Influence of hydrophobic and hydrophilic spacer-containing enzyme conjugates on functional parameters of steroid immunoassay

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ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 373 (2008) 18–25 www.elsevier.com/locate/yabio

Influence of hydrophobic and hydrophilic spacer-containing enzyme conjugates on functional parameters of steroid immunoassay Seema Nara a

a,b

, Vinay Tripathi a,b, Shail K. Chaube a, Kiran Rangari a, Harpal Singh b, Kiran P. Kariya c, Tulsidas G. Shrivastav a,*

Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi 110067, India b Center for BioMedical Engineering, Indian Institute of Technology, New Delhi 110016, India c Department of Chemistry, Vardhaman Mahavir College, Nagpur 444008, India Received 16 June 2007 Available online 4 November 2007

Abstract Introduction of spacers in coating steroid antigen or enzyme conjugates or immunogen is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We have introduced hydrophobic and hydrophilic spacers between enzyme and steroid moieties and studied their effects on functional parameters of enzyme immunoassays, using cortisol as a model steroid. Cortisol-3-O-carboxymethyloxime–bovine serum albumin (F-3-O-CMO-BSA) was used as immunogen to raise the antiserum in New Zealand white rabbits. Three enzyme conjugates were prepared using cortisol-21-hemisuccinate (F-21-HS) as carboxylic derivative of cortisol and horseradish peroxidase (HRP) as an enzyme label. These were F-21-HS-HRP (without spacer), F-21-HS-adipic acid dihydrazide-HRP (adipic acid dihydrazide as hydrophobic spacer), and F-21-HS-urea-HRP (urea as hydrophilic spacer). The influence of hydrophobic and hydrophilic spacers on the functional parameters of assays such as lower detection limit, ED50, and specificity was studied with reference to enzyme conjugate without spacer. The results of the present investigation revealed that the presence of a hydrophilic spacer in the enzyme conjugate decreases the lower detection limit, decreases the ED50, and marginally improves the specificity of assays. These improvements in functional parameters of assays may be due to the decreased magnitude of the overall hydrophobic interactions existing between the spacer in enzyme conjugate and the antigen binding site of the antibody.  2007 Elsevier Inc. All rights reserved. Keywords: Urea; ADH; Cortisol ELISA; Hydrophilic and hydrophobic spacer

The extraordinary specificity and high binding affinity interaction between antigen and antibody is the keystone for the development of sensitive and specific immunoassays. The molecular recognition process that occurs between an antibody and its antigen depends on the antibody binding site composition and spatial structure. However, this molecular recognition process depends also on the antigen molecular structure, especially, in enzyme immunoassays of hapten [1]. Considerable variations in the affinity, owing to the hapten structures, have been reported between the homologous and the heterologous *

Corresponding author. Fax: +91 11 26101623. E-mail addresses: [email protected], [email protected] (T.G. Shrivastav). 0003-2697/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.10.042

enzyme immunoassays (EIA)1 [2–6]. For homologous EIAs, with no difference in the hapten derivative used for conjugation with carrier protein (immunogen) to generate the antibody and enzyme to prepare the enzyme conjugate, less sensitivity has been observed due to the strong interactions between antibody and hapten coupled to enzyme.

1 Abbreviations used: ADH, adipic acid dihydrazide; BSA, bovine serum albumin; EDAC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; EIA, immunoassay; F-3-O-CMO-BSA, cortisol-3-O-carboxymethyloxime–bovine serum albumin; F-3CMO, cortisol-3-CMO; HS, cortisol-21-hemisuccinate; HRP, horseradish peroxidase; IgG, immunoglobulin G; LDD, lower detection dose; mAb, monoclonal antibody; NHS, N-hydroxysuccinimide; NRS, normal rabbit serum; OEG, oligoethylene glycol.

Influence of spacer-containing enzyme conjugates on steroid immunoassay / S. Nara et al. / Anal. Biochem. 373 (2008) 18–25

Steroids are being measured by immunoassay using the competitive inhibition principle. There are two kinds of assay formats, with either immobilized antigen or antibody. Incorporation of slight differences at the level of bridge, site, or hapten in coating steroid antigen or enzyme conjugate or immunogen is known to improve the sensitivity of steroid EIA. For the antigen-immobilized format, varying lengths of hydrophilic heterobifunctional linker6-aminocaproic acid were coupled by homologation between progesterone and ovalbumin to prepare coating steroid antigen, for colorimetric and surface plasmon resonance (SPR) biosensing end-point detection using an ELISA reader and BIAcore instrument, respectively [7]. The effect of increasing length of linker on assay sensitivity has been reported to improve in the SPR BIAcore system, whereas the sensitivity remained unaffected in conventional ELISA [8]. On the contrary, when progesterone was conjugated to ovalbumin with an oligoethylene glycol (OEG) linker to form protein conjugate, which was immobilized on a mixed self-assembled monolayer (mSAM) surface for SPR, signal enhancement by anti-rat immunoglobulin G (IgG) and IgG/nanogold (10 nm) reduced the required anti-progesterone monoclonal antibody (mAb) concentration and significantly improved the sensitivity of the assay [9]. Furthermore, when a 15-atom-long hydrophilic linker-OEG was coupled through the fourth position of progesterone and estradiol derivative and covalently immobilized on a dextran surface in the biacore biosensor, signal enhancement by anti-rat IgG and gold-streptavidin attached to biotinylated-mAb (10 and 20 nm) reduced the required mAb concentration and significantly improved the sensitivity of the assay [10,11]. The use of these long flexible chains of highly hydrophilic OEG linker facilitates the aqueous solubility of the steroid, thereby greatly improving the protein conjugation. Moreover, the use of a hydrophilic linker helps in projecting the steroid in the aqueous phase, leading to enhanced antibody binding signal and improved sensitivity of the assay. Further, the antibody formation is reduced if the OEG linkers are used in immunogen [12,13]. In the antibody-immobilized format, linkers have been coupled between steroid derivative and label protein to minimize the conventional bridge recognition effects and to overcome steric constraints between the two high molecular weight proteins, i.e., antibody and label [14–16]. A mass-action model has been proposed to theoretically predict such behavior of assays showing differential affinities [17]. Bridge heterology in steroid immunoassay improves the assay sensitivity if 125I, enzymes, and chemiluminescent compounds are used as label [18–21]. The effect of linkers in enzyme conjugates has been studied so far with respect to their increasing length. However, there is a relative dearth of literature discussing the effect of hydrophilicity of linkers on the assay sensitivity when incorporated in enzyme conjugates. In the present study, we have incorporated two homobifunctional molecules with different atomic lengths, and differential solubilities in water, as linkers in the enzyme

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conjugates. Adipic acid dihydrazide (ADH) and urea have been coupled with F-21-HS and HRP. In this study, we tried to explain the role played by the hydrophobic and hydrophilic nature of the spacers and how and in what manner this characteristic of the spacers is influencing the sensitivity and specificity of site heterologous assay of cortisol. Experimental Chemical and reagents All solvents, chemicals, and salts used in the present study are of analytical grade. Bovine serum albumin (BSA), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC), complete Freund’s adjuvant, gelatin, thimerosal and horseradish peroxidase (lot numbers 15H9520 and 28H7848) were purchased from Sigma Chemical Company (St. Louis, MO). All the steroids used for the synthesis and cross-reactivity were obtained from Sterloids Inc. (Newport, RI). Tetramethylbenzidine/H2O2 solution was purchased from Bangalore Genei (Bangalore, India). Microtiter plates were procured from Greiner, Germany. The buffers were as follows: A. coating buffer, the most frequently used buffer was 10 mM phosphate (10 mM PB), pH 7.0, containing 0.9% NaCl (10 mM PBS) and 0.1% NaN3; B. enzyme dilution buffer, 10 mM acetate buffer (10 mM AB), pH 5.6, containing 0.1% thimerosal and dextran T-70, 0.3% BSA, and 6 lg/ml of danazol; C. antibody dilution buffer, 10 mM PB containing BSA (2 g/L) and 0.01% thiomerosal as preservative; D. blocking or stabilizing buffer, 10 mM PB containing 0.9% NaCl, 0.2% BSA, 0.1% gelatin, thimerosal, dextran T-70, ethylene diaminetetraacetic acid:dipotassium salt (EDTA:K salt), and 0.01% gentamicin sulfate. Methodology Immunogen (F-3-CMO-BSA) preparation Cortisol-3-CMO (F-3CMO) was coupled with BSA by an N-hydroxysuccinimide ester method with modification, described elsewhere [22]. To 5 mg of F-3CMO, 200 ll each of dioxan and dimethylformamide was added. To this solution 100 ll of water containing 10 mg NHS and 20 mg EDAC was added; the reaction mixture was activated for 24 h at 4 C. Activated F-3CMO solution was added to the aqueous solution of BSA (1 mg/0.3 ml), vortex-mixed, and kept for 24 h at 4 C. The F-3CMO-BSA conjugate was dialyzed against 3–4 changes of water. The dialyzate was frozen, lyophilized, and kept at 4 C in aliquots of (1 mg) for immunization. Immunization The antibody against F-3CMO-BSA was generated in New Zealand white rabbits according to the Shrivastav et al. protocol [23]. An emulsion of 0.5 ml of Freund’s complete adjuvant in 0.5 ml of saline containing 1 mg of F-

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Influence of spacer-containing enzyme conjugates on steroid immunoassay / S. Nara et al. / Anal. Biochem. 373 (2008) 18–25

3CMO-BSA-immunogen was prepared freshly. The amount of 250 ll of emulsion was injected intramuscularly in each limb of New Zealand white rabbits. The intramuscular injections were repeated on 7, 14, and 21 days following the initial injection, followed by booster every 30 days thereafter. Blood was collected14 days after the first booster injection and every 30 days thereafter. Antiserum was collected after centrifugation at 2500 rpm for 10 min and stored at 30 C. Preparation of enzyme conjugate All the conjugation reactions were carried out as previously described by an active ester method with modification [22]. Without spacer (F-21-HS-HRP). To 5 mg of F-21-HS, 200 ll each of dimethylformamide and dioxan was added.

To this solution 100 ll of water containing 10 mg NHS and 20 mg EDAC was added; the reaction mixture was activated for 24 h at 4 C. Activated F-21-HS solution was added to the aqueous solution of 1 mg/ml HRP (lot number 15H9520) and kept at 4 C for 24 h. After incubation the reaction mixture was passed through a G-25 column, previously equilibrated with 10 mM PBS containing 0.1% thimerosal. The brown-colored fractions containing enzyme activity were pooled and, to it, 1% (w/v) of sucrose, ammonium sulfate, BSA, and an equal volume of ethylene glycol were added. The solution was kept at 30 C in aliquots for future use.

With spacer (F-21-HS-ADH-HRP and F-21-HS-ureaHRP). These conjugates were prepared according to the reaction scheme in Fig. 1.

Fig. 1. Reaction scheme for the preparation of F-21-HS-ADH-HRP and F-21-HS-urea-HRP.

Influence of spacer-containing enzyme conjugates on steroid immunoassay / S. Nara et al. / Anal. Biochem. 373 (2008) 18–25

(a) Coupling of spacers (ADH/urea) to HRP: To the 4 mg/200 ll aqueous solution of HRP (lot number 28H7848), NHS (8 mg) and EDAC (16 mg) were added and the reaction mixture was kept at 4 C for overnight to activate the carboxylic group of HRP. After activation, the reaction mixture was divided in to two portions. To one portion, 20 mg of ADH/ 300 ll of ammonium carbonate was added and to another portion 20 mg urea/300 ll of water was added. The solutions were kept at 4 C for 24 h to allow the HRP-CO-NH-spacer bond formation. The HRP-ADH and HRP-urea solutions were then dialyzed separately against 3–4 changes of water. (b) Coupling of F-21-HS to HRP-ADH and HRP-urea: To 4 mg of F-21-HS, 200 ll each of dioxan and dimethylformamide was added. Thereafter, 100 ll of water containing 10 mg NHS and 20 mg EDAC was added and the reaction mixture was kept for 24 h at 4 C for activation. After activation F-21-HS solution was divided in two portions, one portion was added to the HRP-ADH solution and another to HRP-urea solution. The reaction mixtures were vortex-mixed and incubated at 4 C for overnight. After incubation the reaction mixtures were passed through G-25 columns, previously equilibrated with 10 mM PBS containing 0.1% thimerosal. The brown-colored fractions containing enzyme activity were pooled and, to it, an equal volume of ethylene glycol, 1% (w/v) of sucrose, ammonium sulfate, and BSA was added. The solutions were kept at 30 C in aliquots for future use. Checkerboard assay Coating of microtiter plates. The 96-well microtiter plates were coated using the immunobridge technique for primary antibody immobilization described elsewhere [24]. In brief, 250 ll of the Normal rabbit serum (NRS) diluted (1:250) in buffer A was dispensed into each well and incubated at 37 C overnight. Following incubation, the plate was washed under running tap water. To the NRS-coated wells, 250 ll of 1:1000 diluted goat anti-rabbit gamma globulin (ARGG) was added and incubated for 2 h at 37 C. After incubation, the contents of the plate were decanted and washed under running tap water. To the ARGG-coated microtiter plates, 200 ll of serially diluted (1:500, 1:1000, 1:2000, and 1:4000) F-3-CMO-BSA antiserum in buffer C was dispensed (one dilution per 8 wells). For nonspecific binding 200 ll of buffer C was added in the separate 8-well strip. The plate was left at 37 C for 2 h. Unadsorbed antibody was then washed off and 250 ll of buffer D was then added to block the unoccupied sites of the plate. The plate was kept at 37 C for 1 h. The contents were decanted and the plate was dried at room temperature (RT) and kept at 4 C for future use. Determination of optimum dilution of antibody and enzyme conjugates. To determine the amount of immobilized primary antibody and enzyme conjugates required to develop the assay, 100 ll of serially diluted (1:500, 1:1000, 1:2000,

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and 1:4000) three enzyme conjugates (F-21-HS-HRP, F-21-HS-ADH-HRP, and F-21-HS-urea-HRP) were added in the above coated plates for respective assays (one dilution per two wells in vertical fashion). The plates were incubated at RT for 1 h. Unbound contents were washed off and the bound enzyme activity was measured by adding 100 ll of TMB/H2O2 substrate to each well and incubated at RT for 15 min. The reaction was stopped by adding 100 ll of 0.5 M H2SO4 and the color intensity was measured at 450 nm in a Tecan-spectra ELISA plate reader. The dilutions of antiserum and enzyme conjugate showing maximum zero binding and least nonspecific binding were selected for each combination for assay development. Standard preparation A parent stock solution of cortisol was prepared in ethanol and kept at 30 C. A working stock of 600 lg/dl was made after air drying the desired amount of parent stock and reconstituting it in buffer A. The eight standards of 0, 0.5, 1, 3 10, 30, 60, and 90 lg/dl were made from the working stock in same buffer and kept at 4 C for further use. ELISA procedure To the antibody-coated wells, 25 ll of cortisol standards (0–90 lg/dl) was added along with 100 ll of suitably diluted F-21-HS-HRP/F-21-HS-ADH-HRP/F-21-HSurea-HRP enzyme conjugates in duplicate. The plate was further processed as described under determination of optimum dilution of antibody and enzyme conjugates. Recovery To 10-ml aliquots of six serum pools, we added 0, 0.5, 1, 2, 4, and 6 lg of cortisol (corresponds to 0, 5, 10, 20, 40, and 60 lg/dl). The percentage recovery is calculated as O/ E ·100, where O is the observed concentration and E is the sum of the amount of analyte added and inherent concentration in the matrix. Precision Serum pools of low, medium, and high cortisol concentrations were utilized for determining the level of imprecision in assay by estimating cortisol in each pool for six times in assay and in six different assays. The percentage coefficient of variation is calculated as (SD/mean ·100). Correlation coefficient The correlation coefficient was calculated by comparing serum cortisol concentrations as determined by developed ELISA with an established radioimmunoassay (RIA) kit (Immunotech, France). Results Dose–response study The dose–response studies of the three enzyme conjugates F-21-HS-HRP, F-21-HS-ADH-HRP, and F-21-HS-

22

Influence of spacer-containing enzyme conjugates on steroid immunoassay / S. Nara et al. / Anal. Biochem. 373 (2008) 18–25

urea-HRP were carried out with the antibody raised against F-3CMO-BSA. Fig. 2 depicts the composite standard curves of six assays each, using three enzyme conjugates, where concentrations of cortisol are plotted on the x axis and bound fraction (A/A0%) on the y axis. The slope and intercept of the curves were calculated by logit-log transformation of the data.

Table 1 Slope, intercept, ED50 and lower detection dose of cortisol assay, using F-3-CMO-BSA antibody in combination with different F-21-HS-enzyme conjugates Enzyme conjugate

Lower detection ED50 ± SD dose (ng/ml) (ng/ml)

F-21-HS-HRP 17.7 ± 0.4 F-21-HS-ADH-HRP 0.740 ± 0.04 F-21-HS-urea-HRP 0.520 ± 0.01

Slope (m)

Intercept (c)

303.6 ± 0.03 1.86 2.76 102.0 ± 0.02 1.33 1.34 68.20 ± 0.06 1.42 1.19

Sensitivity The assay sensitivity is usually expressed in terms of its lower detection dose (LDD) and the effective displacement at 50% (ED50). The LDD is the lowest concentration of analyte (A) giving a response statistically different from that observed in the absence of analyte (A0). It is calculated as A0 – 2·SD, after 20-fold determination of A0. The ED50 is the effective concentration at which 50% of inhibition in the binding of enzyme conjugates occurs in assays in the presence of analyte. It is calculated as ED50 ± SD, after 20 times determination of ED50. Table 1 represents the LDD and the ED50 of the three assays. The insertion of urea bridge in the enzyme conjugate has improved the LDD to 0.520 ng/ml and the ED50 to 68.2 ng/ml.

Specificity The specificity of the F-3CMO-BSA antibody was estimated as the percentage of cross-reaction with 49 commercially available C18, C19, C21, and C27 steroids of analogous molecular structure. Table 2 presents percentage crossreactivity of cross-reactants in respective assays. The incorporation of hydrophilic spacer in the enzyme conjugate has decreased the degree of cross-reaction as compared to the reference assay (F-21-HS-HRP) and that having the hydrophobic spacer in the label. Since the assay with F-21-HS-urea-HRP gave better sensitivity and specificity, this combination was further studied for analytical variables like recovery, precision, and correlation coefficients with RIA. Recovery Table 3 represents the recovery profile of the assay that is in the range of 89.7–97.5%. Precision Table 4 depicts the inter- and intraassay variation. The coefficient of variation (CVs) of three serum pools for Table 2 The percentage cross-reaction of main cross reactants in cortisol assay using F-3-CMO-BSA antiserum with F-21-HS-HRP, F-21-HS-ADHHRP and F-21-HS-urea-HRP enzyme conjugates Cross reactant

F-21-HSHRP

F-21-HS-ADHHRP

F-21-HSurea-HRP

Dexamethasone Corticosterone Progesterone Cortisone 17a-OH-progesterone

0.8 3.9 0.63 11.2 17.5

1.6 0.3 <0.1 10.6 12.3

0.8 0.45 <0.1 6.8 11.6

Table 3 Recoveries of cortisol from exogenously spiked pooled serum

Fig. 2. Dose–response curves of cortisol using F-3-CMO antibody and F21-HS-HRP, F-21-HS-ADH-HRP, and F-21-HS-urea-HRP enzyme conjugates. Data points are mean ± SD of 6 assays (in duplicate). The CV at each concentration is shown in parentheses.

Serum pools

Cortisol Cortisol Cortisol %Recovery added (lg/dl) observed (lg/dl) expected (lg/dl)

Pool Pool Pool Pool Pool Pool

— 5.0 10.0 20.0 40.0 60.0

A B C D E F

6.3 10.3 14.59 23.01 44.2 58.6

— 11.3 15.3 25.3 45.3 65.3

— 91.1 95.3 90.9 97.5 89.7

Influence of spacer-containing enzyme conjugates on steroid immunoassay / S. Nara et al. / Anal. Biochem. 373 (2008) 18–25 Table 4 Inter- and intra-assay variation of cortisol of three internal control samples as estimated by developed ELISA Sample Pool

Low Medium High

Intra-assay variation

Inter-assay variation

Mean

Standard deviation (n = 6)

%CV

Mean

Standard deviation (n = 6)

%CV

13.92 20.61 24.72

0.7 0.4 1.3

5.0 1.9 5.2

13.91 20.58 23.42

0.74 1.0 1.22

5.3 4.8 5.2

Fig. 3. Regression graph of correlation between the serum cortisol concentrations as estimated by the developed ELISA and an established RIA kit (plotted by Prism 3 software).

intra- and interassay variation (n = 6, replicate of each pool) was <5.3%. Correlation coefficient The correlation coefficient for values of cortisol in human serum samples (n = 65) measured both by RIA kit (Immunotech, France) and ELISA using F-21-HSurea-HRP was calculated and found to be 0.95, i.e., r = 0.95. The linear regression curve of the correlated data (plotted by GraphPad Prism version 3.00 for Windows, GraphPad Software, San Diego, CA) is given in Fig. 3. Discussion We described for the first time the use of urea as a novel spacer in enzyme conjugates. Three enzyme conjugates were prepared using F-21-HS as a carboxylic derivative of cortisol and HRP as an enzyme label. These were F21-HS-HRP (without any spacer), F-21-HS-ADH-HRP (ADH as hydrophobic spacer), and F-21-HS-urea-HRP (urea as hydrophilic spacer). The influence of hydrophobic and hydrophilic spacers on the functional parameters of site heterologous cortisol ELISA was studied with reference to the assay with no spacer in the enzyme conjugate. In the present study, the conjugation reactions have been carried out using two batches of HRP, having the lot numbers 15H9520 and 28H7848, from Sigma Chemical

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Company. The HRP with former lot number was able to couple with the carboxylic derivative of steroid through NHS-mediated carbodiimide reaction. However, the HRP with latter lot number was unable to couple because of the nonavailability of –NH2 groups. This nonavailability of amino group in the second batch of HRP may be due to changes in extraction conditions employed during purification of HRP from the roots of the horseradish plant. During its purification allyl isothiocyanates are released, which form stable thioureas with the enzyme’s amine group, making the enzyme unreactive toward the activated steroid carboxylic derivative [25]. Changes in extraction conditions are likely to lead to batch-to-batch variation in the number of reactive amines that survive the process. Such differences in amine availability have been observed, which makes it difficult to establish reaction conditions, which can be used for more than one batch of HRP. ADH is a 10-atom homobifunctional molecule having a long aliphatic chain in it. The insertion of ADH in enzyme conjugates has increased the sensitivity of the site heterologous assay (F-3-CMO-BSA/F-21-HS-HRP) from 17.7 to 0.740 ng/ml and decreased the ED50 from 303.6 to 108.2 ng/ml. On the contrary, urea is only a 3-atom-long molecule and has achieved a sensitivity of 0.52 ng/ml and ED50 of 68.2 ng/ml. This differential behavior of ADH and urea linkers toward the sensitivity and ED50 of assays might be due to the difference in the magnitude of overall forces of attraction between the antibody and the enzyme conjugates. Wilson and co-workers have detailed the threedimensional structure of the antisteroid Fab 0 fragment, which predominantly consists of hydrophobic residues like TrpH47, TrpH50, and PheH100b [26]. Since, we have used polyclonal antibodies in contrast to the monoclonal Fab 0 fragment of Wilson et al., different amino acid sequences could constitute the Fab 0 fragment owing to the varying affinity constants of a mixture of antibodies in polyclonal serum. But, as the steroid molecule is basically a nonpolar structure, hydrophobic bonds are formed between the large areas of steroid and the aliphatic and aromatic side chains of the protein [27]. Therefore, the aliphatic and aromatic amino acids are likely to constitute the Fab 0 fragment. It may be suggested that the long and flexible aliphatic chain of the ADH molecule in the label is also contributing toward these interactions with the antibody, whereas the urea molecule being nonaliphatic, hydrophilic, and rigid in nature due to the presence of double bond in it lacks these interactions. The present finding suggests that the nature of the spacer (hydrophilic, hydrophobic, flexible, or rigid) is related to assay sensitivity and not to spacer length. It has also been previously reported that the spacer length does not bear any correlation with the assay sensitivity in ELISA [8,25] as it does have an effect in some other immunoassay and auxiliary binding systems. The specificity of the steroid immunoassays is mainly determined by the coupling position in the steroid moiety

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Influence of spacer-containing enzyme conjugates on steroid immunoassay / S. Nara et al. / Anal. Biochem. 373 (2008) 18–25

during immunogen preparation. The heterologous site labeling is believed to increase the sensitivity but at the cost of specificity [28–30]. In our heterologous assay system (no spacer in enzyme conjugate) there were 11.5 and 17.2% cross-reactions with cortisone and 17-OH-P, respectively. The specificity of this site heterologous assay improved to some extent with incorporation of the spacer in label. Moreover, the percentage cross-reaction for cortisone and dexamethasone is relatively less with urea as compared to ADH bridge in the label. This decrease in the percentage cross-reaction could be explained on the basis of some difference in the orientations of the two labels in the antibodybinding pocket. The cross-reaction pattern depends on how good is the shape complementarity between the analogous steroid and the antibody-binding pocket, in the presence of the specific antibody-ligand interactions [31,32]. It could be suggested that the 3-D orientation of F-21-HS-urea-HRP in the Ag-Ab complex is such that either it is masking some of the amino acid residues of the binding pocket or is preventing the cross-reacting steroid from interacting with these residues due to a nonaliphatic and rigid nature because of double bond in it. In the present study, we have used danazol as a steroid displacer to improve the assay recovery. The steroids like danazol and dexamethasone have high affinities for corticosteroid binding globulin (CBG) and have been used to displace major steroids (cortisol/progesterone) from CBG [33]. Moreover, these displacing agents also prevent the binding of labeled antigen with the plasma protein. The disadvantage of the use of these displacing agent is that in their blocking concentration, some of them reduce the specific binding of the antigen with antibody. In some cases, these displacing agents also cross-react with the antibody. In several cases a mixture of different nonrelated steroids is also used as displacing agents in direct assays of steroids like testosterone and estradiol. These exogenous steroids are potential sources of cross-reaction. The complex mixtures of these displacing agents in direct immunoassay interfere with the performance of the assay and the sensitivity is affected [34]. From present study, we conclude that the physicochemical nature of the bridge is determinental because of the assay parameters and not the spacer length. Urea being a biomolecule, cheaply available, and hydrophilic and rigid in nature could be tried for other steroids/hapten assays and its role as a spacer needs to be studied more deeply at the structural level. Future studies could also be designed using other more hydrophilic molecules such as oligoethylene glycol as spacers in enzyme conjugates. Acknowledgments This study was supported by the National Institute of Health and Family Welfare, New Delhi, India. We are grateful to Prof. Deoki Nandan, Prof. N. K. Sethi, and Prof. K. Kalaivani for their keen interest and encouragement throughout the study.

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