Hapten Versus Competitor Design Strategies for Immunoassay

Chapter 10

Hapten Versus Competitor Design Strategies for Immunoassay Development

Downloaded by UNIV OF NEW ENGLAND on February 12, 2017 | http://pubs.acs.org Publication Date: March 23, 1995 | doi: 10.1021/bk-1995-0586.ch010

Robert E. Carlson ECOCHEM Research, Inc., Suite 510, 1107 Hazeltine Boulevard, Chaska, MN 55318-1043

Separate design strategies for immunizing haptens and labeled competitor reagents are critical to the development of sensitive and specific immunoassays. Immunoassay development has generally focused on hapten and competitor designs which faithfully mimic the target analyte and vary mainly in the attachment site, chain length and functional groups of the spacer arm. We have investigated the concept that while the immunizing hapten defines assay specificity, it is the labeled competitor which determines sensitivity. Thus it is not an inherent assay development requirement that the labeled competitor substantially duplicate the analyte. The development of a polychlorinated biphenyl (PCB) immunoassay is described which uses a "core" heterology approach, in which the competitor is a structural fragment of the analyte, to maximize analyte sensitivity while retaining assay specificity. The basis of immunoassay is the competitive interaction of an antibody with the analyte and a labeled or conjugated derivative of the analyte (7). In the absence of analyte, the labeled or conjugated competition reagent is fully bound to the antibody. When analyte is present, the degree of competitor to antibody binding is reduced in proportion to the analyte concentration. This differential interaction results in a measurable colorimetric, fluorescent, etc. signal which is proportional to analyte concentration. HAPTEN DERIVED ANTIBODY ANALYTE CONCENTRATION -interaction produces->PROPORTIONAL SIGNAL LABELED COMPETITOR Thus, the properties of the assay depend on its key components: the hapten derived antibody and the labeled or conjugated competition reagent. To differentiate the labeled competitor reagent from the immunizing hapten and to reflect its role in the competition step of the assay, we refer to these materials as "competitor reagents" or "competitor conjugates". The simplest approach to assay development is to use the

0097-6156/95/0586-0140$12.00/0 © 1995 American Chemical Society

Nelson et al.; Immunoanalysis of Agrochemicals ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


10. CARLSON

Hapten Versus Competitor Design Strategies

141

immunizing hapten as the competitor in a homologous (competitor = hapten) assay format. However, it has long been recognized (2) that the preference of the antibody for the hapten usually results in significantly less than optimum analyte sensitivity. Consequently, heterologous assays utilize competitors which are closely related but non-identical to the hapten (2). The basic principle upon which our assay development program is based is that the hapten defines specificity while the competitor determines sensitivity. This approach suggests that each component plays a key role in the assay. The role of the hapten is to develop a binding pocket in the antibody which closely reflects the structure of the analyte. However, the sensitivity of the assay depends on the affinity of the antibody for the analyte in relation to the affinity of the antibody for the competitor. Thus hapten and competitor performance criteria are different. Consequently, hapten and competitor design should be approached as independent processes. METHODS Experimental details on the development of the Aroclor directed PCB immunoassay will be described in detail elsewhere (Carlson, R.E. et al., in preparation). Synthesis and Conjugation. Hapten synthesis utilized Cadogen (3) coupling of a trichloroaniline with a chloroanisole to produce a methoxytetrachlorobiphenyl intermediate which was converted to the hapten. Competitor synthesis was based on the addition of the anion of di- or trichlorotoluene to a 4-bromobutyrate synthon. Bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) conjugate preparation was based on l-ethyl-3-(3-dimethylaminopropyl)carbodiimide / nhydroxysuccinimide activation of the pendant carboxyl group. Antisera Development. Antisera were prepared in female New Zealand White rabbits using a KLH conjugate of the hapten. The immunization schedule used 300 ug of conjugate on day 1 in complete Fruend's adjuvant, 200 ug of conjugate in incomplete adjuvant on day 14, followed by 100 ug of conjugate in incomplete adjuvant on days 21, 28, 42, 56, 84, 112 etc. Rabbits were bled at 14 day intervals beginning on day 26. Serum samples were stored at -20° C until analyzed. Immunoassay. Standard enzyme linked immunosorbent assay (ELISA) procedures were followed (4). Microtiter plates (Dynatech Immulon-2) were coated with BSA hapten or competitor conjugates. Titer determinations were performed by incubating the coated wells with antisera diluted in a phosphate buffered saline buffer which contained BSA / dimethylformamide / Triton X-100. Bound antibody was measured using alkaline phosphatase labeled anti-rabbit IgG second antibody. The same format, with the addition of methanol solutions of the Aroclors or specific PCB congeners, was used to determine the analyte response of the assay. Reported part-per-million values reflect concentration of the analyte in the assay solution. RESULTS / PCB IMMUNOASSAY DEVELOPMENT An example of our approach to hapten and competitor design is the development of a polychlorinated biphenyl immunoassay. The PCBs are a class of chloroaromatics produced by the chlorination of biphenyl to give complex mixtures that can theoretically contain as many as 209 mono- to decachlorobiphenyl congeners (5).



142

IMMUNOANALYSIS OF AGROCHEMICALS

The commercial PCB formulations (e.g., Aroclor 1221, 1248, 1260) each contain fewer than 100 congeners at a concentration greater than 0.1 mole% (5-8). Consequently, for maximum sensitivity, a successful assay must be responsive to those congeners which are most prevalent in the common, target Aroclor formulations (i.e., Aroclors 1248, 1254 and 1260). Previous studies on the development of PCB immunoassays have been reported by Luster, et al. (9), Newsome and Shields (70), Franek, et al. (77) and Mattingly, et al. (72). None of these studies led to the development of a commercially successful assay. In particular, these assays exhibited marginally useful sensitivity and a strong preference for the immunizing hapten. The goal of this assay development program was to achieve useful Aroclor sensitivity. Moreover, the assay must be specific for PCBs and non-responsive to all other potential environmental co-contaminants. Hapten Design. Based on the notion that the hapten defines specificity, the design of the immunizing hapten should closely duplicate the structure of the analyte within the limits set by the incorporation of a linker moiety for immunizing conjugate preparation. Three factors were considered to be critical to the design of the PCB hapten: 1. Congener specific analysis (6-8) has shown that although 81 congeners are present at >0.1 mole% concentration in Aroclor 1254, only 13 of the 81 congeners are greater than 3.0 mole%. However, these 13 congeners comprise 66 mole% of this Aroclor. This suggests that a hapten can be designed which will represent the most prevalent congeners in the Aroclor mixtures. 2. The 2,4,5- substitution pattern and its 2,4- and 2,5- subsets represent a significant portion of the mole% composition of these complex Aroclor mixtures ( 6-8). For example, the six congeners with a common 2,4,5- substitution pattern (e.g., 2,2' ,4,4',5-pentachloro-, 2,2',4,4',5,5'-hexachloro-, 2,2',3,4,4',5,5'-heptachloro-) account for 32 mole% of Aroclor 1254. Addition of the three congeners which are 2,4or 2,5- substituted (e.g., 2,2',3,4,5'-pentachloro-) brings this total to 46 mole%. Furthermore, Figure 1 illustrates that the 6 congeners which are 2,4,5- substituted in one ring are dominated by 2,4- substitution in the second ring. Thus incorporation of the 2,4- / 2,5- / 2,4,5- substitution patterns would be expected to maximize mole% congener binding and assay response to the Aroclor analytes. 3. The dihedral angle between the phenyl moieties of a biphenyl derivative is dependent on the steric effect of the substituents at the 2,2', 6 and 6' (ortho) positions (73). PCB's which are 2,2', 6, and/or 6' substituted are not coplanar. The size of the dihedral angle between the phenyl groups is dependent on the degree of substitution. The 2,2'-chlorine substitution pattern that predominates in the target Aroclors results in a dihedral angle of about 70° (13). Clearly, this angle should be preserved in the design of the hapten. Incorporation of both the dominant patterns of chlorine substitution and the requirement for specific chlorine substitution at the critical 2 and 2' (ortho) positions of the biphenyl nucleus, directs the design of the immunizing hapten to incorporate the 2,4,5-/2',4'- substitution pattern. However, incorporation of a linker moiety for hapten conjugation requires modification of this pattern. Three factors were considered in this step of the design process: 1. The common 2,4,5- substitution pattern should be incorporated in unmodified form into one of the two phenyl moieties.



10. CARLSON


143

2. As discussed above, the presence of 2 chlorines in the 2,4,5-/2',4'substitution pattern which are ortho to the biphenyl bond establishes a particular value (ca. 70°) for the dihedral angle between the two phenyl rings (13 J4). Incorporation of the linker moiety into the 2,2',6 or 6'- positions would be expected to have a detrimental effect on analyte binding to the antibody and assay performance because of the potential for torsional angle and steric differences between the analyte and the hapten derived antibody binding pocket. 3. The functional group which serves as the attachment point between the linker and the phenyl group should be a good "chlorine mimic" (9) to increase the potential for recognition of congeners with chlorine substitution in the same position as the linker attachment site. These criteria led to the design of PCB/Hapten I (Figure 2). PCB/Hapten I incorporates both 2,4,5- and 2,2'- chlorine substitution with an ether moiety based linker as the chlorine mimic. The linker has been placed in the 4'- attachment site to minimize the impact of the linker on the biphenyl directed antibody binding pocket. In addition, the 4'- linker site is attractive because any flexibility in the antibody binding pocket due to linker steric requirements would be expected to improve cross-reaction within the target congener group (e.g., 2,4,5-/2',4'- versus 2,4,5-/2',5'- versus 2,4,5/2',4',5'-). Figure 3 allows comparison of a molecular model of PCB/Hapten-I with its analogous PCB congener, 2,2',4,4',5-pentachlorobiphenyl (14). The hapten duplicates the analyte, both in substitution pattern and spatially, except for the distal chlorine-to-ether linker moiety exchange. Figure 3 also compares the pentachloro congener with a representative, 2'-amide based hapten of the type used in prior PCB immunoassay development studies (9-11). The dihedral angle difference between the analyte, PCB/Hapten I and the amide hapten is relatively small (dihedral angles of 70°, 68° and 67° respectively ( 74)). However, comparison of the molecular model of the amide hapten and the model of the PCB congener clearly illustrates that the ortho positioned, sterically bulky amide linker would be expected to lead to the development of an antibody binding pocket which is different from the spatial requirements of the PCB congener. Homologous Assay Evaluation. A KLH conjugate of PCB/Hapten I produced a strong anti-hapten titer in rabbits. However, inhibition ELISA using a BSA conjugate of PCB/Hapten I as the coating conjugate gave a poor PCB response using Aroclor 1248 as the analyte. As shown in Table I, the minimum sensitivity of the assay for Aroclor 1248 was >10 ppm. Thus, this homologous (hapten as competitor) assay would not be sufficiently sensitive for the development of a useful environmental matrix PCB immunoassay. More importantly, Table I compares the sensitivity of the assay to PCB/Hapten I versus Aroclor 1248. The assay was significantly more responsive to the hapten than to the analyte. This result indicates that the hapten-KLH immunizing conjugate has successfully elicited a PCB hapten directed antibody and that the homologous assay is strongly biased toward the hapten's functional groups. These observations are consistent with: 1) prior PCB immunoassay development experience based on amide and ether haptens as described by Luster, et.al. (amide (9)), Newsome and Shields (amide (70)), Franek, et.al. (amide (77)) and Mattingly, et.al. (ether (72)) which all demonstrated strong anti-hapten assay bias, 2) the expectation, based on the subjective notion that an ether is a stronger epitope than the chloroaryl moiety, that the relative affinity of the antisera for the hapten ether moiety would be greater than its affinity for the chloroaryl moiety and 3) the accepted notion (2) that hapten designs which are optimum for the generation of an anti-hapten / antianalyte antibody often are not optimum as competitors for assay development.



144


RELATIVE CHLORINE 3 SUBSTITUTION

2

3 4 5 POSITION ON THE PHENYL RING

6

Figure 1. Relative Phenyl Substitution Pattern of the 2,4,5Substituted PCB Congeners. Bar height equals relative chlorine frequency among the 6 selected congeners. The 2-position is chlorinated in 5 of the 6 congeners, 3- in 3 of 6, 4- in 4 of 6, 5- in 2 of 6 and 6- in 1 of 6.

OCH2CH2CH2CH2CH2COOH

Figure 2.

Structure of PCB.Hapten-I.



145


10. CARLSON

Figure 3. Molecular Model Comparison of PCB Haptens and a PCB Congener.


146



Table I. Response of the Homologous ELISA to Hapten and PCB Analytes.

ANALYTE

110 (ppm)

PCB/HAPTEN I (R = -O-linker) AROCLOR 1248 (R = Cl ) n

0.18 11

I50 (ppm)

6.3 200 (estimated)

Heterologous Assay Development. The definitions of VanWeeman and Shuurs (2) summarize the principles of heterologous assay design: 1. Hapten Heterology - the competitor is structurally related, but not identical to the hapten. 2. Bridge Heterology - only the linking chain of the hapten is varied. 3. Site Heterology - only the attachment site of the linking chain to the hapten is varied. These definitions, particularly as systematically evaluated by Jung, et al. (75), Harrison, et al. ( 76), Jockers, et al (7 7) and Karu, et al. ( 78) form the current basis for the rational development of sensitive and specific immunoassays. Clearly, the optimum competitor should incorporate a combination of the standard hapten, linker and/or site heterologies (2,15-18) to maximize the relative difference in competitor versus analyte binding to the antibody (77). However, the strongly hapten biased response of tins assay suggests that modest enhancement in the interaction of the analyte with the antibody relative to a hapten derived heterologous competitor would not significantly improve on analyte sensitivity. For example, in Luster, et.al. (9), the assay was 8-fold more sensitive to a hapten analog than to a comparable PCB congener and » 1 0 - f o l d more sensitive to a hapten analog than to Aroclor 1248. It seemed probable that relatively minor variations in the competitor would not be sufficient to achieve the required improvement in sensitivity. "Core" Heterology Design. The development of heterologous competitors has traditionally focused on preserving the structure of the analyte (2,15-18). We hypothesized that although the design of the hapten must faithfully represent the analyte in order to produce an anti-analyte antibody binding pocket, the competitor need only bind to the antibody sufficiently to produce an adequate, analyte competitive assay signal. As long as the assay has an adequate signal, the most sensitive assays would



10. CARLSON


147

be expected to result from a competitor which has the lowest antibody affinity relative to the analyte. To achieve this goal, the competitor may only have to be a fragment of the analyte. We have termed this concept "Core Heterology". "Core" heterologous competitor design for the PCB assay was based on five factors: 1. A polychlorobenzene would be the logical core fragment for a polychlorinated biphenyl. (core heterology) 2. The functional group that serves as the attachment point between the linker and the phenyl group should not be an ether, (bridge heterology) 3. Linker chain length heterology should be incorporated through the use of a shorter spacer, (bridge heterology) 4. Various chlorine substitution patterns that mimic the "2,4,5-" pattern should be evaluated, (site, hapten heterology) 5. The site of linker attachment should be consistent with or in addition to the "2,4,5-" pattern, (site, hapten heterology) A variety of polychlorobenzene based competitors could be designed to satisfy these criteria. For example, the attachment functional group could be an amide, carbonyl, amine or methylene moiety. Additionally, the linker could incorporate amide or polyether groups and the aromatic nucleus could incorporate various 2,4-, 2,5- or 2,4,5- substitution patterns. However, we initially focused on alkyl based linkers and "2,4,5-" substitution as the most direct approach to incorporation of the above factors. This focus led the the synthesis of Competitors 1, 2 and 3 (Figure 4) and their evaluation in the ELISA format with Aroclor 1248 as the analyte. Table II compares antiserum titer and ELISA sensitivity for assays based on these competitors to the homologous hapten based assay. The titer results, which may be an indirect measure of antibody to competitor affinity, indicate the relatively weak antiserum binding of these competitors compared to the hapten. The improvement in assay sensitivity over the homologous system using these three competitors was 6- to 150-fold at the minimum detection limit (Ιχο) and 70 to 220-fold at the assay mid-point (I50). These results are consistent with our expectation that lower competitor titers will be reflected in a significant improvement in relative competitor versus analyte binding and, most importantly, analyte sensitivity. Assay Characterization. The results in Table II indicated that competitors 1, 2 or 3 could be used to develop a sensitive PCB immunoassay. However, the utility of the assay is dependent on both sensitivity and specificity. Three aspects of assay performance were evaluated, using the competitor 3 based ELISA, to define specificity: 1. The congener specificity data in Table Ilia shows that there is a general loss of cross-reactivity relative to 2,4,5-/2,4,5- substitution as the 4- and 5- chlorines are removed. Table Illb demonstrates the loss of cross-reaction relative to 2,4,5-/2,4,5substitution as ortho chlorination is altered through either the loss of 2- position chlorines or the addition of chlorines at the 6-position. These results indicate that the assay is responsive, as would be expected from the design of the immunizing hapten, to 2,4-, 2,5- and 2,4,5- substitution and ortho chlorination. 2. The Aroclor cross-reaction data in Table IV demonstrate that the assay is broadly responsive to the Aroclor 1242, 1248, 1254 and 1260 analytes. The response of the assay was within a factor of 2 for Aroclor 1242 to 1260, in agreement with the significance of 2,4- / 2,5- / 2,4,5- substitution in these congener mixtures. Crossreaction to Aroclor 1221 was only l/10th that of Aroclor 1248. This result is also in agreement with expectation because Aroclor 1221 is composed of congeners (e.g., 2monochloro, 4-monochloro, 2,4'-dichloro) which are

Hapten Versus Competitor Design Strategies for Immunoassay

Recommend Documents