Recombinant Antibodies for Pesticide Immunoanalysis - ACS

Chapter 38

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Recombinant Antibodies for Pesticide Immunoanalysis Karl Kramer and Bertold Hock Department of Botany at Weihenstephan, Technical University of München, D-85350 Freising, Germany

Standardized immunochemical methods for food analysis and environmental monitoring have profited from the use of monoclonal antibodies. However, this approach is restricted by the length of time required for antibody production to new analytes. The recombinant antibody technology is expected to compensate for these limitations. After a discussion of recombinant antibody state of the art, two approaches are shown based on the herbicidal s-triazines. Single-chain Fvs directed against different s-triazines were expressed as fusion proteins on the surface of M13 phages. In addition, a recombinant Fab for atrazine was produced in the pASK85-based expression system, which exhibited similar binding and displacement kinetics as the original monoclonal antibodies. The feasibility to obtain mutant antibodies may eventually replace the circumstantial approach to obtain new antibodies by new immunizations. Immunochemical analysis has become an important tool for the detection of toxicants and pesticides in foods, water and other environmental samples. A major factor has been the use of hybridoma technology. The ability to produce monoclonal antibodies (Mab) means that techniques are available for the unlimited production of antibodies of the same isotype exhibiting constant properties. Although this technique is expected to remain in the future, a technology revolution has taken place during the last few years, which utilizes bacteria to display a diverse library of antibodies. This approach is equivalent to the mammalian immune repertoire and thereby avoids timeconsuming immunizations and the use of live animals. Novel protein engineering strategies are used that rely on bacterial expression systems rather than mice for the rapid selection of antibodies. This topic has been reviewed intensively, e.g. by Winter and Milstein (/) and Irving and Hudson (2). A comparison of the hybridoma and the recombinant approach shows interesting parallels. Both strategies use immortilization processes as central methods. The

0097-6156/96/0621-0471S15.00/0 © 1996 American Chemical Society Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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hybridoma method is based on the immortilization of B-lymphocytes, the antibody producing cells, whereas the recombinant antibody (Rab) approach relies on the immortilization of antibody genes. Both strategies require screening processes, although they are carried out at different levels during the production of antibodies. The recombinant antibody approach The basic technology of Rab production has been developed in the medical field and is now mainly targeting medical areas including cancer and H I V research. The principle of Rab production in bacteria was pioneered in 1988 by Better et al (3) and Skerra and Pluckthun (4). Further improvements were introduced by the groups of Winter (5), Lerner (6), Borrebaeck (7), Pluckthun (8) and Skerra (9). These improvements included the isolation of light and heavy chain encoding D N A sequences by the polymerase chain reaction (PCR) technique, better screening and purification procedures, as well as the modification of Rab. A broadening of the field of Rab was observed during the last few years. It resulted in refined strategies for cloning and expression of antibody genes. Table I summarizes some of the important steps of this development. Figure 1 (right part) gives a scheme for the production of Rab. It outlines a few general steps shared by different protocols. First, mRNA is isolated from hybridomas or B-lymphocytes. Following the synthesis of the complementary DNA-strand (cDNA), the desired immunoglobulin sequences are amplified by the PCR, which represents a central element in Rab production. Since one PCR cycle duplicates the included D N A sequence, up to 2 - 2 copies are obtained after 25-35 repetitions of the temperature regime. The primer mixtures, deduced from data bases (14), facilitate the synthesis of any antibody encoding sequence without requiring knowledge of the specific nucleotide sequence. The primers are designed to hybridize with conserved frame regions of the variable domains and the constant domains, respectively, due to species-specific sequence homology. After insertion of restriction sites at the 3'- and 5'-end, the PCR amplificates are ligated with an appropriate vector and introduced in the host culture, most commonly E. coli. Each individual transformed bacterium containing an antibody encoding moiety propagates the foreign D N A by replication and transfers it to its descendants by cell division. These represent a recombinant bacterial clone. If a hybridoma cell line is used as starting material, the bacteria exclusively contain the sequence of the corresponding Mab. However, if B-lymphocytes (which represent a heterogeneous cell population) are used as the source for the mRNA, the whole repertoire of antibody encoding D N A sequences is distributed to different clones and thus constitute a gene library. Subsequently, the individual heavy and light chain fragments can associate by random combination forming heterodimers that are expressed as functional Rab. These can be purified from culture supernatants or cell lysates. 25

35

Recombinant antibodies for environmental analysis A few groups are already involved in Rab synthesis for pesticide and toxicant analysis, among them the laboratories of Hammock, Karu, and Stanker (See chapter by

Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


S e c r e t i o n of monoclonal antibodies

H y b r i d o m a culture

Light c h a i n library

vector

Figure 1. Synthesis of antibody fragments utilizing Mab (left) or Rab (right). Only the Rab approach allows the production of scFv. Antigen recognition takes place a t the complementarity determining regions (CDR) of the variable region. In contrast to the adjacent frame regions (FR), the CDRs are characterized by sequence diversity.

E x p r e s s i o n in b a c t e r i a

/

L i b r a r y with p a i r e d L- a n d Η - c h a i n f r a g m e n t s

combination

^

Transformation of E. coli

L i g a t i o n with p l a s m i d

S e p a r a t e a m p l i f i c a t i o n of d e f i n e d H - a n d L - c h a i n r e g i o n s

Random

H e a v y c h a i n library

B-lymphocytes

cDNA-synthesis

m R N A - i s o l a t i o n from

474


Table I: Landmarks in recombinant antibody technology.

1984

chimeric antibodies

Neuberger et al (10)

1986

humanized antibodies

Jones et al (11)

1988

Rab in bacteria

Betters al (3)

1988

Fv-fragments in bacteria

Skerra and Pluckthun (4)

1992

combinatorial library

Barbas et al (12)

1992

affinity maturation by chain shuffling

Marks et al (13)


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Recombinant Antibodies for Pesticide Immunoanalysis

475

Kamps-Holtzapple and Stanker, this volume) in the USA, Harris in Scotland, Morgan in England and our group in Germany (Table II). It can be foreseen that Rab will become a common tool in environmental monitoring. Hammock's group is focused on Rab expression in baculovirus. This eukaryotic expression system is well suited for the synthesis of larger proteins, e.g. complete antibodies as they are secreted by B-lymphocytes. In contrast to prokaryotic expression in bacteria, the baculovirus system enables the glycosylation of folded antibodies as it occurs in mammalians. This goal was achieved by starting with the synthesis of the appropriate PCR primers for the isolation of antibody D N A sequences and the development of a suitable expression vector (75). This part was restricted to E. coli, because of the vast knowledge available for Rab synthesis in this expression system. On the basis of pGEM5fZ(-) (Promega Corp.) the plasmid vector pGEMEB was constructed to allow separate cloning of H - and L-chain PCR ampliflcates derived from the hybridoma cell line AM7B2.1 (20). AM7B2.1 secretes Mab against the herbicide atrazine. In order to express the Rab derived from this Mab, the positive H - and L-chain clones were subcloned into the expression vector pET3d (27) to form a functional Fab fragment after the induction of bacteria. Although the light chain exhibits sequence deviations from the original Mab AM7B2.1, the recombinant Fab binds to an atrazine-phosphatase conjugate used as a tracer in a dot blot assay in like manner to the original antibody. Most importantly, binding did not take place to the recombinant Fab if the antibodies were desensitized by atrazine. The screening used in this approach is based on the classical white/blue selection, which depends on a substrate turnover initiated by transformed bacterial clones. However, if screening of libraries with a large repertoire of different Rab is intended, this screening method is limited. Therefore, more efficient selection strategies were developed in recent years, most notably the phage display of Rab (22-24). This system allows the discrimination of desired Rab clones at an early state of production (Figure 2). Amplified antibody sequences are inserted into a phage or a phagemid vector. The vector includes the gene coding for the major (g8p) or minor (g3p) phage coat protein, which is placed after the Rab sequence. Phages bearing Rab sequences display the corresponding Rab as a fusion product with the coat protein on their surface. This enables the selection of desired clones in panning devices with immobilized target molecules. The primary advantage of the phage display system consists in the idea that the affinity and specificity of the immunoreactants is the dominating selection criterion. Karu (16) utilized a phage display system developed by Lerner to express recombinant Fab fragments against the phenylurea herbicide diuron. The Fab encoding sequences were isolated from hybridoma cells and subsequently amplified by PCR. H - and L-chains were combined in the phagemid vector pComb3. After the transformation of E. coli and infection by helper phages, complete phage particles were synthesized presenting specific Fab fragments on their surfaces. These positive phages were enriched by diuron-BSA conjugates immobilized to a solid phase. After four rounds of selection, desorption of bound phages at pH 2.2 and reinfection of bacteria, four out of several hundred enriched clones reacted specifically with an alkaline phosphatase-labeled diuron tracer. Soluble Fab were derived from these positive phages by excising the vector sequence, which codes for the phage coat


476


Table Π. Recombinant antibodies in pesticide and toxicant analysis.

a

1993

recombinant atrazine Fab

Ward et al (IS)

1994

recombinant diuron Fab

Karu et al (16)

1994

scFv against organic pollutants

Sheltonera/. (17)

1994

scFv against aflatoxin diacetoxyscirpenol, and parathion

Morgan

1995

recombinant scFv for s-triazines

Hock etal (18)

1995

recombinant scFv for glycoalkaloids

Kamps-Holtzapple and Stanker (19)

a

(Morgan, R. A. M . , Norwich Laboratory, Norwich, U K , personal communication, 1994)


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Recombinant Antibodies for Pesticide Immunoanalysis

477

Assembling of amplified variable regions with peptide linker linker

•

ι Ligation with phagemid scFv-DNA fragment

Phagemid-DNA

1 Transformation of E. coli and expression in M13-phage g8p

jooooooooooooœoooooooooi °ooooooooooooooooooooœœoocooœooo(

!

1 Panning of specific phage antibodies

* ?? ?? Figure 2. Phage display of recombinant antibodies. Due to technical reasons, the scheme gives an abstracted image of in vivo conditions. The length of filamentous M13-phage depends on the size of enclosed DNA (estimated size of scFv presenting M13 phage: 1 pm length, 15-20 Â diameter).


478


protein. The recombinant Fab exhibited the same binding properties as the Fab fragments obtained by papain digestion from the corresponding Mab 481. The Rab as well as the Fab prepared by proteolytic cleavage showed increased sensitivities by almost two orders of magnitude in the competitive, indirect enzyme-linked immunosorbent assay (ELISA) for the hapten diuron. In addition, Karu (16) investigated the semi-synthetic combinatorial library approach to select Rab with significant affinity and specificity towards diuron. The libraries, which were provided by Barbas (25,26), differed only in their CDR3 domains. After five rounds of panning on diuron carrier conjugates, 600 colonies in the positive fraction were tested in the direct ELISA. Nine percent of these clones bound diuron-alkaline phosphatase tracer with low affinity and a single clone showed weak competition with free diuron. We used two different approaches for the expression of Rab, recombinant single chain Fv (scFv) and recombinant Fab. The phage display of scFv was based on the work of Winter (27). mRNA from our hybridoma cell lines K1E4, K4E7 and P6A7 (28-30) was isolated in order to synthesize scFvs against the s-triazine herbicides terbutryn, atrazine and terbuthylazine, respectively. The scFv molecule consists of the heavy and light chain variable region connected by a (Gly Ser) peptide linker (Figure 1). Covalent binding prevents dissociation of the paired V and V domains caused by the varying stability of the heterodimer due to the structure-dependent differences of the binding energy between the heavy and light chain moiety in individual antibodies. scFvs are expressed as g3p coat protein conjugates on the surface of the phage particle. Our efforts have been focused on providing an efficient screening procedure, which facilitates the rapid isolation of appropriate clones. Since selection by panning suffers from significant contamination of positive clones with unspecific ones, repeated cycles of selection are required, especially when large libraries are handled. Using immunomagnetic screening for phage particles derived from the hybridoma cell line K1F4, we obtained 184 out of 1.48xl0 transformed clones in the positive fraction by an unique selection step using the solid phase of the triazine-coated paramagnetic beads. Almost 80% of the positive fraction (143 clones) reacted specifically with the corresponding terbutryn conjugate. Comparison of the phage-conjugated scFv with the corresponding Mab showed similar concentration-dependent binding to the immobilized triazine-ovalbumine conjugate in the non-competitive ELISA (Figure 3). Unspecific binding was excluded because incubation with uncoupled ovalbumine resulted in very low absorbance measurements. However, phage-associated scFv bound to the immobilized analyte could not be replaced by the free analyte. This common effect may be due to the stickiness of tagged phage particles on the microtiter plate surface after binding to the coat-conjugate. Soluble scFv are presently being tested by E L I S A and surface plasmone resonance. Initial results indicate that soluble scFv are replaced by free analytes. The scFv contains an artificial linker sequence to connect the variable domains. For certain applications, e.g. structure analysis imaging the binding pockets of natural antibodies, Fab fragments are the molecules of choice. Dissociation of the heterodimer is avoided by the stabilizing disulfide bonds between the heavy and light chain constant domains. Once this Fab fragment is obtained it can be fused into 4

3

H

L

6


38. KRAMER & HOCK

Recombinant Antibodies for Pesticide Immunoanalysis 479

j

b

•

Mab K1F4

E3

Rab 6C

•

Rab 2B

Β

RablOE

ι

100 μς/πιί

OVA

100

33

, . 10

3,3

terbutryn-OVA

Figure 3. Mab and Rab in the non-competitive, indirect ELISA.


480


vectors to religate an Fc region to generate full antibody sequence. In our second approach we used a Fab expression system developed by Skerra (57). The principle of Fab synthesis is shown in Figure 4. It is based on the vector pASK85, which contains the constant domains C 1 and C of a murine antibody. Therefore, P C R amplification of the antibody genes is again reduced to the variable regions of the antibody of interest. This strategy rninimizes sequence deviations generated during PCR because the accuracy of PCR is inversely proportional to the length of the amplified D N A product. After separate cloning of the H and L variable regions into the vector, the complete light chain is excised and cloned into the corresponding heavy chain vector. Following transformation of E. coli and induction of expression, the chains are secreted into the periplasm of the transformants. In this bacterial compartment protein folding, association of a functional heterodimer and formation of disulfide bonds is accomplished. Expressed Fab fragments are purified from cell lysates by affinity chromatography using immobilized Z n . This strategy takes advantage by a histidine hexamer extension at the C-teiminus of the heavy chain exhibiting an affinity for Z n ions. Calibration curves obtained with competitive ELISAs indicate the concentration-dependent displacement of the recombinant Fab by the analyte similar to the original Mab K4E7 (Figure 5). The absorption values ranged between 1.5 for the zero control and 0.08 for the analyte excess. H

L

2+

2+

Perspectives It can be foreseen that the Rab technology will be readily established in environmental research. This approach is considered as an improvement in comparison with the Mab technology. A significant difference between Mab and Rab is that the latter is accessible for selection and modification of antibody properties without requiring new immunization. Methods are now provided to rapidly isolate desired clones in antibody libraries and to manipulate individual Rab by genetic engineering. Thereby, the specificity and affinity of Rab can be designed to match the specific demands of food and environmental analysis. Two different sources of antibody genes, natural and synthetic ones, could be used to reach this goal. Natural antibody genes are isolated from nonimmunized or immunized donors to synthesize antibody libraries. The Rab of the former library are not biased toward a particular antigen, thus providing a highly diverse antibody repertoire. Since this library type corresponds to an early stage of lymphocyte development, the affinity of the Rab obtained is very low. Utilizing the antibody genes derived from immunized donors for the production of a library, the antibody repertoire is shifted towards the immunogen. However, these Rab are characterized by increased association constants. This is due to the affinity maturation of stimulated B-lymphocytes, which accompany the immunization process. Libraries on the basis of synthetic antibody genes are commonly using an existent antibody as a backbone. Diversity and refinement of the library is accomplished by varying the CDRs (Figure 1). CDR sequences can be randomized by means of P C R Synthetic primers are applied, which generate the whole repertoire of possible sequence variations. This repertoire is theoretically limited only by the length of the targeted sequence. The engineering of a single CDR region was first applied by Barbas et al (72). The complete heavy chain CDR3 was randomized in a length of 48 nucleotides to


38. KRAMER & HOCK

Recombinant Antibodies for Pesticide Immunoanalysis 481

Ligation of amplified variable regions with plasmid

CD

Γ Ρ

Plasmid pASK85 containing constant domains

\

ι Transformation of E. coli

ι

i

Subcloning of light chain fragment into heavy chain clone

v

H

c i

v

H

L

C

l

^ ' ^

\ Transformation of E. coli

Expression of Fab-fragments

Figure 4. Recombinant Fab synthesis using the approach of Skerra (9).


482


atrazine

Figure 5. Mab and recombinant Fab in the competitive, indirect ELISA.


38.

KRAMER & HOCK

Recombinant Antibodies for Pesticide Immunoanalysis 483 7

yield a library of 5xl0 clones. Since the representation of all possible sequence variations would necessitate more than 10 clones in this experiment, it is obvious that the potential size of the synthetic library exceeds the number of bacterial clones in the generated library by several orders of magnitude. This is because the achievable transformation rates in bacteria are limited by the available techniques, e.g. electroporation. If this restriction can be overcome, the extent of sequence variations included in a library will always surpass the possible variations of Rab specificities and affinities toward a defined target analyte. This is due to the fact that not every sequence variation generates structural changes, which are relevant for the immune reaction. Therefore, molecular modelling studies of structural binding sites will become an indespensible tool in antibody engineering. However, the above mentioned libraries provide a pool of antibody diversity from which particular clones are isolated for further affinity improvement by advanced genetic manipulation, e.g. chain shuffling (73) or CDR walking (32). Work is in progress to apply this strategy for the improvement and diversification of our Rab against .s-triazines. This approach takes advantage of the high level of optimization already reached by the first generation of our Rab. 20

Acknowledgments The authors wish to thank Dr. Arne Skerra for his advice in recombinant Fab production and for generously providing the pASK85-based Fab system. We thank the Deutsche Forschungsgemeinschaft for a grant (Ho 383/29-1). We are indebted to Dr. R. Beier and Dr. B. Hammock for reading the manuscript. Literature Cited 1. Winter, G.; Milstein, C. Nature 1991, 349, 293-299. 2. Irving, R.; Hudson, P. In Monoclonal antibodies. The second generation; Zola, H., ed.; BIOS Scientific Publishers Ltd.: Oxford, UK, 1995; pp 119-139. 3. Better, M.; Chang, C. P.; Robinson, R. R.; Horwitz, A. H. Science 1988, 240, 1041-1043. 4. Skerra, Α.; Plückthun, A. Science 1988, 240, 1038-1041. 5. Orlandi, R.; Gussow, D. H.; Jones, P. T.; Winter, G. Proc. Natl.Acad.Sci. USA 1989, 86, 3833-3837. 6. Sastry, L.; Alting-Mees, M.; Huse, W. D.; Short, J. M.; Sorge, J. Α.; Hay, B. N.; Janda, K. D.; Benkovic, S.J.;Lerner, R. A. Proc. Natl.Acad.Sci. USA 1989, 86, 5728-5732. 7. Larrick, J. W.; Danielsson, L.; Brenner, C. Α.; Abrahamson, M.; Fry, Κ. E.; Borrebaeck, C. A. K. Biochem. Biophys. Res. Commun. 1989, 160, 1250-1256. 8. Pack, P.; Kujau, M.; Schroeckh, V.; Knüpfer, U.; Wenderoth, R.; Riesenberg, D.; Plückthun, A. Bio/Technology 1993, 1271-1277. 9. Skerra, A. Gene 1994, 141, 79-84. 10. Neuberger, M.. S.; Williams, G. T.; Fox, R. O. Nature 1984, 312, 604-608. 11. Jones, P. T.; Dear, J. H.; Foote,J.;Neuberger, M. S.; Winter, G. Nature 1986, 321, 522-525.


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12. Barbas III, C. F.; Bain, J. D.; Hoekstra, D. M.; Lerner, R. A. Proc. Natl. Acad. Sci. USA 1992, 89, 4457-4461. 13. Marks, J. D.; Griffiths, A. D.; Malmqvist, M.; Clarkson, T. P.; Bye, J. M.; Winter, G. Bio/Technol. 1992, 10, 779-783. 14. Kabat, Ε. Α.; Wu, T. T.; Perry, H.; Gottesman, K. S.; Foeller, C. In Sequences of Proteins of Immunological Interest; US Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. 15. Ward, V. K.; Schneider, P. G.; Kreißig, S. B.; Hammock, B. D.; Choudary, P. V. Prot. Eng. 1993, 6, 981-988. 16. Karu, A. E.; Scholthof, K.-B. G.; Zhang, G.; Bell, C. W. Food Agric. Immunol. 1994, 6, 277-286. 17. Shelton, Α.; Graham, B.; Byrne, F.; Learmonth, D.; Porter, Α.; Harris, W. ICHEME Industrial Immunology, Biotechnology '94: Brighton, UK, 1994; pp 8-10. 18. Hock, B.; Dankwardt, Α.; Kramer, K.; Marx, A. Anal. Chim. Acta 1995, in press. 19. Kamps-Holtzapple, C.; Stanker, L. H., 209th ACS Meeting & Exposition: Anaheim, CA, 1995. 20. Karu, Ε. Α.; Harrison, R. O.; Schmidt, D. J.; Clarkson, C. E.; Grassman, J.; Goodrow, M. H.; Lucas, Α.; Hammock, B. D.; Van Emon, J. M.; White,R.J. In Immunoassays for Trace Chemical Analysis; Vanderlaan, M.; Stanker, L. H.; Watkins, Β. E.; Roberts, D. W., eds.; ACS Symposium Series 451, American Chemical Society: Washington, DC, 1991; pp 59-77. 21. Studier, F. W.; Rosenberg, A. H.; Dunn, J.J.;Dubendorf, J. W. Methods Enzymol. 1990, 185, 60-89. 22. Smith, G. P. Science 1985, 228, 1315-1316. 23. Huse, W. D.; Sashy, L.; Iverson, S. Α.; Kang, A. S.; Alting-Mees, H.; Burton, D. R.; Benkovic, J. S.; Lerner, R. A. Science 1989, 246, 1275-1281. 24. Hoogenboom, H. R.; Griffiths, A. D.; Johnson, K. S.; Chiewell, D.J.;Hudson, P.; Winter, G. Nucl. Acids Res. 1991, 19, 4133-4137. 25. Barbas III, C. F.; Lerner, R. A. In Methods: A Companion to Methods in Enzymology; Lerner, R. Α.; Burton, D. R., eds.; Academic Press: Orlando, FL, 1991; pp 119-124. 26. Barbas III, C. F.; Rosenblum, J. S.; Lerner, R. A. Proc. Natl.Acad.Sci. USA 1993, 90, 6385-6389. 27. Winter, G.; Griffiths, A. D.; Hawkins, R. E.; Hoogenboom, H. R. Annual Rev. Immunol. 1994, 12, 433-455. 28. Giersch, T.; Hock, B. Food Agric. Immunol. 1990, 2, 85-97. 29. Giersch, T. J. Agric. Food Chem. 1993, 41, 1006-1011. 30. Giersch, T.; Kramer, K.; Weller, M. G.; Hock, B. Acta Hydrochim. Hydrobiol. 1993, 21, 312-315. 31. Skerra, A. Gene 1994, 151, 131-135. 32. Barbas III, C. F.; Hu, D.; Dunlop, N.; Sawyer, L.; Cababa, D.; Hendry, R. M.; Nara, P. L.; Burton, D. R. Proc. Natl.Acad.Sci. USA 1994, 91, 3809-3813. RECEIVED July 31, 1995


Recombinant Antibodies for Pesticide Immunoanalysis - ACS

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