- Email: [email protected]
Antibody production: Low dose immunogen vs. low incorporation hapten using salmeterol as a model
a n a l y t i c a c h i m i c a a c t a 6 3 7 ( 2 0 0 9 ) 328–332
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/aca
Antibody production: Low dose immunogen vs. low incorporation hapten using salmeterol as a model Terence L. Fodey ∗ , Nuala M. Greer, Steven R.H. Crooks Agri-Food and Biosciences Institute, Veterinary Sciences Division, Stoney Road, Belfast BT4 3SD, UK
a r t i c l e
i n f o
a b s t r a c t
Article history:
Haptens are low molecular weight compounds that are non-immunogenic and so must be
Received 19 June 2008
conjugated to carrier molecules to elicit an immune response.
Received in revised form
Doses of 50–1000 g protein conjugate have been suggested for immunisation of rabbits
3 September 2008
with hapten–protein immunogens. Although larger doses may give a faster response, lower
Accepted 9 September 2008
doses may result in higher affinity antibodies. The amount of hapten presented to the host’s
Published on line 17 September 2008
immune system can be controlled by varying either the amount of immunogen administered or the quantity coupled to a fixed amount of protein. This study compares the two
Keywords: Antibody production
approaches for the production of antibodies to the -agonist salmeterol as a model. A range of salmeterol–HSA conjugates was prepared, with varying molar ratios of hap-
Immunogen dose
ten:protein (80:1, 15:1, 7.5:1, 4:1), for use as immunogens. The 80:1 immunogen was
Hapten incorporation
administered to different animals at four concentrations (0.5, 0.2, 0.1 and 0.05 mg dose−1 )
Affinity
while the remaining three immunogens were administered at 0.5 mg dose−1 . The effects
Titre
of immunogen dose and hapten incorporation on the titre and affinity of the antibodies produced to salmeterol were investigated. It was found that both approaches resulted in the production of more sensitive antibodies, although reducing the degree of hapten incorporation brought about a larger reduction in antibody titre. Reducing the degree of hapten incorporation also produced a more consistent pattern of results regarding antibody sensitivity (after the sixth immunisation in this study) which may make it easier to predict the most suitable time for antibody harvesting. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
The proficiency of an immunoassay used for the detection of a low molecular weight compound is usually gauged by its sensitivity to the analyte. The sensitivity of the assay is, in turn, conferred by the relative affinity (to the analyte) of the antibody employed. Compounds of low molecular weight (haptens) are not immunogenic and so do not induce the production of specific antibodies when introduced to the immune system of
a host animal; a weight of at least 5 kDa is required to elicit an immune response [1]. To overcome this problem a hapten must be conjugated to a carrier molecule, usually a protein, which is of sufficient mass to produce an immune response. When the compound has been conjugated to the carrier, the complex is usually referred to as an immunogen, and not an antigen, which is capable of binding to an antibody but not necessarily eliciting an immune response. The immunogen can be administered to the host at a range of doses, with respect to the amount of carrier protein. Doses
Abbreviations: ELISA, enzyme linked immunosorbent assay; HRP, horseradish peroxidase; HSA, human serum albumin. Corresponding author. Tel.: +44 28 90525784; fax: +44 28 90525840. E-mail address: [email protected] (T.L. Fodey). 0003-2670/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2008.09.025 ∗
329
a n a l y t i c a c h i m i c a a c t a 6 3 7 ( 2 0 0 9 ) 328–332
of 0.05–1 mg [2] and 0.1–0.5 mg [3] have been suggested for the immunisation of rabbits with immunogens prepared from hapten–protein conjugates. Although a larger dose may bring about a faster response it has been observed that lower doses result in the production of relatively higher affinity antibodies [4]. When the antigen interacts with cellular antibody the cells bearing it are stimulated to proliferate and differentiate into plasma cells. If antigen is present in critical concentrations it will stimulate those cells which bind it most efficiently. Large doses may stimulate cells bearing antibody of low affinity for long periods of time because of the persistence of a high concentration of the antigen [5]. The generation of antibodies with increasing affinity with time after immunisation (affinity maturation) is due to a change in the structure of the antibody being synthesised. This change is brought about by somatic mutation of germ-line encoded genes [6]. Furthermore, antigen-selected B cells first proliferate in germinal centres in an unmutated form and then hypermutate and die unless selected by antigen to differentiate further into plasma cells and memory cells [7]. Therefore while high dose immunisation gives rise to a larger accumulation of mutations, selection for high affinity antibodies is less stringent. Low dose immunisation promotes selection of a memory cell pool displaying a more focused set of mutations because the genes with the most relevant mutations are selected more efficiently [8]. The dose of antigen exposed to the immune system can be controlled through the amount of immunogen administered [4,5] but also by manipulation of the molar ratio of incorporated hapten to carrier [9]. A high degree of incorporation can lead to a stronger primary than secondary response generating antibodies of lower affinity. High titres of antibody with moderate affinity may be produced with intermediate hapten incorporation while a lower incorporation induces a slower response but with the production of higher affinity antibodies [9]. The aim of this study was to compare the two approaches of delivering a low dose of low molecular weight hapten to produce sensitive antibodies: (A) an immunogen was prepared for salmeterol with a 80× molar excess of hapten to carrier protein and administered to rabbits at a dose of 0.5 mg (controls), 0.2, 0.1 and 0.05 mg; (B) three further immunogens were prepared with molar excesses of 30×, 15× and 7.5× of hapten to carrier protein and administered at a dose of 0.5 mg.
2.2.
Procedures
2.2.1.
Preparation of salmeterol immunogens
The primary amine groups on HSA were modified using a suitable cross-linker before coupling to salmeterol xinafoate. Salmeterol was added to the protein at a range of molar excesses (80×, 30×, 15×, and 7.5×). All immunogen preparations were purified by dialysis against normal saline.
2.2.2.
Preparation of salmeterol enzyme label
An enzyme label was prepared by coupling salmeterol xinafoate to a modified form of HRP. Purification was achieved by gel filtration using a PD-10 Sephadex G-25 column with 1 mM sodium acetate solution.
2.2.3. Preparation of immunogenic emulsion using Montanide ISA 50V adjuvant The immunogen (in sterile saline) was added slowly to an equal volume of Montanide ISA 50V adjuvant with vortexing to produce an emulsion. The emulsions were thickened by passing them between two syringes connected by a narrow bored Perspex block.
2.2.4.
Immunisation and blood sampling of animals
The immunogens were administered at a range of doses as shown in Table 1. The rabbits were immunised every 4 weeks and the emulsions were introduced subcutaneously into four separate sites. Blood samples were collected 10 days after each immunisation.
2.2.5.
Assessment of antibody affinity by ELISA in buffer
Serum samples obtained from each animal after the second and subsequent immunisations were analysed by a competitive ELISA to determine IC50 s (half maximal inhibitory concentration) as a measure of antibody affinity. Antiserum (100 L) in 1 mM sodium acetate solution was coated onto a microtitre plate (Falcon; 353070) at a dilution which had been predetermined by a checkerboard titration and was allowed to incubate overnight at room temperature. The unbound antibody solution was tapped out before addition of 50 L of each salmeterol (base) standard (0, 1, 2, 10, 20, 50, 100 and 200 ng mL−1 ), in triplicate, in 1 mM sodium acetate solution. Horseradish peroxidase-labelled salmeterol (50 L) in blocking buffer (2 mg mL−1 bovine serum albu-
2.
Experimental
Table 1 – Molar excess of hapten:protein and dose of immunogen administered to each rabbit
2.1.
Reagents and chemicals
Rabbit
Salmeterol xinafoate (S-5068), salmeterol base (S-2692) and human serum albumin (HSA; A-1887) were obtained from Sigma (Poole, Dorset, UK) and horseradish peroxidase (HRP; 814407) was purchased from Roche Diagnostics (Lewes, East Sussex, UK). PD-10 Gel filtration columns (17-0851-01) were obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK) and the Montanide ISA 50V adjuvant was obtained from Seppic (Paris, France).
C1 C2 A1 A2 A3 B1 B2 B3
Hapten:protein molar excess 80:1 80:1 80:1 80:1 80:1 30:1 15:1 7.5:1
Dose of protein (mg) 0.5 0.5 0.2 0.1 0.05 0.5 0.5 0.5
330
a n a l y t i c a c h i m i c a a c t a 6 3 7 ( 2 0 0 9 ) 328–332
min in 1 mM sodium acetate solution) was also added at a dilution which had been predetermined by checkerboard titration. The plate was allowed to incubate overnight at 4 ◦ C. After washing with wash buffer (0.9% sodium chloride, 0.1% Tween 20), antibody bound peroxidase was reacted with the colorimetric substrate, 3,3 ,5,5 -tetramethylbenzidine (100 L, TMB/E; Chemicon International ES001). Colour development was stopped after 12 min by the addition of 2.5 M sulphuric acid (25 L) and the absorbance of each well was read at 450 nm on a Bio-Tek plate reader. The average absorbance of all the standard wells containing salmeterol was normalised, by expressing it as a percentage of the mean absorbance measured in the zero standard. The normalised response of each standard was then plotted against concentration to create a standard curve. The IC50 was calculated as the concentration of analyte that caused a 50% reduction in binding of the enzyme-labelled form of the analyte and was used as a comparative indicator of sensitivity.
2.2.6.
Assessment of antibody titre by ELISA in buffer
The antibodies obtained from each animal were also compared by their titre. A range of eight dilutions (1/1000–1/128,000) was prepared for each serum sample, obtained from each animal, after the second and subsequent immunisations. The diluted sera were coated onto a microtitre plate as already described. One dilution of enzyme label was chosen, after evaluation of checkerboard titrations, to use with all sera samples. The particular dilution of antibody that gave an absorbance of 0.5 was determined and plotted against time, for each rabbit, to observe changes in antibody titre over the course of immunisations.
3.
Results and discussion
When preparing an immunogenic conjugate of a low molecular weight compound coupled to a carrier protein, the extent of hapten incorporation is limited by the number of functional groups on the protein. In this study HSA was the chosen carrier protein and primary amino groups were used as the site of conjugation to salmeterol. HSA contains 59 lysine residues [10] plus the N-terminal amino acid to give a total of 60 primary amino groups per molecule, although not all of these will be available for derivatisation [11]. A molar excess of 80× was chosen to produce an immunogen with the highest incorporation of salmeterol. The remaining immunogens were prepared with lower molar excesses of salmeterol (30×, 15× and 7.5×). Two control rabbits were administered the immunogen with maximal salmeterol incorporation at a dose of 0.5 mg (HSA). Three rabbits received the same immunogen but at the lower doses of 0.2, 0.1, and 0.05 mg (HSA) as a means of reducing the amount of salmeterol presented to the immune system but with the consequential side effect of also reducing the amount of HSA presented. A further three rabbits were immunised with immunogens at the same dose as the controls (0.5 mg HSA) but produced with lower molar excesses of salmeterol over HSA.
3.1.
Antibody affinity
Antibody affinity was compared by calculating the IC50 s (Table 2) of the antisera from each rabbit after the second and subsequent immunisations. The two rabbits receiving 0.5 mg of the 80:1 immunogen (C1 and C2) produced their most sensitive antisera after five and seven immunisations respectively. The three rabbits receiving 0.2 mg (A1), 0.1 mg (A2) and 0.05 mg (A3) of the same immunogen produced their most sensitive antisera after eight, seven and seven immunisations respectively. Two of these antisera (A2 and A3) displayed lower IC50 s than those from the higher dose rabbits (C1 and C2). The rabbits immunised with the 30:1 (B1) and 15:1 (B2) hapten:protein immunogens provided, after eight immunisations, the most sensitive antibodies produced in the study. The final rabbit (B3), administered the 7.5:1 hapten:protein immunogen produced its highest affinity antibody after seven immunisations but it was the least sensitive antibody of the study. This was probably because the amount of salmeterol presented to the immune system had fallen below a critical level.
3.2.
Antibody titre
The anti-salmeterol antibody titres obtained from each rabbit over the course of the trial are shown in Fig. 1. The highest titres were obtained from the rabbits receiving immunogens with maximal incorporation of hapten, these titres being obtained after 5 (1:76,000 for C2), 6 (1:99,000 for C1; 1:96,000 for A1 and 1:69,000 for A3) or 7 (1:98,000 for A2) immunisations. A reduction in dose from 0.5 to 0.2 mg and 0.1 mg had little effect on the maximal titre of anti-salmeterol antibody obtained and only when reduced to 0.05 mg was a reduction observed. The rabbits receiving 0.5 mg of the immunogens with reduced salmeterol incorporation displayed correspondingly reduced maximal titres (1:20,000 for B1; 1:3000 for B2 and 1:<1000 for B3), a finding supported by previous studies [12,13]. It therefore appears that, when the concentrations of both hapten and carrier protein are reduced in the administered immunogen, the titre of antibody to the hapten remains relatively high contrasting with a substantial decrease in antibody titre when the concentration of hapten only is reduced.
3.3.
Affinity–titre correlation
The relationship between antibody affinity and titre is not proportional [13,14] and while rabbit A3 produced anti-salmeterol
Fig. 1 – Development of anti-salmeterol antibody titre with number of immunisations for each rabbit.
331
a n a l y t i c a c h i m i c a a c t a 6 3 7 ( 2 0 0 9 ) 328–332
Table 2 – IC50 s for antibodies from each rabbit after the second and subsequent immunisations IC50 s (ng mL−1 )
No. of immunisations
2 3 4 5 6 7 8
C1
C2
A1
A2
A3
B1
B2
B3
>200 82.2 28.1 7.9 10.6 11.4 21.3
168.0 78.4 17.6 17.6 14.4 6.6 10.6
>200 37.2 21.5 25.3 18.4 8.0 7.2
111.2 34.6 15.2 13.2 9.6 6.0 42.7
56.0 13.4 15.2 14.0 10.0 4.8 26.4
37.3 11.0 12.6 11.8 5.8 5.4 4.7
88.8 8.8 11.4 7.6 5.2 6.3 4.5
90.4 46.6 48.7 75.0 39.8 13.4 427
The antibody with optimum sensitivity for each rabbit is italized.
antibody titres lower than the other group A rabbits, it provided the most sensitive antibody of this group. This was corroborated in the study by the finding that rabbits B1 and B2 produced the most sensitive antibodies overall with relatively the lowest antibody titres (except B3); although still adequate for use in an immunoassay. For the screening ELISAs (particularly those used for the detection of prohibited and unauthorised substances) used in our laboratory, sensitivity, and not titre, is generally regarded as the most important criterion in governing the selection of an antibody for implementation into the statutory testing programmes. Only when an antibody is in very short supply and with a titre of <1:500 would the titre become problematic. Wring et al. [15] found that, when using three different carrier proteins (bovine serum albumin, bovine thyroglobulin and keyhole limpet hemocyanin) conjugated with the same hapten, there was an increase in antibody titre (to the hapten) corresponding to an increase in molecular weight of the protein. This is because the larger molecular weight proteins possess relatively more functional groups allowing for greater incorporation of hapten. However the affinities of the antibodies were “not discernibly different” because the number of moles of hapten per mole of protein were approximately equivalent for each immunogen. Therefore in order to improve antibody affinity, by reducing the concentration of hapten presented to the immune system, it is not sufficient to simply use a relatively lower molecular weight protein, instead the extent of hapten incorporation or the amount of hapten–protein immunogen administered must be reduced. When these two approaches were investigated in this study it was found that both led to the development of higher affinity antibodies (A3, B1 and B2) compared to the controls, but with a corresponding reduction in titre. The greater reduction in titre for the lower incorporation approach (B) was probably due to a reduction in the hapten:protein ratio which did not occur for group A. With a smaller proportion of the whole immunogen comprising of hapten, relatively fewer antibodies would be produced to it. This, in conjunction with the low concentration of hapten, may have led to the selective stimulation of B cells that bound it most efficiently. These cells would then differentiate into plasma cells secreting high affinity antibody. The higher concentration of protein in approach B maintains the immunogenicity of the complex while simultaneously reducing the hapten concentration; making approach B potentially more attractive with respect to producing high affinity antibodies as long as a high titre of antibody is not essential.
It was observed that the IC50 s obtained from the antisera taken after immunisations six, seven and eight varied more for group A than group B rabbits (excluding B3). The standard deviations of the IC50 s for the last three antisera samples were 6.2, 20.2 and 11.3 for A1, A2 and A3 respectively compared to 0.6 and 0.9 for B1 and B2 respectively. The consistency from the group B samples would allow the technician a larger time frame within which to harvest antisera with a superior affinity.
4.
Conclusion
Reducing the dose of an immunogen, fully incorporated with hapten, may bring about a small reduction in the titre of antibody but improve its sensitivity. However reducing the degree of hapten incorporation in the immunogen (up to a point) can also produce an improvement in sensitivity despite a relatively large reduction in antibody titre. An immunogen with a lower incorporation of hapten seems to produce a more consistent pattern of results regarding antibody sensitivity (after the sixth immunisation in this study) which may make it easier to predict the most suitable time for antibody harvesting.
Acknowledgement The authors would like to offer their gratitude to the Department of Agriculture and Rural Development for Northern Ireland (DARDNI) for its financial support.
references
[1] M.M. Kane, J.N. Banks, in: J.P. Gosling (Ed.), Immunoassays, Oxford University Press, Oxford, UK, 2000, p. 26. [2] E. Harlow, D. Lane, Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, New York, U.S.A., 1988, p. 101. [3] E. Liddell, in: D. Wild (Ed.), The Immunoassay Handbook, 2nd edition, Nature Publishing Group, London, UK, 2001, p. 122. [4] K.M. Cooper, A. Caddell, C.T. Elliott, D.G. Kennedy, Anal. Chim. Acta 520 (2004) 79. [5] E.A. Goidl, W.E. Paul, G.W. Siskind, B. Benacerraf, J. Immunol. 100 (1968) 371. [6] G.M. Griffiths, C. Berek, M. Kaartinen, C. Milstein, Nature 312 (1984) 271. [7] Y.J. Liu, D.E. Joshua, G.T. Williams, C.A. Smith, J. Gordon, I.C.M. Maclennan, Nature 342 (1989) 929. [8] A. González-Fernández, C. Milstein, Immunology 93 (1998) 149.
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[9] K.V. Singh, J. Kaur, G.C. Varshney, M. Raje, C.R. Suri, Bioconjug. Chem. 15 (2004) 168. [10] B. Meloun, L. Morávek, V. Kostka, FEBS Lett. 58 (1975) 134. [11] G.T. Hermanson, Bioconjug. Tech., Academic Press, San Diego, California, U.S.A., 1996, p. 423. [12] S. Kontiainen, Int. Arch. Allergy Immunol. 41 (1971) 729.
[13] M.K. Pedersen, N.S. Sorensen, P.M.H. Heegaard, N.H. Beyer, L. Bruun, J. Immunol. Methods 311 (2006) 198. [14] T.L. Fodey, P. Delahaut, C. Charlier, C.T. Elliott, Vet. Immunol. Immunopathol. 122 (2008) 25. [15] S.A. Wring, J.L. Williams, R.M. O’Neill, D.W. Ayres, W.N. Jenner, G.D. Bowers, I. Fellows, R.M. Carr, A.H. Wadsworth, D.R. Sutherland, Anal. Chim. Acta 334 (1996) 225.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/aca
Antibody production: Low dose immunogen vs. low incorporation hapten using salmeterol as a model Terence L. Fodey ∗ , Nuala M. Greer, Steven R.H. Crooks Agri-Food and Biosciences Institute, Veterinary Sciences Division, Stoney Road, Belfast BT4 3SD, UK
a r t i c l e
i n f o
a b s t r a c t
Article history:
Haptens are low molecular weight compounds that are non-immunogenic and so must be
Received 19 June 2008
conjugated to carrier molecules to elicit an immune response.
Received in revised form
Doses of 50–1000 g protein conjugate have been suggested for immunisation of rabbits
3 September 2008
with hapten–protein immunogens. Although larger doses may give a faster response, lower
Accepted 9 September 2008
doses may result in higher affinity antibodies. The amount of hapten presented to the host’s
Published on line 17 September 2008
immune system can be controlled by varying either the amount of immunogen administered or the quantity coupled to a fixed amount of protein. This study compares the two
Keywords: Antibody production
approaches for the production of antibodies to the -agonist salmeterol as a model. A range of salmeterol–HSA conjugates was prepared, with varying molar ratios of hap-
Immunogen dose
ten:protein (80:1, 15:1, 7.5:1, 4:1), for use as immunogens. The 80:1 immunogen was
Hapten incorporation
administered to different animals at four concentrations (0.5, 0.2, 0.1 and 0.05 mg dose−1 )
Affinity
while the remaining three immunogens were administered at 0.5 mg dose−1 . The effects
Titre
of immunogen dose and hapten incorporation on the titre and affinity of the antibodies produced to salmeterol were investigated. It was found that both approaches resulted in the production of more sensitive antibodies, although reducing the degree of hapten incorporation brought about a larger reduction in antibody titre. Reducing the degree of hapten incorporation also produced a more consistent pattern of results regarding antibody sensitivity (after the sixth immunisation in this study) which may make it easier to predict the most suitable time for antibody harvesting. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
The proficiency of an immunoassay used for the detection of a low molecular weight compound is usually gauged by its sensitivity to the analyte. The sensitivity of the assay is, in turn, conferred by the relative affinity (to the analyte) of the antibody employed. Compounds of low molecular weight (haptens) are not immunogenic and so do not induce the production of specific antibodies when introduced to the immune system of
a host animal; a weight of at least 5 kDa is required to elicit an immune response [1]. To overcome this problem a hapten must be conjugated to a carrier molecule, usually a protein, which is of sufficient mass to produce an immune response. When the compound has been conjugated to the carrier, the complex is usually referred to as an immunogen, and not an antigen, which is capable of binding to an antibody but not necessarily eliciting an immune response. The immunogen can be administered to the host at a range of doses, with respect to the amount of carrier protein. Doses
Abbreviations: ELISA, enzyme linked immunosorbent assay; HRP, horseradish peroxidase; HSA, human serum albumin. Corresponding author. Tel.: +44 28 90525784; fax: +44 28 90525840. E-mail address: [email protected] (T.L. Fodey). 0003-2670/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2008.09.025 ∗
329
a n a l y t i c a c h i m i c a a c t a 6 3 7 ( 2 0 0 9 ) 328–332
of 0.05–1 mg [2] and 0.1–0.5 mg [3] have been suggested for the immunisation of rabbits with immunogens prepared from hapten–protein conjugates. Although a larger dose may bring about a faster response it has been observed that lower doses result in the production of relatively higher affinity antibodies [4]. When the antigen interacts with cellular antibody the cells bearing it are stimulated to proliferate and differentiate into plasma cells. If antigen is present in critical concentrations it will stimulate those cells which bind it most efficiently. Large doses may stimulate cells bearing antibody of low affinity for long periods of time because of the persistence of a high concentration of the antigen [5]. The generation of antibodies with increasing affinity with time after immunisation (affinity maturation) is due to a change in the structure of the antibody being synthesised. This change is brought about by somatic mutation of germ-line encoded genes [6]. Furthermore, antigen-selected B cells first proliferate in germinal centres in an unmutated form and then hypermutate and die unless selected by antigen to differentiate further into plasma cells and memory cells [7]. Therefore while high dose immunisation gives rise to a larger accumulation of mutations, selection for high affinity antibodies is less stringent. Low dose immunisation promotes selection of a memory cell pool displaying a more focused set of mutations because the genes with the most relevant mutations are selected more efficiently [8]. The dose of antigen exposed to the immune system can be controlled through the amount of immunogen administered [4,5] but also by manipulation of the molar ratio of incorporated hapten to carrier [9]. A high degree of incorporation can lead to a stronger primary than secondary response generating antibodies of lower affinity. High titres of antibody with moderate affinity may be produced with intermediate hapten incorporation while a lower incorporation induces a slower response but with the production of higher affinity antibodies [9]. The aim of this study was to compare the two approaches of delivering a low dose of low molecular weight hapten to produce sensitive antibodies: (A) an immunogen was prepared for salmeterol with a 80× molar excess of hapten to carrier protein and administered to rabbits at a dose of 0.5 mg (controls), 0.2, 0.1 and 0.05 mg; (B) three further immunogens were prepared with molar excesses of 30×, 15× and 7.5× of hapten to carrier protein and administered at a dose of 0.5 mg.
2.2.
Procedures
2.2.1.
Preparation of salmeterol immunogens
The primary amine groups on HSA were modified using a suitable cross-linker before coupling to salmeterol xinafoate. Salmeterol was added to the protein at a range of molar excesses (80×, 30×, 15×, and 7.5×). All immunogen preparations were purified by dialysis against normal saline.
2.2.2.
Preparation of salmeterol enzyme label
An enzyme label was prepared by coupling salmeterol xinafoate to a modified form of HRP. Purification was achieved by gel filtration using a PD-10 Sephadex G-25 column with 1 mM sodium acetate solution.
2.2.3. Preparation of immunogenic emulsion using Montanide ISA 50V adjuvant The immunogen (in sterile saline) was added slowly to an equal volume of Montanide ISA 50V adjuvant with vortexing to produce an emulsion. The emulsions were thickened by passing them between two syringes connected by a narrow bored Perspex block.
2.2.4.
Immunisation and blood sampling of animals
The immunogens were administered at a range of doses as shown in Table 1. The rabbits were immunised every 4 weeks and the emulsions were introduced subcutaneously into four separate sites. Blood samples were collected 10 days after each immunisation.
2.2.5.
Assessment of antibody affinity by ELISA in buffer
Serum samples obtained from each animal after the second and subsequent immunisations were analysed by a competitive ELISA to determine IC50 s (half maximal inhibitory concentration) as a measure of antibody affinity. Antiserum (100 L) in 1 mM sodium acetate solution was coated onto a microtitre plate (Falcon; 353070) at a dilution which had been predetermined by a checkerboard titration and was allowed to incubate overnight at room temperature. The unbound antibody solution was tapped out before addition of 50 L of each salmeterol (base) standard (0, 1, 2, 10, 20, 50, 100 and 200 ng mL−1 ), in triplicate, in 1 mM sodium acetate solution. Horseradish peroxidase-labelled salmeterol (50 L) in blocking buffer (2 mg mL−1 bovine serum albu-
2.
Experimental
Table 1 – Molar excess of hapten:protein and dose of immunogen administered to each rabbit
2.1.
Reagents and chemicals
Rabbit
Salmeterol xinafoate (S-5068), salmeterol base (S-2692) and human serum albumin (HSA; A-1887) were obtained from Sigma (Poole, Dorset, UK) and horseradish peroxidase (HRP; 814407) was purchased from Roche Diagnostics (Lewes, East Sussex, UK). PD-10 Gel filtration columns (17-0851-01) were obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK) and the Montanide ISA 50V adjuvant was obtained from Seppic (Paris, France).
C1 C2 A1 A2 A3 B1 B2 B3
Hapten:protein molar excess 80:1 80:1 80:1 80:1 80:1 30:1 15:1 7.5:1
Dose of protein (mg) 0.5 0.5 0.2 0.1 0.05 0.5 0.5 0.5
330
a n a l y t i c a c h i m i c a a c t a 6 3 7 ( 2 0 0 9 ) 328–332
min in 1 mM sodium acetate solution) was also added at a dilution which had been predetermined by checkerboard titration. The plate was allowed to incubate overnight at 4 ◦ C. After washing with wash buffer (0.9% sodium chloride, 0.1% Tween 20), antibody bound peroxidase was reacted with the colorimetric substrate, 3,3 ,5,5 -tetramethylbenzidine (100 L, TMB/E; Chemicon International ES001). Colour development was stopped after 12 min by the addition of 2.5 M sulphuric acid (25 L) and the absorbance of each well was read at 450 nm on a Bio-Tek plate reader. The average absorbance of all the standard wells containing salmeterol was normalised, by expressing it as a percentage of the mean absorbance measured in the zero standard. The normalised response of each standard was then plotted against concentration to create a standard curve. The IC50 was calculated as the concentration of analyte that caused a 50% reduction in binding of the enzyme-labelled form of the analyte and was used as a comparative indicator of sensitivity.
2.2.6.
Assessment of antibody titre by ELISA in buffer
The antibodies obtained from each animal were also compared by their titre. A range of eight dilutions (1/1000–1/128,000) was prepared for each serum sample, obtained from each animal, after the second and subsequent immunisations. The diluted sera were coated onto a microtitre plate as already described. One dilution of enzyme label was chosen, after evaluation of checkerboard titrations, to use with all sera samples. The particular dilution of antibody that gave an absorbance of 0.5 was determined and plotted against time, for each rabbit, to observe changes in antibody titre over the course of immunisations.
3.
Results and discussion
When preparing an immunogenic conjugate of a low molecular weight compound coupled to a carrier protein, the extent of hapten incorporation is limited by the number of functional groups on the protein. In this study HSA was the chosen carrier protein and primary amino groups were used as the site of conjugation to salmeterol. HSA contains 59 lysine residues [10] plus the N-terminal amino acid to give a total of 60 primary amino groups per molecule, although not all of these will be available for derivatisation [11]. A molar excess of 80× was chosen to produce an immunogen with the highest incorporation of salmeterol. The remaining immunogens were prepared with lower molar excesses of salmeterol (30×, 15× and 7.5×). Two control rabbits were administered the immunogen with maximal salmeterol incorporation at a dose of 0.5 mg (HSA). Three rabbits received the same immunogen but at the lower doses of 0.2, 0.1, and 0.05 mg (HSA) as a means of reducing the amount of salmeterol presented to the immune system but with the consequential side effect of also reducing the amount of HSA presented. A further three rabbits were immunised with immunogens at the same dose as the controls (0.5 mg HSA) but produced with lower molar excesses of salmeterol over HSA.
3.1.
Antibody affinity
Antibody affinity was compared by calculating the IC50 s (Table 2) of the antisera from each rabbit after the second and subsequent immunisations. The two rabbits receiving 0.5 mg of the 80:1 immunogen (C1 and C2) produced their most sensitive antisera after five and seven immunisations respectively. The three rabbits receiving 0.2 mg (A1), 0.1 mg (A2) and 0.05 mg (A3) of the same immunogen produced their most sensitive antisera after eight, seven and seven immunisations respectively. Two of these antisera (A2 and A3) displayed lower IC50 s than those from the higher dose rabbits (C1 and C2). The rabbits immunised with the 30:1 (B1) and 15:1 (B2) hapten:protein immunogens provided, after eight immunisations, the most sensitive antibodies produced in the study. The final rabbit (B3), administered the 7.5:1 hapten:protein immunogen produced its highest affinity antibody after seven immunisations but it was the least sensitive antibody of the study. This was probably because the amount of salmeterol presented to the immune system had fallen below a critical level.
3.2.
Antibody titre
The anti-salmeterol antibody titres obtained from each rabbit over the course of the trial are shown in Fig. 1. The highest titres were obtained from the rabbits receiving immunogens with maximal incorporation of hapten, these titres being obtained after 5 (1:76,000 for C2), 6 (1:99,000 for C1; 1:96,000 for A1 and 1:69,000 for A3) or 7 (1:98,000 for A2) immunisations. A reduction in dose from 0.5 to 0.2 mg and 0.1 mg had little effect on the maximal titre of anti-salmeterol antibody obtained and only when reduced to 0.05 mg was a reduction observed. The rabbits receiving 0.5 mg of the immunogens with reduced salmeterol incorporation displayed correspondingly reduced maximal titres (1:20,000 for B1; 1:3000 for B2 and 1:<1000 for B3), a finding supported by previous studies [12,13]. It therefore appears that, when the concentrations of both hapten and carrier protein are reduced in the administered immunogen, the titre of antibody to the hapten remains relatively high contrasting with a substantial decrease in antibody titre when the concentration of hapten only is reduced.
3.3.
Affinity–titre correlation
The relationship between antibody affinity and titre is not proportional [13,14] and while rabbit A3 produced anti-salmeterol
Fig. 1 – Development of anti-salmeterol antibody titre with number of immunisations for each rabbit.
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a n a l y t i c a c h i m i c a a c t a 6 3 7 ( 2 0 0 9 ) 328–332
Table 2 – IC50 s for antibodies from each rabbit after the second and subsequent immunisations IC50 s (ng mL−1 )
No. of immunisations
2 3 4 5 6 7 8
C1
C2
A1
A2
A3
B1
B2
B3
>200 82.2 28.1 7.9 10.6 11.4 21.3
168.0 78.4 17.6 17.6 14.4 6.6 10.6
>200 37.2 21.5 25.3 18.4 8.0 7.2
111.2 34.6 15.2 13.2 9.6 6.0 42.7
56.0 13.4 15.2 14.0 10.0 4.8 26.4
37.3 11.0 12.6 11.8 5.8 5.4 4.7
88.8 8.8 11.4 7.6 5.2 6.3 4.5
90.4 46.6 48.7 75.0 39.8 13.4 427
The antibody with optimum sensitivity for each rabbit is italized.
antibody titres lower than the other group A rabbits, it provided the most sensitive antibody of this group. This was corroborated in the study by the finding that rabbits B1 and B2 produced the most sensitive antibodies overall with relatively the lowest antibody titres (except B3); although still adequate for use in an immunoassay. For the screening ELISAs (particularly those used for the detection of prohibited and unauthorised substances) used in our laboratory, sensitivity, and not titre, is generally regarded as the most important criterion in governing the selection of an antibody for implementation into the statutory testing programmes. Only when an antibody is in very short supply and with a titre of <1:500 would the titre become problematic. Wring et al. [15] found that, when using three different carrier proteins (bovine serum albumin, bovine thyroglobulin and keyhole limpet hemocyanin) conjugated with the same hapten, there was an increase in antibody titre (to the hapten) corresponding to an increase in molecular weight of the protein. This is because the larger molecular weight proteins possess relatively more functional groups allowing for greater incorporation of hapten. However the affinities of the antibodies were “not discernibly different” because the number of moles of hapten per mole of protein were approximately equivalent for each immunogen. Therefore in order to improve antibody affinity, by reducing the concentration of hapten presented to the immune system, it is not sufficient to simply use a relatively lower molecular weight protein, instead the extent of hapten incorporation or the amount of hapten–protein immunogen administered must be reduced. When these two approaches were investigated in this study it was found that both led to the development of higher affinity antibodies (A3, B1 and B2) compared to the controls, but with a corresponding reduction in titre. The greater reduction in titre for the lower incorporation approach (B) was probably due to a reduction in the hapten:protein ratio which did not occur for group A. With a smaller proportion of the whole immunogen comprising of hapten, relatively fewer antibodies would be produced to it. This, in conjunction with the low concentration of hapten, may have led to the selective stimulation of B cells that bound it most efficiently. These cells would then differentiate into plasma cells secreting high affinity antibody. The higher concentration of protein in approach B maintains the immunogenicity of the complex while simultaneously reducing the hapten concentration; making approach B potentially more attractive with respect to producing high affinity antibodies as long as a high titre of antibody is not essential.
It was observed that the IC50 s obtained from the antisera taken after immunisations six, seven and eight varied more for group A than group B rabbits (excluding B3). The standard deviations of the IC50 s for the last three antisera samples were 6.2, 20.2 and 11.3 for A1, A2 and A3 respectively compared to 0.6 and 0.9 for B1 and B2 respectively. The consistency from the group B samples would allow the technician a larger time frame within which to harvest antisera with a superior affinity.
4.
Conclusion
Reducing the dose of an immunogen, fully incorporated with hapten, may bring about a small reduction in the titre of antibody but improve its sensitivity. However reducing the degree of hapten incorporation in the immunogen (up to a point) can also produce an improvement in sensitivity despite a relatively large reduction in antibody titre. An immunogen with a lower incorporation of hapten seems to produce a more consistent pattern of results regarding antibody sensitivity (after the sixth immunisation in this study) which may make it easier to predict the most suitable time for antibody harvesting.
Acknowledgement The authors would like to offer their gratitude to the Department of Agriculture and Rural Development for Northern Ireland (DARDNI) for its financial support.
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