat Goat Goat Human IgG - H&L Mouse IgG - F(ab’), pre-adsorbed Mouse IgG - H&L Mouse IgG - H&L Mouse IgG - H&L Mouse IgG - H&L Rabbit IgG - H&L Rabbit IgG - H&L Rabbit IgG - H&L Rat IgG - H&L IgG IgG IgG IgG - H&L H&L H&L H&L Conjugate Applications HRP TRITC TR Biotin Dot, ELISA, EM, IHC, WB Flow Cyt, IM Flow Cyt, IM Dot, ELISA, IHC, IM, WB 1.4nm Gold TRITC EM, WB Flow Cyt, ICC/IF, IHC-P HRP TRITC FITC Dot, ELISA, EM, IHC, WB Flow Cyt, IM Flow Cyt, IM Biotin HRP Biotin Dot, ELISA, IHC, IM, WB Dot, ELISA, EM, IHC, WB Dot, ELISA, IHC, IM, WB www.abcam.com/… ab6667 ab6522 ab6523 ab6578 ab30826 ab51379 ab6668 ab6823 ab6670 ab6669 ab6824 ab7055 ab7171 ab7176 F(ab’)2 fragments Host Target Conjugate Applications Donkey Goat IgG - H&L, pre-adsorbed Rabbit IgG - H&L, pre-adsorbed PE PE Flow Cyt, IHC-Fr, IHC-P, IM Flow Cyt, ICC/IF, IHC-P, IM ab7004 ab7007 Goat Mouse IgG - (Fab)’2, pre-adsorbed Mouse IgG - Fc, pre-adsorbed Mouse IgG - H&L, pre-adsorbed Mouse IgG+IgM+IgA - H&L, pre-adsorbed Mouse IgG+IgM+IgA - H&L, pre-adsorbed Mouse IgM - mu chain Mouse IgM - mu chain Mouse IgM - mu chain Mouse IgM - mu chain Rabbit IgG - (Fab)’2, pre-adsorbed Rabbit IgG - (Fab)’2, pre-adsorbed TR PE PE HRP FITC Biotin HRP AP FITC HRP TRICT Flow Cyt, IM Flow Cyt, IM Flow Cyt, IHC-Fr, IHC-P, IM Dot, ELISA, EM, IHC, WB Flow Cyt, IF Dot, ELISA, Flow Cyt, IHC, IM, WB Dot, ELISA, EM, IHC, IHC-Fr, WB Dot, ELISA, IHC, WB Flow Cyt, IM Dot, ELISA, EM, IHC, WB Flow Cyt, IM ab5884 ab5881 ab7002 ab6006 ab5999 ab5929 ab5930 ab5931 ab5926 ab6112 ab6110 Rabbit Sheep www.abcam.com/… Goat IgG - H&L HRP Dot, ELISA, EM, IHC, WB Goat IgG - H&L FITC Flow Cyt, ICC/IF ab47846 ab5755 Mouse IgG - H&L , pre-adsorbed Cy3® IHC-P ab50502 Engineered antibodies Bivalent diabodies sdAb Single domain antibody Engineered antibodies scFv Single-chain variable fragment Trifunctional antibody Chemically linked F(ab) BITE® Bi-specific T-cell engager • A single-chain variable fragment (scFv) is a powerful tool in phage display, as it can express the antigen-binding domain as a single peptide. ScFv is also used in FACS, IHC and as antigen-binding domains of artificial T cell receptors. • Diabody plays a role in the recruitment of antibody effector functions and cytotoxic T-cell responses. • A single domain antibody (sdAb) consists of a single monomeric variable antibody domain and is involved in pharmaceutical applications. • A trifunctional antibody is a monoclonal antibody with binding sites for two different antigens, typically CD3 and a tumor antigen. Trifunctional antibodies are considered as a type of bispecific monoclonal antibody. • Bi-specific T-cell engagers (BiTE®) is a class of bispecific monoclonal antibody that direct a host's T cells cytotoxic activity against diseased cells. F(ab) and Fc Receptors Non-specific antibody binding F(ab’)2 antibody Antibody D2 D2 D2 D2 FcγRllla Fc receptor names Immune cell FcγRI...............CD64 FcγRIIB2.......CD32 FcεRII..............CD23 FcγRIIA..........CD32 FcγRIIIA ........CD16a FcαRI ..............CD89 FcγRIIB1.......CD32 FcγRIIIB ........CD16b F(ab) and F(ab’)2 antibodies eliminate non-specific binding between Fc portions of antibodies and Fc receptors on cells (such as macrophages, dendritic cells, neutrophils, NK cells, B cells…) Ig structural differences IgD IgG3 IgG1,2+4 light chain k or l N N N N VH VH VL VL S S S S C CHO C CH2 biological activity SS SS CH2 Isotypes CL IgM CL 1 CH CH 1 antigen binding CHO CH3 CH3 IgE C C heavy chain g IgA Isotype: Distinct forms of light and heavy chains which are present in all members of a species. Kappa and lambda are isotypes of light chains; mu, delta, gamma, alpha and epsilon are isotypes of heavy chains. IgG IgG IgG Idiotypes Allotypes IgG Allotype: Allelic variants within the constant region of the immunoglobulin light or heavy chains. Of a given isotype, members of a species differ in function to the particular alleles they have received from their parents. Idiotype: Antigenic specificity of a particular monoclonal immunoglobulin. Secondary antibodies Anti-IgG H&L Host Target Conjugate Applications Donkey Goat Goat Mouse Mouse Mouse Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Goat Mouse Mouse Rat Sheep HRP FITC HRP TRITC AP Biotin Cy3® Cy5® FITC HRP HRP HRP TR HRP HRP Dot, ELISA, IHC-Fr, IHC-P, IM, WB Flow Cyt, ICC/IF, IHC-Fr, IHC-P, IM, WB Dot, ELISA, IHC-Fr, IHC-P, IM, WB Flow Cyt, ICC/IF, IHC-Fr, IHC-P, IM Dot, ELISA, ICC/IF, IHC-Fr, IHC-P, WB Dot, ELISA, IHC-Fr, IHC-P, IM, WB Flow Cyt, ICC, IF, IHC-Fr, IHC-P Flow Cyt, ICC, IF, IHC-Fr, IHC-P Flow Cyt, ICC/IF, IHC-Fr, IHC-P, IM Dot, ELISA, IHC-Fr, IHC-P, IM, WB Dot, ELISA, IHC-Fr, IHC-P, IM, WB Dot, ELISA, IHC-Fr, IHC-P, IM, WB Flow Cyt, IHC-Fr, IHC-P, IM Dot, ELISA, IHC-Fr, IHC-P, IM, WB Dot, ELISA, IHC-Fr, IHC-P, IM, WB Rabbit Datasheet www.abcam.com/… ab6885 ab6785 ab6789 ab6786 ab6722 ab6720 ab6939 ab6564 ab6717 ab6721 ab6741 ab6728 ab6726 ab6734 ab6747 Anti-IgM Host Target Applications Goat Human Human Mouse Mouse (Biotin) Rabbit Sheep (clone [G9]) Human Mouse Rabbit (clone [SB87a]) Dot, ELISA ELISA, ID, Ie, WB ELISA, ID, Ie, WB ELISA ELISA, ICC, WB Conjugation, ELISA Dot, ELISA ELISA, Ie, WB ELISA, Flow Cyt Host Target Applications Chicken Goat Human Human Human Mouse Rabbit Human (clone [hlgA1]) Human Mouse ELISA, ID, Ie, WB Dot, ELISA ELISA, ID, Ie, WB ELISA, ID, Ie, WB ELISA, ICC, WB ELISA, RIA Dot, ELISA ELISA, ID, Ie, WB Mouse Rabbit Rat Datasheet www.abcam.com/… ab8496 ab9160 ab9167 ab49760 ab2754 ab19991 ab8505 ab9175 ab80301 Anti-IgA ab9151 ab8499 ab9157 ab9161 ab2758 ab433 ab8508 ab9170 125-05/10-FF Mouse Rabbit Datasheet www.abcam.com/… Enzymatic detection methods Enzyme Substrates Applications Advantages Disadvantages Horseradish Peroxidase (HRP) Chromogenic, soluble (TMB, ABTS, OPD...) ELISA Easy to use Light sensitive coloration Alkaline Phosphatase (AP) Chromogenic, precipitating (CN, AEC, DAB,...) WB, SB, IHC Easy to use Background in blood samples and some other tissues Staining stability lower than AP Fluorogenic (ADHP/resorufin) Chemiluminescent ELISA High sensitivity Need fluorescence equipment Luminol WB, SB, IHC High sensitivity Need radiographic equipment or light scanner Chromogenic, soluble (pNPP) ELISA Linear kinetic Often more sensitive than HRP Unstable Chromogenic, precipitating (BCIP/NBT,...) WB, SB, IHC Staining stability higher than HRP Interference with nuclear counterstain Fluorogenic (4-MUP) ELISA, IHC Sensitivity Need fluorescence equipment How to choose the right secondary antibody? Guide to help you choose the most appropriate secondary antibody for your application. 1. What is the host species of the primary antibody? The secondary antibody is directed against the species of the primary antibody. If you use a primary antibody raised in rabbit, you will need an antirabbit secondary antibody raised in a species other than rabbit. 2. What do I need to know about the isotype of the primary antibody? The secondary antibody has to be directed against the isotype of the primary antibody. Polyclonal primary antibodies are generally raised in rabbit, goat, sheep or donkey and are an IgG isotype. The secondary antibody will typically be an anti-IgG H&L antibody. Monoclonal primary antibodies are commonly raised in mouse, rabbit and rat. For example, if the primary monoclonal antibody is a mouse IgG1, you will need an anti-mouse IgG or a less specific F(ab) fragment anti-mouse IgG. Human immunoglobulin classes, subclasses, types and subtypes: • Classes or isotypes: IgG (γ heavy chains), IgM (μ), IgA (α), IgE (ε), IgD (δ) • Subclasses: IgG1 (γ1 heavy chains), IgG2 (γ2), IgG3 (γ3), IgG4 (γ4), IgA1 (α1), IgA2 (α2) • Types: κ light chain, λ light chain • Subtypes: λ1, λ2, λ3, λ4 Other type of reactivities: • Polyvalent antibodies react with all classes • Anti-Fc or heavy chain (α, δ, ε, γ, and μ) antibodies react with heavy chain only • Anti-F(ab) or whole molecule antibodies react with heavy and light chains independently of the class • Anti-light chain (κ and λ) antibodies react with all classes since all classes use the same κ and λ light chains 3. Do I need an enzymatic or fluorescent detection? The type of conjugation is application dependent. For enzymatic and biotin detection, e.g. in WB or ELISA, we suggest a secondary antibody conjugated to HRP, AP or biotin. Both avidin and streptavidin bind very strongly to biotin and enable signal amplification, regardless of the host species of the antibody. If a laser light is involved, e.g. in Flow Cytometry, ICC/IF or IHC, we suggest fluorescent detection with a secondary antibody conjugated to a fluorochrome. 4. Do I need a pre-adsorbed secondary antibody? We usually recommend using a secondary antibody, pre-adsorbed with serum, for western blotting, of immunoglobulin-rich tissues and cells. Pre-adsorbed secondary antibodies are less likely to interact with endogenous immunolgobulins and consequently may reduce non-specific background. The secondary antibody should be pre-adsorbed against the same species as the sample on which the detection is performed. For example, a human preadsorbed antibody will be required for detection in human tissue. 5. Do I need an affinity purified antibody or IgG fraction? The advantage of using affinity purified antibodies or IgG fractions will depend on the type of binding expected. Affinity purified antibodies give the lowest amount of non-specific binding whereas IgG fractions contain high affinity antibodies. Indeed, during an affinity purification, high affinity antibodies stay fixed on the matrix and cannot be eluted. 6. Is it necessary to use a F(ab) or F(ab')2 fragment antibody? F(ab) and F(ab')2 fragment antibodies eliminate non-specific binding between Fc portions of antibodies and Fc receptors on cells (such as macrophages, dendritic cells, neutrophils, NK cells and B cells) and penetrate tissues more efficiently due to their smaller size. As fragment antibodies do not have Fc portions, they do not interfere with anti-Fc mediated antibody detection. 7. Do I need an anti-IgG H&L, anti-light chain or anti-F(ab')2 secondary antibody? Our secondary antibodies are supplied in different formats: • Anti-IgG H&L antibodies react with both heavy and light chains of IgG subclasses • Anti-light chain antibodies react with the light chain of primary antibodies which is the same among all classes • Anti-F(ab')2 secondary antibodies react with the F(ab')2 portion of the primary antibody Fluorescent detection methods Spectral properties of DyLight® fluorochromes Ex/Em ε (M-1cm-1) DyLight® 488 493/518 70K DyLight® 549 562/576 150K DyLight® 594 593/618 80K DyLight® 649 654/673 250K Fluorochrome Color Spectrally equivalent dyes Alexa Fluor 488®, Fluorescein, FITC, Cy2® Alexa Fluor 546® or 555®, Cy3®, TRITC Alexa Fluor 594®, Texas Red® Alexa Fluor 647®, Cy5® Ex/Em: Excitation and emission wavelength in nanometers (+/- 4nm) ε: Molar extinction coefficient at the absorption maximum 100% technical support • Thinking of purchasing a secondary antibody but have some technical questions? • Unsure of buffers or incubation times? • Having difficulty with a product? ...Our scientific experts are here to help. All Abcam secondary antibodies are guaranteed to work as specified on the datasheet. • 100% Scientific and Customer Support for any product you buy from Abcam or one of its authorized distributors • We guarantee our products work in the tested species and applications as stated on the datasheet • We will replace or refund products not performing as stated on the datasheet if reported within 120 days of purchase