Life ScienceS in SwitzerLand CHIMIA 2016, 70, No. 12 893

doi:10.2533/chimia.2016.893 Chimia 70 (2016) 893–897 © Swiss Chemical Society Recombinant for Academia: A Practical Approach

Pierre Cosson* and Oliver Hartley

Abstract: After several decades of optimization, technology enables the routine isolation and production of recombinant monoclonal antibodies in vitro. As such it has the potential to provide the academic community with a vast, inexpensive and renewable supply of well-characterized reagents, reducing bottlenecks in basic science, helping increase reproducibility of experiments, and phasing out the use of animals for production and discovery of antibodies. Yet the overwhelming majority of fundamental research laboratories still use incompletely characterized antibodies developed in animals. In order to promote increased use of recombinant antibodies in academia, we have recently initiated an open source recombinant facility in Geneva (http://www.unige.ch/antibodies). Here we describe our experience at the Geneva Antibody Facility: the various techniques involved in isolation and production of antibodies, the strategic choices that we have made, and what we hope will be a bright future for this project as part of a growing movement in the scientific community to replace all animal-derived antibodies with recombinant antibodies. Keywords: Recombinant antibodies

The 3R strategy concerning animal the data generated. Replacement strategies the foreign . It has been known for experiments[1] has three principles: experi- have proven more difficult to implement, centuries that pathogen challenge can be ments involving animals should be refined however, because experimental procedures readily mimicked by , and to decrease animal suffering and increase require constant adaptation as fundamental the natural antibody response in animals the scientific value of the results obtained. research questions evolve. In this respect, has been adopted as a tool to generate an- In doing so, the number of animals should animal immunization for antibody discov- tibodies against a wide range of . be reduced to the minimum necessary to ery and production is a rare exception. The usual strategy is to inject the antigen obtain useful data. Finally, where possible, Antibody generation in animals is no (protein, peptide, or, for non-peptidic anti- animal experiments should be replaced longer either necessary or desirable. Over gens, protein conjugates) into the animal, with alternative non-animal models or the last few decades, new techniques have formulated with adjuvants in order to stim- technologies. been developed to isolate and produce anti- ulate a potent immune response. Notably, In the field of toxicology, replacement bodies entirely in vitro.[3] This provides the the adjuvants generally used for animal im- has recently gathered a great deal of mo- academic community with a unique oppor- munization are not deemed acceptable for mentum: it is likely that integrated in vitro tunity to demonstrate its willingness to ap- use in humans because of the side-effects testing strategies will in the near future re- ply replacement strategies when they are they induce. place much of the mandatory animal tests available. Following immunization, the immune currently performed.[2] For basic science in This article has two main goals. First, system of the animal scans its natural rep- academia, much progress has been made to provide a simple description of the main ertoire of different antibody-producing in refining animal experiments to mini- approaches available for generating anti- cells (107–109, according to the size of the mize suffering and increase the quality of bodies, addressed to non-experts, with a animal) in order to (i) select rare cells that specific focus on antibodies generated in produce antibodies which recognize the vitro. Second, to describe the new Geneva antigen, (ii) induce these cells to multiply, facility dedicated to the in vitro discovery, (iii) stimulate them to evolve higher affin- production and archiving of antibodies for ity binding to the antigen, and (iv) trigger the academic community, stressing the them to produce large quantities of the re- technical choices that guided this project sulting antibodies. and outlining the steps that could be taken From a practical point of view, there to develop its future scalability and sus- are three main methods to generate spe- tainability in the context of other similar cific antibodies. They are referred to here initiatives around the world. as polyclonal, monoclonal or recombinant antibodies (Table 1).

First Part: Antibodies Come in Polyclonal Antibodies Three Flavors Polyclonalantibodiesrepresenttheanti- body mixture that can be purified directly *Correspondence: Prof. Dr. P. Cosson Faculty of Medicine When challenged with a pathogen, from an animal’s serum a few months af- University of Geneva innate immunity represents the first line ter immunization. The term ‘polyclonal’ is Centre Médical Universitaire of defence against infections. This is fol- used because at this time the serum will 1 rue Michel Servet CH-1211 Geneva 4 lowed by adaptive immunity, which in- contain a mixture of different antibodies E-mail: [email protected] cludes the generation of antibodies against directed against the antigen, resulting from 894 CHIMIA 2016, 70, No. 12 Life ScienceS in SwitzerLand

Table 1. Relative advantages of different types of antibodies sequences, and can be readily produced in cultured cells, in a variety of formats, as POLYCLONAL MONOCLONAL RECOMBINANT described below.

Time to isolate and 6 weeks 3–6 months 6 weeks What Constitutes a Useful produce Antibody for Research? Animals required YesYes No A perfect antibody is one that would be suitable for all of the different applications Selection in vivo, by immune in vivo, by immune in vitro, used by the academic research commu- system system independent of nity, (e.g. ELISA, Western blot, immuno- immune system fluorescence microscopy, etc.). According to these criteria there are very few, if any, Defined molecular No YesYes perfect antibodies. All antibodies, whether species polyclonal, monoclonal or recombinant, Reformating No No Yes are initially characterized by testing their possible ability to bind the antigen or antigen frag- ment that is typically presented in a purified Storage Largest Smaller, requires Smallest, and/or concentrated form. Further charac- volume maintenance no maintenance terization is then necessary to determine, for example, (i) whether an antibody raised Sustainable No Yes, unless Yes against a peptide fragment recognizes the production problems with full-length target protein, (ii) if it is capable storage of detecting the antigen at its endogenous Sequence available No No Yes level (usually low), and (iii) with which de- tection procedures it is compatible. For this reason, catalogs of research antibodies for sale typically specify for which application the combined production of a number of all available monoclonal antibodies are of or applications they are best suited. different antibody producing cell clones rodent origin. By the same criteria, very few adequate- that were amplified during the antibody Monoclonal antibodies are technically ly characterized antibodies are worthless: response. Because more blood can be more difficult to produce than polyclonal for example (i) many antibodies that do not drawn from larger animals, typical sourc- antibodies, but as single molecular spe- recognize their target protein by Western es of polyclonal antibodies are rabbits, cies of defined structure they generally blot recognize it in immunofluorescence sheep and goats rather than rats or mice. represent more valuable research reagents. experiments, (ii) an antibody that recog- Polyclonal antibodies are relatively inex- Furthermore, immortalized hybridoma nizes a peptide epitope within a protein but pensive to produce, but the total amount cells outlive the animals from which they is unable to recognize the full-length pro- that can be produced is limited by the were obtained: they can be stored frozen tein may be useful for certain applications, amount of blood that can be drawn from for many years and then, if needed, ex- such as helping to determine the extent to the immunized animal during its lifetime. panded in culture to produce monoclonal which a protein is folded or degraded, or Once a supply of serum from an animal is antibody in amounts greatly in excess of to report on the post-translational modifi- exhausted, a new immunization must be what the immunized animal could natu- cations it has received. For this reason, all performed, necessitating a new animal ex- rally produce in its lifetime. adequately characterized antibodies with periment and resulting in a polyclonal mix- definable specificity and utility should ture that, because of the stochastic nature Recombinant Antibodies ideally be kept in a searchable archive that of the adaptive immune response, cannot To generate recombinant antibodies, is available for use by the scientific com- possibly be identical to the previous one. the whole process of antibody selection munity. Additionally, calls have been made and production is reconstituted in vitro. for sequence information, which uniquely Monoclonal Antibodies In the most widely used technology, an- defines each , to be In 1975, Köhler and Milstein dis- tibody phage display,[3] a large collection deposited as part of the characterization covered a method to immortalize mouse of filamentous bacteriophages (typically procedure.[5] antibody-producing cells.[4] This led to in the range 109–1010) are engineered to the development of a new strategy to pro- encode and display a repertoire of differ- duce monoclonal antibodies, with such ent antibodies. This phage repertoire is Second Part: Recombinant far-reaching consequences that they were then incubated with the chosen antigen Antibodies Are Better awarded the Nobel Prize for Physiology in vitro. Phages displaying antibodies ca- and Medicine in 1984. To generate mono- pable of binding to the antigen are puri- In addition to ethical considerations, clonal antibodies, antibody-producing fied in a ‘panning’ step, then the recombinant antibodies exhibit numerous cells are collected from an immunized encoding the selected antibodies can be practical advantages over animal-derived animal and fused to a tumour cell line to amplified. Over several rounds of panning antibodies. These are outlined below. generate immortalized antibody-produc- and amplification, extremely rare antigen- ing cell lines called hybridomas. Each hy- specific monoclonal antibodies can be Control of Selection Conditions bridoma can be cultured as an individual isolated. These antibodies are referred to When an antigen is used to immu- clone, enabling unique (i.e. monoclonal) as recombinant antibodies, as a reference nize an animal, it is subject to whatever antibodies with the desired characteristics to the recombinant DNA technology used modification and degradation that might to be isolated and produced. Hybridoma to engineer the initial collection of bac- take place before and during its encounter technology has not been widely adopted in teriophages. Recombinant antibodies are with the animal’s adaptive immune sys- species other than rats and mice, so almost initially selected as DNA antibody-coding tem. Furthermore, the immune system has Life ScienceS in SwitzerLand CHIMIA 2016, 70, No. 12 895

evolved complex tolerance mechanisms in bacteria (Fig. 1C).[6] However, since according to the manner in which the anti- that prevent it from generating antibodies monovalent scFv fragments do not benefit body sample was originally produced. directed against structures that resemble from the avidity gain of multimerized im- For polyclonal antibodies, the available self-antigens, and that reduce its capacity munoglobulins, they are often reformatted serum, though limited (typically 100 to 200 to generate antibodies against many anti- into constructs that provide dimerization ml), must be kept frozen, and requires sig- gens. and facilitate detection with secondary nificant freezer space. For example a maxi- These problems are bypassed in the reagents. This can be achieved by produc- mum of 100 typically sized serum samples generation of recombinant antibodies.[3] ing a fusion protein composed of an scFv can be kept in a small –20 °C freezer. Selection of the antibodies is achieved in fused to an Fc domain, a format often re- For monoclonal antibodies, production vitro under conditions that can be tightly ferred to a minibody (Fig. 1D).[7] In most relies on a stock of hybridoma cells kept controlled by the user. For example, en- in vitro experimental settings, minibodies frozen in liquid nitrogen. In addition, each zyme inhibitors can be used to ensure are equivalent in performance to intact hybridoma cell line must be thawed and that a peptide antigen remains intact and immunoglobulins,[8] although for in vivo refrozen every few years, and occasionally retains any user-added modifications (e.g. work it is sometimes necessary to refor- recloned and retested. Many hybridoma phosphorylation) throughout the selection mat into a full immunoglobulin in order to cells have been lost due to improper han- procedure. The use of large naïve or syn- take advantage of the enhanced circulatory dling or storage, or following accidents. thetic antibody libraries bypasses the re- lifetime and effector functions provided by Because of the required time and effort for quirement to use in vivo immune systems, the intact antibody structure. A significant conservation, when a researcher retires his/ enabling the selection of antibodies against advantage of recombinant antibodies is her stock of polyclonal or monoclonal anti- self-antigens and against non-peptidic an- that during reformatting into minibodies bodies is often lost. tigens that generally show low immunoge- the Fc fragment can easily be interchanged Storing recombinant antibodies re- nicity. between that of different species, e.g. hu- quires significantly less space and resourc- Additionally, it is possible to use in man, rabbit or mouse, allowing the use of es. The encoding a given antibody vitro selection approaches to direct the iso- a wide range of secondary antibodies. One can be stored in the form of highly stable lation of recombinant antibodies towards disadvantage of minibody reformatting is plasmid DNA, or even in silico, in a da- specific structures within an antigen, for that for efficient production, minibodies tabase of recombinant antibody genes that example by alternating positive panning require expression in mammalian cells, in- can readily be resynthesized at a later date steps against the antigen with negative volving procedures that are more complex and at a different site to enable production panning steps against a structural variant and expensive than production in bacteria. of the encoded antibody. There are essen- of the antigen lacking the target structure. Finally, the range of possibilities for tially no limits to the number of recombi- Such approaches are impossible using im- reformatting recombinant antibodies is nant antibodies that can be stored in this munized animals. not restricted to variations on natural anti- way, and accidental loss is easily avoided. Finally, once an antibody with prom- body formats. This presents a wide range In summary, because recombinant anti- ising binding affinity has been identified, of attractive research options (e.g. fusion bodies bypass the need for animal experi- using recombinant antibody technology it with enzymes, linkage of several different mentation, they can be selected in a more is possible to generate a second-generation antigen-binding domains, etc.). One such controlled and less biased manner than repertoire using targeted mutagenesis to application, where scFv are fused to fluo- animal-derived antibodies. Like monoclo- modify its sequence, and then perform fur- rescent proteins for expression in the cyto- nal antibodies they are unique and defined ther selection on this repertoire in order to sol of a mammalian cell as intrabodies, has molecular species that can be produced in isolate new antibodies with improved af- been successfully used to enable detection unlimited amounts, but they can be stored finity and/or specificity for the target. of the corresponding proteins during live safely in much higher numbers, readily se- cell imaging.[9] quenced and can be produced in a wide va- Formatting riety of formats according to the intended Antibody proteins have a modular Production and Storage use. With all of these key advantages, we structure (Fig. 1A) featuring variable re- All characterized antibody samples are believe that the widespread replacement of gions, responsible for engaging antigens, potentially valuable, and should in prin- animal-derived antibodies with recombi- and constant regions (Fc), which do not ciple be stored in a searchable archive, but nant antibodies is inevitable: no longer a engage antigen but whose physiologi- there are costs and difficulties associated question of if, but when and how? cal roles are (i) to increase the valency of with archiving, and these vary significantly the antigen-binding variable domains, (ii) to increase the in vivo stability of the an- tibody molecule, and (iii) to link antigen binding to effector functions in the im- mune system. From a practical point of view, the constant domains are also the tar- gets of secondary reagents used to reveal the presence of an antibody. In antibody phage display, the smallest functional an- tigen binding fragment, known as a single chain Fv (scFv) is most commonly used, with its small format facilitating the clon- ing of large antibody gene libraries and the Fig. 1. Formatting recombinant antibodies. A. Natural IgG antibodies are made of two heavy production of recombinant phage in bac- chains and two light chains. In the variable domains (V), hypervariable regions (orange) form the antigen-binding sites. B. To select recombinant antibodies, the variable domains of the heavy teria (Fig. 1B). After isolation, the DNA and light chains are connected by a linker to form a single-chain Fv (scFv) fused to the p3 protein encoding scFvs selected by phage display at the surface of a phage. C. Selected antibodies can be produced as monovalent scFv. D. The can be readily reformatted for soluble (i.e. minibody format (an scFv fused to the Fc portion of a heavy chain) is dimeric and its Fc portion no longer attached to phage) expression can be recognized by classical secondary detection reagents. 896 CHIMIA 2016, 70, No. 12 Life ScienceS in SwitzerLand

Third Part: Recombinant to antigens of human origin. Necessarily, ferent secondary reagents. Consequently, Antibodies for Academia many of the antibodies generated will be the Geneva Antibody Facility produces used very infrequently, once a year world- antibodies exclusively in the minibody In the pharmaceutical industry, recom- wide or even less. There is an enormous format. Three variants (human, mouse or binant antibody technology has been used academic value in generating and main- rabbit Fc) are currently available. extensively to generate therapeutic anti- taining such a universal archive, but selec- bodies for the treatment of a variety of pa- tion, production, storage, and even funding How We Store and Produce thologies, including inflammatory disease, strategies must be thought out in advance Antibodies autoimmune disease and . Today a in order to set up a framework potentially Long-term storage of huge numbers of sizable portion of the new treatments ap- capable of handling hundreds of thousands antibodies can only be achieved in plas- proved each year is based on the use of of antibodies. mid or in silico form. This requires that, therapeutic recombinant antibodies.[10] on demand, antibodies must be produced There is a recognized need to adopt Why We Select Antibodies on by transient of mammalian recombinant antibodies in academia.[5] Behalf of Users cells. The Facility has developed plasmids Antibodies have been defined as one of We recognize that at present most re- that allow us to reach antibody concentra- the most variable reagent classes used in search laboratories have not mastered the tions of 100 µg/ml in serum-free cell su- fundamental research laboratories, ac- technology to select and produce recom- pernatants. Alternatively, more simple and counting for a significant part of the low binant antibodies, and many will never do cheaper production procedures typically reproducibility of many experiments.[11] so. In addition, high-quality standard naïve yield cell supernatants containing approxi- Recombinant antibodies have the poten- antibody libraries are not easily accessible. mately 1 µg/ml of antibodies. Finally, us- tial to become a huge, reliable and well- A huge step forward in the creation of the ers are welcome to bypass the Facility’s characterized source of reagents for basic Geneva Antibody Facility was to gain ac- production process by either (i) obtaining science,[5] but this has not happened so far, cess to libraries from NovImmune SA, on a sample of the expression plasmid for and currently use of recombinant antibod- the understanding that the main aim of the their own use or (ii) synthesizing the gene ies in academia is rare. facility is to generate fundamental research encoding an antibody of interest and incor- The initial development and imple- reagents for the academic community. porating it into their own expression vec- mentation of recombinant antibody tech- Consequently, the primary aim of the tor. Here again, we envisage that the open nology was strongly driven by industrial Facility is to perform selection and pro- source model is the most appropriate way incentives. Technology for generating and duction of recombinant antibodies for the to provide scalability and sustainability to using recombinant antibody repertoires academic community. This is clearly not a large scale recombinant antibody initia- was protected by patents, and for almost scalable, however, and we believe that the tive. two decades recombinant antibody activ- construction of a truly universal recombi- ity and expertise was largely confined to nant antibody archive will not be possible Incorporation of Previously the industrial sector. It is still very chal- without the input of many different groups, Published Recombinant Antibodies lenging for academic research groups to and will therefore depend upon further Many recombinant antibodies have gain access to high quality naïve antibody spread of recombinant antibody expertise, already been selected and characterized libraries. high quality libraries and know-how into by the academic community, but their se- Recent years have seen a much wider the academic sector. quences have not been centralized into a uptake of phage antibody technology, lead- single database. We plan to launch a new ing to a broader base of scientists qualified Why We Only Do Initial initiative in 2017: the Facility will ensure to use it, together with the movement of Characterization reformatting and production, on demand, some of the early leaders in the field from The antibodies selected by the Geneva of any previously published recombinant industry to academia.[12–14] This has cre- Antibody Facility have all been shown to antibody or sequenced hybridoma. For ated an environment where, for the first specifically recognize their target anti- this, we will only request the sequence of time, recombinant antibody technology gen (often a protein fragment) by ELISA. the antibody, and where applicable a pub- is poised to start catering to the academic Although it has been proposed that recom- lished reference in which it is described. community: reliable binding agents for binant antibody characterization could be all.[5] standardized by specially funded laborato- ries,[5] we believe a more sustainable and Conclusion Antibodies Against Any Target: The scalable solution would be to ensure that Value of the Long Tail further characterization is performed and As much as possible we are trying to The Geneva Antibody Facility selects reported by the scientist end-users them- develop the Geneva Antibody Facility as and produces new recombinant antibodies selves, using an open access knowledge- an open-access, low cost and collaborative for the academic community. The Geneva base attached to the Facility’s website. platform. It is complementary to the other Antibody Facility is not focused on the This knowledgebase would also list pub- large recombinant antibody initiatives generation of research antibodies that are lications describing the use of a particular that are currently underway[16] as well as commercially available, nor was it set up to antibody. those that were carried out in the past,[15] compete with private companies in the gen- with the exception that we believe that a eration of therapeutic antibodies. Instead, Why We Use Minibodies community-driven open source venture its vision is to help create a sustainable sys- Currently, our experience with Facility is more likely to have the scalability and tem to generate a vast searchable archive of users suggests that most researchers wish above all the financial sustainability neces- antibodies against any antigen in any spe- to obtain recombinant antibodies that sary for success. We hope that researchers cies, for the use of the academic research can be used experimentally in the same worldwide will take advantage of this new community. way as intact, animal-derived antibodies. resource and in doing so will contribute Unlike some of the other recombi- Researchers would be reluctant to use un- to its success, providing an important re- nant antibody initiatives,[15] the Geneva usual antibody formats that would neces- source for increasing data reproducibility Antibody Facility’s scope is not restricted sitate the use of different procedures or dif- in biomedical research. Life ScienceS in SwitzerLand CHIMIA 2016, 70, No. 12 897

Finally, from an ethical, regulatory and Minibody: a fusion protein comprised of a [8] S. Moutel, A. El Marjou, O. Vielemeyer, C. political point of view, it is highly desirable scFv and an Fc portion (see Fig. 1) Nizak, P. Benaroch, S. Dubel, F. Perez, BMC scFv: single-chain fragment variable, a Biotechnol. 2009, 9, 14. to operate the switch from antibodies pro- [9] C. Nizak, S. Monier, E. del Nery, S. Moutel, B. duced in animals to antibodies selected and fusion protein consisting of the two immuno- globulin domains of an antibody that comprise Goud, F. Perez, Science 2003, 300, 984. produced entirely in vitro. In this perspec- [10] M. S. Kinch, Drug Discov. Today 2015, 20, 393. the antigen binding site (VH and VL) joined via [11] M. Baker, Nature 2015, 521, 274. tive, this initiative is also aimed at fostering a flexible linker peptide (see Fig. 1) a climate of understanding and cooperation [12] D. J. Schofield, A. R. Pope, V. Clementel, J. Western blot: detection of a proteic antigen Buckell, S. D. J. Chapple, K. F. Clarke, J. S. between academia and civil society. after a protein mixture has been fractionated on Conquer, A. M. Crofts, S. R. Crowther, M. an acrylamide gel and transferred to a nitrocel- R. Dyson, G. Flack, G. J. Griffin, Y. Hooks, Appendix: Glossary lulose membrane. Protein are usually in an un- W. J. Howat, A. Kolb-Kokocinski, S. Kunze, Antigen: an element (protein, lipid, small folded state on the nitrocellulose. C. D. Martin, G. L. Maslen, J. N. Mitchell, molecule, etc.) against which an antibody can Secondary antibodies: antibodies that rec- M. O’Sullivan, R. L. Perera, W. Roake, S. P. be raised ognize a large number of antibodies of a given Shadbolt, K. J. Vincent, A. Warford, W. E. Antibody: a Y-shaped protein (see Fig. 1), Wilson, J. Wie, J. L. Young, J. McCafferty, species via the Fc portions, which are normally Genome Biol. 2007, 8, R254. also known as an immunoglobulin, whose two used in research as conjugates (fluorochrome, [13] J. T. Koerber, M. J. Hornsby, J. A. Wells, J. Mol. identical arms are responsible for antigen bind- enzyme) to reveal the presence of a ‘primary’ Biol. 2015, 427, 576. ing and whose stalk is responsible for interact- (antigen-specific) antibody. [14] M. Hornsby, M. Paduch, S. Miersch, A. Sääf, ing with effector components of the immune T. Matsuguchi, B. Lee, K. Wypisiniak, A. system. The antigen binding sites of different Received: July 26, 2016 Doak, D. King, S. Ustyuk, K. Perry, V. Lu, antibodies are highly variant in sequence. W. Thomas, J. Luke, J. Goodman, R. J. Hoey, Epitope: a small region of an antigen recog- [1] Background to the Three Rs Declaration of D. Lai, C. Griffin, Z. Li, F. J. Vizecoumar, D. Bologna, as adopted by the 3rd World Congress Dong, E. Campbell, S. Anderson, N. Zhomg, nized by a specific antibody on Alternatives and Animal Use in the Life Fc portion: the stalk region of the Y-shaped S. Gräslund, S. Koide, J. Moffat, S. Sidhu, A. Sciences, Bologna, Italy, on 31 August 1999. Kossiakoff, J. Wells, Mol. Cellul. Proteomics antibody molecule that, in contrast to the anti- Altern. Lab. Anim. 2009, 37, 286 2015, 14, 2833. gen-binding sites, is invariant in sequence (see [2] S. Gibb, Reprod. Toxicol. 2008, 25, 136. [15] S. Dubel, O. Stoevesandt, M. J. Taussig, M. Fig. 1). Most secondary antibodies recognize [3] A. R. M. Bradbury, S. Sidhu, S. Dübel, J. Hust, Trends Biotechnol. 2010, 28, 333. the Fc portion of antibodies from a defined spe- McCafferty, Nat. Biotechnol. 2011, 29, 245. [16] K. Groff, J. Brown, A. J. Clippinger, Biotechnol. cies (e.g. anti-mouse Ig) [4] G. Kohler, C. Milstein, Nature 1975, 256, 495. Adv. 2015, 33, 1787. Immunization: Delivery of an antigen to an [5] A. Bradbury, A. Pluckthun, Nature 2015, 518, animal formulated in such a way as to elicit the 27. [6] H. R. Hoogenboom, A. D. Griffiths, K. S. generation of an antibody response Johnson, D. J. Chiswell, P. Hudson, G. Winter, Immuno-fluorescence: detection of a pro- Nucl. Acids Res. 1991, 19, 4133. tein by fluorescent antibodies, after fixation and [7] S. Hu, L. Shively, A. Raubitschek, M. Sherman, permeabilization of a cell or tissue. Proteins are L. E. Williams, J. Y. Wong, Cancer Res. 1996, usually in a folded state in fixed cells. 56, 3055.