Antibody Conjugation Methods, Techniques & Protocols



Conjugation of Monoclonal Antibodies by Mario Roederer - In this series of web pages, protocols, notes, and various illustrations are given to aid in the conjugation of proteins--principally monoclonal antibodies--to fluorescent dyes. These conjugation procedures are commonly performed in our laboratory--we have conjugated several hundred different monoclonals using almost all of the various dyes listed. The procedures are relatively straightforward and require only minimal familiarity with standard laboratory techniques (gel filtration and spectrophotometry are the most difficult!).

Quick and Simple Method for Conjugation mAbs - Note 1: as the method for coupling both FITC and Biotin are very similar we usually make both conjugates at the same time (unless antibody is in short supply or bought commercially at great expense!). Note 2: small amounts of antibody (ie. 100mcg to 1mg) can be conjugated directly in a "Slide-A-Lyzer 10K" dialysis cassette (Pierce).

Antibody Conjugation Protocol - The Following Protocol can be used to couple either FITC or Biotin to IgG. The same method is used for both, and when discrepancies occur, they will be noted.

Rapid Fluorescent-Antibody Conjugation Procedure - This note describes a technique developed for rapid preparation of specific and brilliant conjugates for direct staining of rickettsial agents in yolk-sac, tick, and vertebrate tissues.


Published Antibody Conjugation Methods, Techniques & Protocols


Sapsford KE, Soto CM, Blum AS, Chatterji A, Lin T, Johnson JE, Ligler FS, Ratna BR.

A cowpea mosaic virus nanoscaffold for multiplexed antibody conjugation: application as an immunoassay tracer.
Biosens Bioelectron. 2006 Feb 15;21(8):1668-73. Epub 2005 Oct 10.
PMID: 16216488 [PubMed - indexed for MEDLINE]


Asai T, Trinh R, Ng PP, Penichet ML, Wims LA, Morrison SL.

A human biotin acceptor domain allows site-specific conjugation of an enzyme to an antibody-avidin fusion protein for targeted drug delivery.
Biomol Eng. 2005 Feb;21(6):145-55. Epub 2004 Dec 9.
PMID: 15748688 [PubMed - indexed for MEDLINE]


Thomas AC, Campbell JH.

Conjugation of an antibody to cross-linked fibrin for targeted delivery of anti-restenotic drugs.
J Control Release. 2004 Dec 10;100(3):357-77.
PMID: 15567502 [PubMed - indexed for MEDLINE]


Jeong JM, Lee J, Paik CH, Kim DK, Lee DS, Chung JK, Lee MC.

Site-specific 99mTc-labeling of antibody using dihydrazinophthalazine (DHZ) conjugation to Fc region of heavy chain.
Arch Pharm Res. 2004 Sep;27(9):961-7.
PMID: 15473668 [PubMed - indexed for MEDLINE]


Wu G, Barth RF, Yang W, Chatterjee M, Tjarks W, Ciesielski MJ, Fenstermaker RA.

Site-specific conjugation of boron-containing dendrimers to anti-EGF receptor monoclonal antibody cetuximab (IMC-C225) and its evaluation as a potential delivery agent for neutron capture therapy.
Bioconjug Chem. 2004 Jan-Feb;15(1):185-94.
PMID: 14733599 [PubMed - indexed for MEDLINE]


Li J, Lee SE, Belciug M, Ring DB, Kwok CS.

Chemical conjugation of a novel antibody-interleukin 2 immunoconjugate against c-erbB-2 product.
Chin Med J (Engl). 2000 Feb;113(2):151-3.
PMID: 11775541 [PubMed - indexed for MEDLINE]


Klegerman ME, Hamilton AJ, Huang SL, Tiukinhoy SD, Khan AA, MacDonald RC, McPherson DD.

Quantitative immunoblot assay for assessment of liposomal antibody conjugation efficiency.
Anal Biochem. 2002 Jan 1;300(1):46-52.
PMID: 11743691 [PubMed - indexed for MEDLINE]


Sudhan Shaik M, Kanikkannan N, Singh M.

Conjugation of anti-My9 antibody to stealth monensin liposomes and the effect of conjugated liposomes on the cytotoxicity of immunotoxin.
J Control Release. 2001 Oct 19;76(3):285-95.
PMID: 11578743 [PubMed - indexed for MEDLINE]


Stubenrauch K, Gleiter S, Brinkmann U, Rudolph R, Lilie H.

Conjugation of an antibody Fv fragment to a virus coat protein: cell-specific targeting of recombinant polyoma-virus-like particles.
Biochem J. 2001 Jun 15;356(Pt 3):867-73.
PMID: 11389696 [PubMed - indexed for MEDLINE]


Fu Q, Gowda DC.

Carbohydrate-directed conjugation of cobra venom factor to antibody by selective derivatization of the terminal galactose residues.
Bioconjug Chem. 2001 Mar-Apr;12(2):271-9.
PMID: 11312689 [PubMed - indexed for MEDLINE]


Lundberg BB, Griffiths G, Hansen HJ.

Conjugation of an anti-B-cell lymphoma monoclonal antibody, LL2, to long-circulating drug-carrier lipid emulsions.
J Pharm Pharmacol. 1999 Oct;51(10):1099-105.
PMID: 10579680 [PubMed - indexed for MEDLINE]


Bendas G, Krause A, Bakowsky U, Vogel J, Rothe U.

Targetability of novel immunoliposomes prepared by a new antibody conjugation technique.
Int J Pharm. 1999 Apr 20;181(1):79-93.
PMID: 10370205 [PubMed - indexed for MEDLINE]


Qu Z, Sharkey RM, Hansen HJ, Shih LB, Govindan SV, Shen J, Goldenberg DM, Leung SO.

Carbohydrates engineered at antibody constant domains can be used for site-specific conjugation of drugs and chelates.
J Immunol Methods. 1998 Apr 15;213(2):131-44.
PMID: 9692846 [PubMed - indexed for MEDLINE]


Harapanhalli RS, Matalka KZ, Jones PL, Mahmood A, Adelstein SJ, Kassis AI.

Lysine-directed conjugation of ethidium homodimer to B72.3 antibody: retention of immunoreactivity but altered tumor targeting.
Nucl Med Biol. 1998 Apr;25(3):267-78.
PMID: 9620633 [PubMed - indexed for MEDLINE]


Ozzello L, Blank EW, De Rosa CM, Ceriani RL, Tolo H, Kauppinen HL, Cantell K.

Conjugation of interferon alpha to a humanized monoclonal antibody (HuBrE-3vl) enhances the selective localization and antitumor effects of interferon in breast cancer xenografts.
Breast Cancer Res Treat. 1998 Mar;48(2):135-47.
PMID: 9596485 [PubMed - indexed for MEDLINE]


Muzykantov VR.

Conjugation of catalase to a carrier antibody via a streptavidin-biotin cross-linker.
Biotechnol Appl Biochem. 1997 Oct;26 ( Pt 2):103-9.
PMID: 9357106 [PubMed - indexed for MEDLINE]


Safavy A, Sanders A, Qin H, Buchsbaum DJ.

Conjugation of unprotected trisuccin, N-[tris[2-[(N-hydroxyamino)carbonyl]ethyl]methyl]succinamic acid, to monoclonal antibody CC49 by an improved active ester protocol.
Bioconjug Chem. 1997 Sep-Oct;8(5):766-71.
PMID: 9327144 [PubMed - indexed for MEDLINE]


Goulet AC, Goldmacher VS, Lambert JM, Baron C, Roy DC, Kouassi E.

Conjugation of blocked ricin to an anti-CD19 monoclonal antibody increases antibody-induced cell calcium mobilization and CD19 internalization.
Blood. 1997 Sep 15;90(6):2364-75.
PMID: 9310487 [PubMed - indexed for MEDLINE]


Stalteri MA, Mather SJ, Belinka BA, Coughlin DJ, Chengazi VU, Britton KE.

Site-specific conjugation and labelling of prostate antibody 7E11C5.3 (CYT-351) with technetium-99m.
Eur J Nucl Med. 1997 Jun;24(6):651-4.
PMID: 9169572 [PubMed - indexed for MEDLINE]


Eisenhut M, Lehmann WD, Becker W, Behr T, Elser H, Strittmatter W, Steinstrasser A,

Baum RP, Valerius T, Repp R, Deo Y.

Bifunctional NHS-BAT ester for antibody conjugation and stable technetium-99m labeling: conjugation chemistry, immunoreactivity and kit formulation.
J Nucl Med. 1996 Feb;37(2):362-70.
PMID: 8667077 [PubMed - indexed for MEDLINE]


Lau A, Berube G, Ford CH.

Conjugation of doxorubicin to monoclonal anti-carcinoembryonic antigen antibody via novel thiol-directed cross-linking reagents.
Bioorg Med Chem. 1995 Oct;3(10):1299-304.
PMID: 8564395 [PubMed - indexed for MEDLINE]


Leung S, Losman MJ, Govindan SV, Griffiths GL, Goldenberg DM, Hansen HJ.

Engineering a unique glycosylation site for site-specific conjugation of haptens to antibody fragments.
J Immunol. 1995 Jun 1;154(11):5919-26.
PMID: 7751635 [PubMed - indexed for MEDLINE]


Werlen RC, Lankinen M, Rose K, Blakey D, Shuttleworth H, Melton R, Offord RE.

Site-specific conjugation of an enzyme and an antibody fragment.
Bioconjug Chem. 1994 Sep-Oct;5(5):411-7.
PMID: 7849070 [PubMed - indexed for MEDLINE]


Wilbur DS, Stray JE, Hamlin DK, Curtis DK, Vessella RL.

Monoclonal antibody Fab' fragment cross-linking using equilibrium transfer alkylation reagents. A strategy for site-specific conjugation of diagnostic and therapeutic agents with F(ab')2 fragments.
Bioconjug Chem. 1994 May-Jun;5(3):220-35.
PMID: 7918742 [PubMed - indexed for MEDLINE]


Kurth M, Pelegrin A, Rose K, Offord RE, Pochon S, Mach JP, Buchegger F.

Site-specific conjugation of a radioiodinated phenethylamine derivative to a monoclonal antibody results in increased radioactivity localization in tumor.
J Med Chem. 1993 Apr 30;36(9):1255-61.
PMID: 8487262 [PubMed - indexed for MEDLINE]


Sekhar MC, Sharma GL, Gangal S, Joshi AP, Sarma PU.

Dimethyl suberimidate as an effective crosslinker for antibody-enzyme conjugation.
Prep Biochem. 1991;21(4):215-27.
PMID: 1723521 [PubMed - indexed for MEDLINE]


Sinkule JA, Rosen ST, Radosevich JA.

Monoclonal antibody 44-3A6 doxorubicin immunoconjugates: comparative in vitro anti-tumor efficacy of different conjugation methods.
Tumour Biol. 1991;12(4):198-206.
PMID: 1651554 [PubMed - indexed for MEDLINE]


Comeau RD, Liberatore FA, Cocuzza AJ, Emond GY, Dicker IB.

Conjugation of a monoclonal antibody with a DTPA modified random copolymer of hydroxyethyl methylacrylate and methyl methacrylate.
Int J Rad Appl Instrum B. 1990;17(3):321-9.
PMID: 2341288 [PubMed - indexed for MEDLINE]


Jeanson A, Cloes JM, Bouchet M, Rentier B.

Comparison of conjugation procedures for the preparation of monoclonal antibody-enzyme conjugates.
J Immunol Methods. 1988 Jul 22;111(2):261-70.
PMID: 3135335 [PubMed - indexed for MEDLINE]


Brown BA, Drozynski CA, Dearborn CB, Hadjian RA, Liberatore FA, Tulip TH, Tolman GL, Haber SB.

Conjugation of metallothionein to a murine monoclonal antibody.
Anal Biochem. 1988 Jul;172(1):22-8.
PMID: 3189766 [PubMed - indexed for MEDLINE]


Kinsey BM, Macklis RM, Ferrara JM, Layne WW, Burakoff SJ, Kassis AI.

Efficient conjugation of DTPA to an IgM monoclonal antibody in ascites fluid.
Int J Rad Appl Instrum B. 1988;15(3):285-92.
PMID: 2898458 [PubMed - indexed for MEDLINE]


Borlinghaus KP, Fitzpatrick DA, Heindel ND, Mattis JA, Mease BA, Schray KJ, Shealy DJ, Walton HL Jr, Woo DV.

Radiosensitizer conjugation to the carcinoma 19-9 monoclonal antibody.
Cancer Res. 1987 Aug 1;47(15):4071-5.
PMID: 3496956 [PubMed - indexed for MEDLINE]


Tsuruta J, Yamamoto T, Kozono K, Kambara T.

Application of a new method of antibody-enzyme conjugation with maleimide derivative for immunohistochemistry: hepatocellular production, interstitial tissue distribution, and renal cell reabsorption of plasma albumin in guinea pig.
J Histochem Cytochem. 1985 Aug;33(8):767-77.
PMID: 3926865 [PubMed - indexed for MEDLINE]


Gadkari DA, Fields HA, Maynard JE.

Enzyme-antibody conjugation by a heterobifunctional reagent and its application in enzyme-linked immunosorbent assay (ELISA) for the detection of hepatitis B surface antigen.
J Virol Methods. 1985 Mar;10(3):215-24.
PMID: 3886682 [PubMed - indexed for MEDLINE]


Yamada H, Sano Y.

The biotinylation of the rabbit serotonin antibody and its application to immunohistochemical studies using the two-step ABC method.
Histochemistry. 1985;83(4):285-9.
PMID: 2415494 [PubMed - indexed for MEDLINE]


Kato Y, Umemoto N, Kayama Y, Fukushima H, Takeda Y, Hara T, Tsukada Y.

A novel method of conjugation of daunomycin with antibody with a poly-L-glutamic acid derivative as intermediate drug carrier. An anti-alpha-fetoprotein antibody-daunomycin conjugate.
J Med Chem. 1984 Dec;27(12):1602-7.
PMID: 6209394 [PubMed - indexed for MEDLINE]


Kato Y, Tsukada Y, Hara T, Hirai H.

Enhanced antitumor activity of mitomycin C conjugated with anti-alpha-fetoprotein antibody by a novel method of conjugation.
J Appl Biochem. 1983 Aug-Oct;5(4-5):313-9.
PMID: 6207159 [PubMed - indexed for MEDLINE]


Pelham JM, Gray JD, Flannery GR, Pimm MV, Baldwin RW.

Interferon-alpha conjugation to human osteogenic sarcoma monoclonal antibody 791T/36.
Cancer Immunol Immunother. 1983;15(3):210-6.
PMID: 6577942 [PubMed - indexed for MEDLINE]


Kato K, Hamaguchi Y, Fukui H, Ishikawa E.

Enzyme-linked immunoassay. Conjugation of rabbit anti-(human immunoglobulin G) antibody with beta-D-galactosidase from Escherichia coli and its use for human immunoglobulin G assay.
Eur J Biochem. 1976 Feb 16;62(2):285-92.
PMID: 943288 [PubMed - indexed for MEDLINE]


Thrasher SG, Bigazzi PE, Yoshida T, Cohen S.

The effect of fluorescein conjugation on Fc-dependent properties of rabbit antibody.
J Immunol. 1975 Feb;114(2 pt 2):762-4.
PMID: 1112979 [PubMed - indexed for MEDLINE]


Peacock M, Burgdorfer W, Ormsbee RA.

Rapid Fluorescent-Antibody Conjugation Procedure.
Infect Immun. 1971 Feb;3(2):355-357.
PMID: 16557977 [PubMed - as supplied by publisher]





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