Understanding Secondary Antibodies

Understanding IgG secondary antibodies

Fragment antigen binding antibodies 2 and isotypes D D2

F(ab’)2 after pepsin cleavage www.abcam.com/secondary_antibody

F Antibody Antigen binding sites structure VH VH

VL S S VL S S

and F(ab) S S S CH CH 1 1 S e r u t

c u S S

antibodies r t

CL S S CL

s S S S

S S

y S

d S S o b i t The light chain (LH) folds n Hinge CH CH A 2 2 into a variable domain (VL) S S and a constant domain S S (CL) whereas the heavy CH CH chain is composed of one 3 3

variable domain (VH) and S S three (IgG and IgA) or four S S constant domains (IgE). s e i d o b i t n a

) b a ( F

F(ab) F(ab’)2 F(ab’) after papain cleavage after pepsin cleavage 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') fragm ents have two antigen-binding F(ab) 2 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 Conjugate Applications www.abcam.com/…

Donkey Goat IgG - H&L HRP Dot, ELISA, EM, IHC, WB ab6667 Goat IgG - H&L TRITC Flow Cyt, IM ab6522 Goat IgG - H&L TR Flow Cyt, IM ab6523 Goat IgG - H&L Biotin Dot, ELISA, IHC, IM, WB ab6578

Goat Human IgG - H&L 1.4nm Gold EM, WB ab30826 Mouse IgG - F(ab’) , pre-adsorbed TRITC Flow Cyt, ICC/IF, IHC-P ab51379 Mouse IgG - H&L ab6668 Mouse IgG - H&L HRP Dot, ELISA, EM, IHC, WB ab6823 Mouse IgG - H&L TRITC Flow Cyt, IM ab6670 Mouse IgG - H&L FITC Flow Cyt, IM ab6669 Rabbit IgG - H&L ab6824 Rabbit IgG - H&L Biotin Dot, ELISA, IHC, IM, WB ab7055 Rabbit IgG - H&L HRP Dot, ELISA, EM, IHC, WB ab7171 Rat IgG - H&L Biotin Dot, ELISA, IHC, IM, WB ab7176

F(ab’) 2 fragments

Host Target Conjugate Applications www.abcam.com/…

Donkey Goat IgG - H&L, pre-adsorbed PE Flow Cyt, IHC-Fr, IHC-P, IM ab7004 Rabbit IgG - H&L, pre-adsorbed PE Flow Cyt, ICC/IF, IHC-P, IM ab7007

Goat Mouse IgG - (Fab) ’2, pre-adsorbed TR Flow Cyt, IM ab5884 Mouse IgG - Fc, pre-adsorbed PE Flow Cyt, IM ab5881 Mouse IgG - H&L, pre-adsorbed PE Flow Cyt, IHC-Fr, IHC-P, IM ab7002 Mouse IgG+IgM+IgA - H&L, pre-adsorbed HRP Dot, ELISA, EM, IHC, WB ab6006 Mouse IgG+IgM+IgA - H&L, pre-adsorbed FITC Flow Cyt, IF ab5999 Mouse IgM - mu chain Biotin Dot, ELISA, Flow Cyt, IHC, IM, WB ab5929 Mouse IgM - mu chain HRP Dot, ELISA, EM, IHC, IHC-Fr, WB ab5930 Mouse IgM - mu chain AP Dot, ELISA, IHC, WB ab5931 Mouse IgM - mu chain FITC Flow Cyt, IM ab5926

Rabbit IgG - (Fab) ’2, pre-adsorbed HRP Dot, ELISA, EM, IHC, WB ab6112 Rabbit IgG - (Fab) ’2, pre-adsorbed TRICT Flow Cyt, IM ab6110

Rabbit Goat IgG - H&L HRP Dot, ELISA, EM, IHC, WB ab5755 Goat IgG - H&L FITC Flow Cyt, ICC/IF ab47846

Sheep Mouse IgG - H&L , pre-adsorbed Cy3 ® IHC-P ab50502

See more species and isotype reactivity at www.abcam.com/search_secondary Eng ine ered an tib odies

scFv Bivalent diabodies sdAb Single-chain Single domain variable fragment antibody s e i d o b i t n a

d e r e e n i g n E

Trifunctional BITE® antibody Chemically linked F(ab) 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

F(ab’)2 antibody Antibody g n i d n i b

y d o b i t 2 n D a D2 c i f i

c D2 e p

s D2 - n o N

FcγRllla

Immune cell s e

m Fc γRI ...... CD64 Fc γRIIB2 ...... CD32 Fc εRII ...... CD23 a n

r o t ......

p Fc γRIIA CD32 Fc γRIIIA CD16a Fc αRI CD89 e c e r ......

c Fc γRIIB1 CD32 Fc γRIIIB CD16b F

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

IgG3 IgD IgG1,2+4 light chain ␬ or ␭ NN N N

V H H V V L L V antigen binding C 1

H H 1 C C L L S S S C

s S

e S S p IgM S S y CC t biological o activity C s 2 I H H

CHO 2 CHO

IgE C 3 H H 3 C heavy chain ␥

C C

IgA

Isotype: Distinct forms of light and heavy chains which are present in all T members of a species. K appa and lambda are isotypes of light chains; mu, delta, gamma, alpha and epsilon are isotypes of heavy chains.

I IgG IgG IgG IgG s s e e p p y y t t o o l i l d I A

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. A A S A n n n M C H R G G R G M H D H R R a a a a h o o o o o o o e o o t t t b i b b n t s s s a a a u u c b b b k t t t t t t s s i i i k e i i i e e e - - - t t t y c n I I I g g g o A M G S R R M R M M M R R R M T G R G a

h a a a a a a a o o o o o o o n r e b b b b b b t H a a u u u u u g

e b b b b b b t t s s s s s e

p i i i i i i e e e e e t t t t t t t

d L H H T R H M H H M M M H H M R S T H R a a h a a a u u u u u u u u a o o o o o r r e b m m m m m b m m b m u u u u u g g e b b b s s s s s e e a a a a a a a a p i i i e e e e e t t t t t n n n n n n n n

( (

( ( c c B c l l o o i l o o y H H T C T F H B H H F C H H C A n n t n R R I I i e e i P R R y R R R y R R o n o T T e

3 5 I n ) C C P P P P P P P t [ [

T i ® ® S G [ j n h

C u B 9 l g g 8 ] a ) a A 7 t 1 a e ] ] ) ) n t F F D F F F D F D A D D D D D D l l l l l l p o o o o o o o o o o o o o o o i t t t t t t t t t p w w w w w w , , , , , , , , ,

l b

E E E E E E E E E i C C C C C C c L L L L L L L L L a y y y y y y E E E A D E D E E E E E D C E A E D E I I I I I I I I I t t t t t t t S S S S S S S S S , , , , , , i L L L L L L L L L L L L p p o o o o o

o A A A A A A A A A o I I I I I I I I I I I I I I I I I I t t t n t p p H C C C C C S S S S S S S S S S S S n , , , , , , , , , , , , ,

l l

u E E E E s A A A A A A A A A A A A C C C C C C i i I I I I I I I I I c c H H H H C H H H H g L L L L , , , , , , , , , , , , , - / / / a a

a I I I F C C C C C C C C C I I I I I I R I I I I I I I F F F F I I d t t S S S S D C D D D D D C e t F F r , , , i i - - - - / - - - - , i l

I , , , o o o o I

F F F F F F F F A A A A , , , , , , C A C

I I I

F I

H H H I I n w W n n H r r r r r r r r I I I I I I , H H , , , , , , , , , , e e e e e e

C C C s s

I C

I I I I I I I I W W , , , , , , C C B H C E H H H H H H H H

i - - - - W W W W W W - - F F F C y L P B B C C C C C C C C F F t , r r r e I - B B B B B B , , ,

r r ------S

, , F I P P P P P P P P

I I I M A , , , , , , , , H H H I I r ,

H H

I I I I I I I I C C C I M M M M M M M M C C H s - - - , , , , , , , , - - P P P C

P P , , , W W W W W W W W

- I I I P M M M B B B B B B B B ,

W ,

W B D D D B a a a t a t t a a s s s h h h e e e e e e t

t t w

w w w w w w w w . a . . a a b b b c c c a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a b b b m m m a b b b b b b 4 b b b b b 1 b b b b b b b b b b b b b b b b b 8 . c b 2 8 9 8 9 9 9 9 9 9 8 6 9 6 2 6 6 6 6 6 6 6 6 6 6 6 6 6 9 8 0 . . c c o 4 7 5 1 4 1 1 7 1 1 1 4 7 7 9 7 7 7 7 7 7 7 7 5 9 7 8 7 7 1 5 3 o o m 3 5 0 5 9 7 5 6 6 6 6 9 4 5 9 3 2 2 2 2 8 4 2 6 3 8 8 8 1 7 0 0 m m 3 8 8 1 9 0 7 0 1 7 0 6 7 4 1 4 0 6 8 2 9 1 1 4 9 5 5 6 7 5 5 1 / … / / … …

125-05/10-FF Enzymatic detection methods

Enzyme Substrates Applications Advantages Disadvantages

Horseradish Chromogenic, soluble Light sensitive Peroxidase ELISA Easy to use (TMB, ABTS, OPD...) coloration (HRP)

Background in blood Chromogenic, samples and some precipitating WB, SB, IHC Easy to use other tissues (CN, AEC, DAB,...) Staining stability lower than AP

Fluorogenic Need fluorescence (ADHP/resorufin) ELISA High sensitivity equipment Chemiluminescent

Need radiographic Luminol WB, SB, IHC High sensitivity equipment or light scanner

Alkaline Linear kinetic Chromogenic, Phosphatase ELISA Often more sensitive Unstable soluble (pNPP) (AP) than HRP

Chromogenic, Staining stability Interference with precipitating WB, SB, IHC higher than HRP nuclear counterstain (BCIP/NBT,...)

Fluorogenic Need fluorescence ELISA, IHC Sensitivity (4-MUP) equipment

WB: Western Blot, SB: Southern Blot, IHC: Immunohistochemistry, ELISA: Enzyme Linked Immunosorbent Assay How to choose the right seconda

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 anti- rabbit 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, ary antibody?

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 pre- adsorbed 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 For more information and links to all our downloads please visit: www.abcam.com/secondary_antibody Fluorescent detection methods

Spectral properties of DyLight ® fluorochromes

Fluorochrome Color Ex/Em ε (M -1 cm -1 ) Spectrally equivalent dyes

DyLight ® 488 493/518 70K Alexa Fluor 488 ®, Fluorescein, FITC, Cy2 ®

DyLight ® 549 562/576 150K Alexa Fluor 546 ® or 555 ®, Cy3 ®, TRITC

DyLight ® 594 593/618 80K Alexa Fluor 594 ®, Texas Red ®

DyLight ® 649 654/673 250K Alexa Fluor 647 ®, Cy5 ®

Ex/Em: Excitation and emission wavelength in nanometers (+/- 4nm) ε: Molar extinction coefficient at the absorption maximum

More information at www.abcam.com/DyLight

DyLight® is a trademark of Thermo Fisher Scientific Inc. and its subsidiaries. Abpromise ® Our promise to you

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

Read more about our Abpromise: www.abcam.com/abpromise