Antibody Production Methods, Techniques & Protocols



How Lymphocytes Produce Antibody - The scanning electron micrograph (right) shows a human macrophage (gray) approaching a chain of Streptococcus pyogenes (yellow). Riding atop the macrophage is a spherical lymphocyte. Both macrophages and lymphocytes can be found near an infection, and the interaction between these cells is important in eliminating infection. Below is an animation that illustrates the basic cell-cell interactions that lead to antibody production can be seen in the accompanying animation.

Guidelines for the Use of Adjuvants And Antibody Production - Many methodologies exist for polyclonal antibody production in laboratory animals. Institutional guidelines governing animal use and procedures relating to these methodologies are generally oriented around humane considerations and appropriate conduct for adjuvant use. This includes adjuvant selection, routes and sites of administration, injection volumes per site and number of sites per animal.

Immunological Techniques Making Monoclonal Antibodies - Monoclonal antibodies are great! Why? Specificity! Each one recognizes only one site of the antigen structure. Why else? Immortality! Mass cultures can be generated from a single clone. Mice are small! They require less antigen than a rabbit to get an antibody reaction. The antigen travels through the circulation system. Blood is channeled through the spleen where B lymphocyte cells recognize the foreign bodies and produce antibodies.

Preparation Of Peptide-KLH Conjugates For Immunization By Claire Walczak
January 1996
- These are basically Tim's procedures and have been used successfully by numerous members of our lab and by others around us.

Testing Rabbit Bleeds By Elisa By Claire Walczak January 1996 - You can test whether or not you have gotten an immune response to the peptide and how strong that immune response is by doing ELISAs against peptide conjugated to BSA. By conjugating to BSA, you will eliminate any signal for antibodies generated to KLH during immunization.

Large Scale Production of Monoclonal Antibodies - The methods described here are most suited to the 1-10g production range of a normal rat or mouse hybridoma. We would recommend the following methods as a rough guide for different scales of operation.

Notes on Making Rat x Y3 Monoclonal Antibody Producing Hybridomas - This note is not intended as a full protocol for making monoclonal antibodies, but is to give a few hints and tips for the use of Y3/Ag1.2.3. Generally speaking the methodology is similar to making murine (eg. with NS0) monoclonal antibodies, but there are a few critical differences which are commented on below.

Monoclonal Antibody Production (1999) - Institute for Laboratory Animal Research - Committee on Methods of Producing Monoclonal Antibodies

Polyclonal Antibody Production - Very useful for rapid and simple generation of antibodies for western blots, ELISA assays, and immunoprecipitation

Polyclonal and Monoclonal Antibodies - Production of polyclonal and monoclonal antibodies (The Minion Lab, College of Veterinary Medicine at Iowa State University).

How Your Body Makes Antibodies After Vaccination - Since the introduction of widespread vaccination programmes, millions of people have been protected against potentially fatal diseases, and countless lives have been saved.

Antibody Production - Frequently asked questions about antibody production

Making Antibodies to Synthetic Peptides - You'll need to couple the synthetic peptide hapten to a protein carrier, with a bifunctional reagent.

Published Antibody Production Methods, Techniques & Protocols


Lateef SS, Gupta S, Jayathilaka LP, Krishnanchettiar S, Huang JS, Lee BS.

An Improved Protocol for Coupling Synthetic Peptides to Carrier Proteins for Antibody Production Using DMF to Solubilize Peptides.
J Biomol Tech. 2007 Jul;18(3):173-6.
PMID: 17595313 [PubMed - in process]


Mahalakshmi B, Therese KL, Madhavan HN, Biswas J.

Diagnostic value of specific local antibody production and nucleic acid amplification technique-nested polymerase chain reaction (nPCR) in clinically suspected ocular toxoplasmosis.
Ocul Immunol Inflamm. 2006 Apr;14(2):105-12.
PMID: 16597540 [PubMed - indexed for MEDLINE]


Bootz F, Sieber I, Popovic D, Tischhauser M, Homberger FR.

Comparison of the sensitivity of in vivo antibody production tests with in vitro PCR-based methods to detect infectious contamination of biological materials.
Lab Anim. 2003 Oct;37(4):341-51.
PMID: 14609003 [PubMed - indexed for MEDLINE]


Bowering LC, Bracewell DG, Kesharvarz-Moore E, Hoare M, Weir AN.

Comparison of techniques for monitoring antibody fragment production in E. coli fermentation cultures.
Biotechnol Prog. 2002 Nov-Dec;18(6):1431-8.
PMID: 12467481 [PubMed - indexed for MEDLINE]


Wan HZ, Kaneshiro S, Frenz J, Cacia J.

Rapid method for monitoring galactosylation levels during recombinant antibody production by electrospray mass spectrometry with selective-ion monitoring.
J Chromatogr A. 2001 Apr 13;913(1-2):437-46.
PMID: 11355842 [PubMed - indexed for MEDLINE]


Peterson NC, Peavey JE.

Comparison of In Vitro Monoclonal Antibody Production Methods with an In Vivo Ascites Production Technique.
Contemp Top Lab Anim Sci. 1998 Sep;37(5):61-66.
PMID: 12456135 [PubMed - as supplied by publisher]


Peterson NC, Peavey JE.

Comparison of In Vitro Monoclonal Antibody Production Methods with an In Vivo Ascites Production Technique.
Contemp Top Lab Anim Sci. 1998 Sep;37(5):61-66.
PMID: 12456135 [PubMed - as supplied by publisher]


de Wildt RM, Steenbakkers PG, Pennings AH, van den Hoogen FH, van Venrooij WJ, Hoet RM.

A new method for the analysis and production of monoclonal antibody fragments originating from single human B cells.
J Immunol Methods. 1997 Aug 22;207(1):61-7.
PMID: 9328587 [PubMed - indexed for MEDLINE]


Schlebusch H, Reinartz S, Kaiser R, Grunn U, Wagner U.

Production of a single-chain fragment of the murine anti-idiotypic antibody ACA125 as phage-displayed and soluble antibody by recombinant phage antibody technique.
Hybridoma. 1997 Feb;16(1):47-52.
PMID: 9085128 [PubMed - indexed for MEDLINE]


Hill SL, Lappin MR, Carman J, Collins JK, Reif JS, Spilker M, Jensen C.

Comparison of methods for estimation of Toxoplasma gondii-specific antibody production in the aqueous humor of cats.
Am J Vet Res. 1995 Sep;56(9):1181-7.
PMID: 7486396 [PubMed - indexed for MEDLINE]


Zeng QS, Takeyama H, Kanda S, Irie RF.

Serum preparation and methods for the large-scale production of IgG monoclonal antibody.
Hum Antibodies Hybridomas. 1994;5(1-2):75-80.
PMID: 7858185 [PubMed - indexed for MEDLINE]


Fu P, Barford JP.

Methods and strategies available for the process control and optimization of monoclonal antibody production.
Cytotechnology. 1994;14(3):219-32. Review.
PMID: 7765592 [PubMed - indexed for MEDLINE]


Salgar SK, Paape MJ, Alston-Mills B.

Enzyme-linked immunosorbent assay and flow cytometric methods to screen hybridoma culture supernatants for antibodies to bovine neutrophil surface antigens, and monoclonal antibody production and characterization.
Am J Vet Res. 1993 Sep;54(9):1415-25.
PMID: 8239127 [PubMed - indexed for MEDLINE]


Ermeling BL, Steffen EK, Fish RE, Hook RR Jr.

Evaluation of subcutaneous chambers as an alternative to conventional methods of antibody production in chickens.
Lab Anim Sci. 1992 Aug;42(4):402-7. Erratum in: Lab Anim Sci 1993 Feb;43(1):114.
PMID: 1434503 [PubMed - indexed for MEDLINE]


Federspiel G, McCullough KC, Kihm U.

Hybridoma antibody production in vitro in type II serum-free medium using Nutridoma-SP supplements. Comparisons with in vivo methods.
J Immunol Methods. 1991 Dec 15;145(1-2):213-21.
PMID: 1765654 [PubMed - indexed for MEDLINE]


Ho BC, Chew LM, Singh M, Yap EH.

Course of antibody production by the DIG-ELISA method in neonatally infected and juvenile infected rats after primary infection with Breinlia booliati (Filarioidea: Onchocercidae).
Southeast Asian J Trop Med Public Health. 1992 Jun;23(2):304-11.
PMID: 1439986 [PubMed - indexed for MEDLINE]


Gauny SS, Andya J, Thomson J, Young JD, Winkelhake JL.

Effect of production method on the systemic clearance rate of a human monoclonal antibody in the rat.
Hum Antibodies Hybridomas. 1991 Jan;2(1):33-8.
PMID: 1873495 [PubMed - indexed for MEDLINE]


Pancino GF, Osinaga E, Vorauher W, Kakouche A, Mistro D, Charpin C, Roseto A.

Production of a monoclonal antibody as immunohistochemical marker on paraffin embedded tissues using a new immunization method.
Hybridoma. 1990 Aug;9(4):389-95.
PMID: 2210779 [PubMed - indexed for MEDLINE]


Pancino GF, Osinaga E, Vorauher W, Kakouche A, Mistro D, Charpin C, Roseto A.

Production of a monoclonal antibody as immunohistochemical marker on paraffin embedded tissues using a new immunization method.
Hybridoma. 1990 Aug;9(4):389-95.
PMID: 2210779 [PubMed - indexed for MEDLINE]


Marquis CP, Harbour C, Barford JP, Low KS.

A comparison of different culture methods for hybridoma propagation and monoclonal antibody production.
Cytotechnology. 1990 Jul;4(1):69-76.
PMID: 1366723 [PubMed - indexed for MEDLINE]


Heath C, Dilwith R, Belfort G.

Methods for increasing monoclonal antibody production in suspension and entrapped cell cultures: biochemical and flow cytometric analysis as a function of medium serum content.
J Biotechnol. 1990 Jul;15(1-2):71-89.
PMID: 1366689 [PubMed - indexed for MEDLINE]


Tamaki Y, Kishida T, Fukuda M.

Practical method for production of monoclonal antibody to human IgG allotype G1M F and its applications in ELISAs and dot immunobinding.
J Immunol Methods. 1990 Mar 27;128(1):75-9.
PMID: 2324505 [PubMed - indexed for MEDLINE]


Adamus G, Zam ZS, Emerson SS, Hargrave PA.

A practical method for rescuing desired hybridomas during monoclonal antibody production.
In Vitro Cell Dev Biol. 1989 Dec;25(12):1141-6.
PMID: 2691498 [PubMed - indexed for MEDLINE]


Rej R, Keese CR, Giaever I.

A screening technique for monoclonal antibody production: application of an indium slide immunoassay.
Hybridoma. 1988 Oct;7(5):457-64.
PMID: 3198134 [PubMed - indexed for MEDLINE]


Shibier O, Hampton SM, Marks V.

An immunisation protocol which enhances the frequency of antigen-specific monoclonal antibody production.
J Immunol Methods. 1988 Nov 10;114(1-2):49-52.
PMID: 2460563 [PubMed - indexed for MEDLINE]


Lycke N.

A sensitive method for the detection of specific antibody production in different isotypes from single lamina propria plasma cells.
Scand J Immunol. 1986 Oct;24(4):393-403.
PMID: 3764344 [PubMed - indexed for MEDLINE]


Wang KC, Leung BS.

Fluorometric ELISA method for rapid screening of anti-estrogen receptor antibody production in hybridoma cultures.
J Immunol Methods. 1985 Nov 28;84(1-2):279-90.
PMID: 4067317 [PubMed - indexed for MEDLINE]


Gardner JS, Chiu AL, Maki NE, Harris JF.

A quantitative stability analysis of human monoclonal antibody production by heteromyeloma hybridomas, using an immunofluorescent technique.
J Immunol Methods. 1985 Dec 27;85(2):335-46.
PMID: 3908567 [PubMed - indexed for MEDLINE]


Lane RD.

A short-duration polyethylene glycol fusion technique for increasing production of monoclonal antibody-secreting hybridomas.
J Immunol Methods. 1985 Aug 2;81(2):223-8.
PMID: 4020150 [PubMed - indexed for MEDLINE]


Hofmann H, Heinz FX, Dippe H, Kunz C.

Tick-borne encephalitis: a simple method for detection of antibody production in the brain.
Zentralbl Bakteriol Mikrobiol Hyg [A]. 1985 Aug;260(1):132-8.
PMID: 3904279 [PubMed - indexed for MEDLINE]


Sovova V, Cerna H, Dostalova V, Hlozanek I.

Use of the hybridoma technique for antibody production to group-specific antigens of avian oncornaviruses.
Folia Biol (Praha). 1980;26(5):366-8.
PMID: 6160063 [PubMed - indexed for MEDLINE]





   Related Resources