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Technical Briefs |
1 Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi-110067, India
2 Department of Chemistry, Vardhamon Mahovir College, Nagpur-444008, India
aauthor for correspondence; fax 91-11-26101623, e-mail mrbasu{at}rediffmail.com
The direct coupling of the carboxylic derivative of steroids to the amino group of enzymes is a well-established method in steroid enzyme immunoassays for making enzyme conjugates (1). Horseradish peroxidase (HRP), containing six lysine residues in the sequence, is a widely used enzyme in enzyme immunoassays; in practice, however, only one or two of these are generally available for reaction (2). This variation in amino group content is caused by changes in the extraction conditions used for the isolation of HRP from the roots of the horseradish plant (2)(3). The low yield of HRP coupled to the IgG by the use of bifunctional reagents, namely glutaraldehyde, carbodiimide, cyanuric chloride, bis-diazotized O-dianisidine, and P-P'-difluoro-m, m-dinitro-diphenyl sulfone (FNPS), and so forth, prompted Nakane and Kawaoi to investigate another method (periodate method) for the conjugation of HRP to IgG (4). Comparative coupling efficiency studies were carried out with the use of glutaraldehyde, periodate, and N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) as cross-linking reagents (5)(6) for the preparation of HRP-IgG conjugates. These studies revealed that the most efficient HRP-IgG conjugate was obtained by the periodate method. In practice, the difference in amino group availability in different batches of commercial preparations of HRP makes it difficult to establish standard reaction conditions that could be applicable for more than one batch. Therefore, adipic acid dihydrazide (ADH) has been coupled to HRP with the use of the periodate method reaction to provide the necessary amino group for the preparation of hapten enzyme conjugate with the use of the carbodiimide method.
We describe for the first time the use of ADH as a linking reagent between glycoenzyme (HRP) and a steroid carboxylic derivative to prepare enzyme conjugate for ELISA. The conjugation of HRP to cortisol through ADH as the link is described below.
To 10 mg of HRP (HRP type VI, lot no. 28H7848; Sigma Chemical Company; HRP of this lot failed to conjugate by the direct method) per 1 mL of water, 10 µL of freshly prepared 0.1 M aqueous solution of sodium meta-periodate was added to oxidize the vicinal hydroxy group of carbohydrate moieties of HRP to generate HRP-aldehyde (Fig. 1A
). The reaction mixture was vortex-mixed and kept at room temperature for 30 min in the dark. Activated HRP (HRP-aldehyde) was passed through a Sephadex G-25 column previously equilibrated with 10 mM ammonium carbonate, pH 9.3. The brown colored fraction of activated HRP was directly collected in a vial containing 100 mg of ADH; the reaction mixture was vortex-mixed and kept overnight at 4 °C to form a hydrazone bond (Fig. 1B
). After overnight incubation, 10 µL of 5 M sodium cyanoborohydride in 1 mol/L NaOH was added, reaction mixture was vortex-mixed and kept for 3 h at 4 °C. The reaction mixture was passed through a Sephadex G-25 column previously equilibrated with 10 mM PBS. The brown-colored fraction containing HRP-ADH conjugate was collected and kept at -30 °C for future use. Cortisol-21-hemisuccinate (F-21-HS), procured from Sigma Chemical Company, was conjugated to HRP-ADH as described below. To 5 mg of F-21-HS, 200 µL of dimethyl formamide, 200 µL of dioxan, and 100 µL of distilled water containing 10 mg of N-hydroxysuccinimide and 20 mg of 1-ethyl-3-(3-dimethyl-amino-propyl) carbodiimide-HCL were added; the reaction mixture was vortex-mixed and kept overnight at 4 °C. After overnight incubation, the activated steroid reaction mixture was added to 1 mL (
1 mg of enzyme) of ADH-HRP solution. Reaction mixture was vortex-mixed and incubated for 24 h at 4 °C to form the diimide bond (Fig. 1C
). The conjugate was passed through Sephadex G-25 column. The fractions containing enzyme activity were pooled, and 1 g/L sucrose, ammonium sulfate, and BSA were added. To the above conjugate solution, an equal volume of ethylene glycol was added and kept at -30 °C in aliquots for future use.
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We compared the displacement ELISAs using F-21-HS-ADH-HRP and F-21-HS-HRP. Therefore, HRP (HRP type VI, lot no. 16H9520 from Sigma) was conjugated directly with F-21-HS as follows. To 5 mg of F-21-HS, 200 µL of dimethyl formamide, 200 µL of dioxan and 100 µL of distilled water containing 10 mg of N-hydroxysuccinimide and 20 mg of 1-ethyl-3-(3-dimethyl-amino-propyl) carbodiimide–HCL were added; reaction mixture was vortex-mixed and kept overnight at 4 °C. One mg of HRP was dissolved in 1 mL of distilled water. Activated steroid solution was added to aqueous solution of HRP and was incubated for 24 h at 4 °C. The conjugate was passed through a Sephadex G-25 column. The fractions containing enzyme activity were pooled and 1 g/L sucrose, ammonium sulfate, and BSA were added. To the above conjugate solution, an equal volume of ethylene glycol was added and kept at -30 °C in aliquots for future use.
The study was approved by the Institute’s animal ethics committee. Cortisol antiserum was generated against cortisol-3-O-CMO-BSA as immunogen in New Zealand White rabbits (7). Microtiter plates were coated with cortisol-3-O-CMO antibody, diluted in 10 mM PBS (8). F-21-HS-ADH-HRP or F-21-HS-HRP was diluted in 10 mM sodium acetate buffer, pH 5.6, containing 1 g/L dextran T-70, thimerosal, sodium salicylate, and 3 g/L BSA. Six cortisol calibrators (0 µg/100 mL, 1 µg/100 mL, 3 µg/100 mL, 10 µg/100 mL, 30 µg/100 mL and 60 µg/100 mL) were prepared in stripped serum (7). To the cortisol antibody-coated wells, 25 µL of cortisol calibrators or samples along with 100 µL of either F-21-HS-ADH-HRP conjugate or F-21-HS-HRP enzyme conjugate were added in all of the wells and incubated for 1 h at 37 °C. After incubation, the contents of the wells were flicked out and washed in running tap water. To measure the bound enzyme activity, 100 µL of substrate solution (tetramethyl benzidine/H2O2, Bangalore Genei, India; 1:20 diluted in water) was added to all the wells and was incubated for 20 min at 37 °C. The enzyme reaction was stopped by adding 100 µL of 0.5 M H2SO4, and the color intensity was measured at 450 nm. The logit-log transformation of the calibration curve of cortisol ELISA yielded the following equations:
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The lower detection limit was calculated by the following formula: ALD = B0 - 2 SD, where B0 = mean A at 0 dose; SD = SD of 30-fold A at 0 dose, ALD = absorbance at lower detection limit). The lower detection limit was obtained by interpolating the ALD value from the calibration curve. The lower detection limit of the assay with F-21-HS-ADH-HRP conjugate was 0.05 µg/100 mL; as compared with 0.28 µg/100 mL, with F-21-HS-HRP conjugate. We have assayed 42 random human samples by ELISAs utilizing F-21-HS-ADH-HRP and F-21-HS-HRP as conjugates. There was a good correlation between the results obtained by ELISAs using the two above enzyme conjugates (r = 0.97).
For the first time, the preparation of HRP-ADH reagent, using periodate-oxidation-mediated reaction and its potential application in immunoassay for the preparation of enzyme conjugate is described. This is mainly to overcome the problem of direct conjugation of HRP to immunoreactant with bifunctional coupling reagents. This obstacle is caused by the nonavailability of amino groups, because a majority of 
and 
amino groups of most of the commercially purified HRPs are blocked by allylisothiocyanate (4). To introduce amino groups in HRP, we coupled ADH to it. ADH is a bifunctional cross-linking reagent-containing hydrazide group at both ends. The reagent provides a 10-atom bridge length between cross-linked molecules after conjugation. ADH has been coupled to affinity matrices (9), 96-well microtiter plate, (10), beads (11), avidin and streptavidin(12), and enzymes (13) to produce a hydrazide derivative for coupling to aldehyde ligands. For coupling ADH to HRP, we have used carbohydrate moieties of HRP. HRP contains 20% of carbohydrate by weight. The carbohydrate of HRP principally consists of mannose, N-acetyl glucosamine, xylose, and fucose (14). These carbohydrate residues are good sites for derivatization with periodate-oxidation without any substantial loss in enzyme activity (15). We exploited these carbohydrate sites for generating HRP-aldehyde by periodate oxidation reaction and, subsequently, by coupling with one of the hydrazide groups of ADH by forming a hydrazone bond. The HRP-ADH reagent provides the free amino groups for coupling to a immunoreactant with bifunctional coupling reagents or aldehyde ligands. In the present work, HRP-ADH has been conjugated to the carboxylic group of cortisol by the activated ester method for developing competitive direct ELISA. The hydrazide-containing reagents provide a built-in spacer to accommodate greater steric accessibility (16). The present finding revealed that the use of a linking reagent in the enzyme conjugate increases the detection limit of the competitive immunoassay. The improvement in the detection limit may be caused by the linker group preventing steric hindrance. The lower detection limit of the competitive immunoassay with the use of a HRP-ADH reagent may be treated as an added advantage apart from overcoming the hurdles of direct conjugation with HRP.
The HRP-ADH reagent may also be used for coupling to nucleic acid for nucleic acid hybridization assays and to proteins for preparing enzyme conjugates for immunoassay and immunochemistry, and may also be useful for staining glycoproteins and other glycoconjugates on protein blots after periodate oxidation.
References
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