1410 Technical Briefs

Preparation of Enzyme Conjugate through Adipic Acid mixed and kept overnight at 4 °C to form a hydrazone Dihydrazide as Linker and Its Use in Immunoassays, bond (Fig. 1B). After overnight incubation, 10 ␮Lof5m Anupam Basu,1 Tulsidas G. Shrivastav,1* and Kiran P. Kariya2 sodium cyanoborohydride in 1 mol/L NaOH was added, (1 Department of Reproductive Biomedicine, National In- reaction mixture was vortex-mixed and kept for 3 h at stitute of Health and Family Welfare, Munirka, New 4 °C. The reaction mixture was passed through a Seph- Delhi-110067, India; 2 Department of Chemistry, Vard- adex G-25 column previously equilibrated with 10 mm hamon Mahovir College, Nagpur-444008, India; * author PBS. The brown-colored fraction containing HRP-ADH for correspondence; fax 91-11-26101623, e-mail conjugate was collected and kept at Ϫ30 °C for future use. [email protected]) Cortisol-21-hemisuccinate (F-21-HS), procured from Sigma Chemical Company, was conjugated to HRP-ADH The direct coupling of the carboxylic derivative of steroids as described below. To 5 mg of F-21-HS, 200 ␮Lof to the amino group of enzymes is a well-established dimethyl formamide, 200 ␮L of dioxan, and 100 ␮Lof method in steroid enzyme immunoassays for making distilled water containing 10 mg of N-hydroxysuccini- enzyme conjugates (1). Horseradish peroxidase (HRP), mide and 20 mg of 1-ethyl-3-(3-dimethyl-amino-propyl) containing six lysine residues in the sequence, is a widely carbodiimide-HCL were added; the reaction mixture was used enzyme in enzyme immunoassays; in practice, how- vortex-mixed and kept overnight at 4 °C. After overnight ever, only one or two of these are generally available for incubation, the activated steroid reaction mixture was reaction (2). This variation in amino group content is added to 1 mL (ϳ1 mg of enzyme) of ADH-HRP solution. caused by changes in the extraction conditions used for Reaction mixture was vortex-mixed and incubated for the isolation of HRP from the roots of the horseradish 24 h at 4 °C to form the diimide bond (Fig. 1C). The plant (2, 3). The low yield of HRP coupled to the IgG by conjugate was passed through Sephadex G-25 column. the use of bifunctional reagents, namely glutaraldehyde, The fractions containing enzyme activity were pooled, carbodiimide, cyanuric chloride, bis-diazotized O-dianisi- and 1 g/L sucrose, ammonium sulfate, and BSA were dine, and P-PЈ-difluoro-m, m-dinitro-diphenyl sulfone added. To the above conjugate solution, an equal volume (FNPS), and so forth, prompted Nakane and Kawaoi to of ethylene glycol was added and kept at Ϫ30 °C in investigate another method (periodate method) for the aliquots for future use. conjugation of HRP to IgG (4). Comparative coupling We compared the displacement ELISAs using F-21-HS- efficiency studies were carried out with the use of glutar- ADH-HRP and F-21-HS-HRP. Therefore, HRP (HRP type aldehyde, periodate, and N-succinimidyl 3-(2-pyridyl- VI, lot no. 16H9520 from Sigma) was conjugated directly dithio) propionate (SPDP) as cross-linking reagents (5, 6) with F-21-HS as follows. To 5 mg of F-21-HS, 200 ␮Lof for the preparation of HRP-IgG conjugates. These studies dimethyl formamide, 200 ␮L of dioxan and 100 ␮Lof revealed that the most efficient HRP-IgG conjugate was distilled water containing 10 mg of N-hydroxysuccini- obtained by the periodate method. In practice, the differ- mide and 20 mg of 1-ethyl-3-(3-dimethyl-amino-propyl) ence in amino group availability in different batches of carbodiimide–HCL were added; reaction mixture was commercial preparations of HRP makes it difficult to vortex-mixed and kept overnight at 4 °C. One mg of HRP establish standard reaction conditions that could be ap- was dissolved in 1 mL of distilled water. Activated steroid plicable for more than one batch. Therefore, adipic acid solution was added to aqueous solution of HRP and was dihydrazide (ADH) has been coupled to HRP with the use incubated for 24 h at 4 °C. The conjugate was passed of the periodate method reaction to provide the necessary through a Sephadex G-25 column. The fractions contain- amino group for the preparation of hapten enzyme con- ing enzyme activity were pooled and 1 g/L sucrose, jugate with the use of the carbodiimide method. ammonium sulfate, and BSA were added. To the above We describe for the first time the use of ADH as a conjugate solution, an equal volume of ethylene glycol linking reagent between glycoenzyme (HRP) and a ste- was added and kept at Ϫ30 °C in aliquots for future use. roid carboxylic derivative to prepare enzyme conjugate The study was approved by the Institute’s animal ethics for ELISA. The conjugation of HRP to cortisol through committee. Cortisol antiserum was generated against cor- ADH as the link is described below. tisol-3-O-CMO-BSA as immunogen in New Zealand To 10 mg of HRP (HRP type VI, lot no. 28H7848; Sigma White rabbits (7). Microtiter plates were coated with Chemical Company; HRP of this lot failed to conjugate by cortisol-3-O-CMO antibody, diluted in 10 mm PBS (8). the direct method) per 1 mL of water, 10 ␮L of freshly F-21-HS-ADH-HRP or F-21-HS-HRP was diluted in prepared 0.1 m aqueous solution of sodium meta-perio- 10 mm sodium acetate buffer, pH 5.6, containing 1 g/L date was added to oxidize the vicinal hydroxy group of dextran T-70, thimerosal, sodium salicylate, and 3 g/L carbohydrate moieties of HRP to generate HRP-aldehyde BSA. Six cortisol calibrators (0 ␮g/100 mL, 1 ␮g/100 mL, (Fig. 1A). The reaction mixture was vortex-mixed and 3 ␮g/100 mL, 10 ␮g/100 mL, 30 ␮g/100 mL and 60 kept at room temperature for 30 min in the dark. Acti- ␮g/100 mL) were prepared in stripped serum (7).Tothe vated HRP (HRP-aldehyde) was passed through a Seph- cortisol antibody-coated wells, 25 ␮L of cortisol calibra- adex G-25 column previously equilibrated with 10 mm tors or samples along with 100 ␮L of either F-21-HS-ADH- ammonium carbonate, pH 9.3. The brown colored fraction HRP conjugate or F-21-HS-HRP enzyme conjugate were of activated HRP was directly collected in a vial contain- added in all of the wells and incubated for1hat37°C. ing 100 mg of ADH; the reaction mixture was vortex- After incubation, the contents of the wells were flicked Clinical Chemistry 49, No. 8, 2003 1411

Fig. 1. Conjugation of HRP with ADH and its use in contrast ELISA. (A), oxidation of vicinal hydroxyl groups of carbohydrate moieties of HRP by meta-periodate and, subsequently, formation of HRP aldehyde; (B), formation of a hydrazone bond between HRP-aldehyde and ADH; (C), conjugation of cortisol-21-HS with ADH-HRP to form amide bond; (D), dose-response curves (in semi-log scale) of cortisol ELISA, using cortisol-21-HS-ADH-HRP and cortisol-21-HS-HRP as enzyme conjugate.

out and washed in running tap water. To measure the where X is the concentration of cortisol in log dose and Y bound enzyme activity, 100 ␮L of substrate solution is the logit value of absorbency. (tetramethyl benzidine/H2O2, Bangalore Genei, India; The lower detection limit was calculated by the follow- ϭ Ϫ ϭ 1:20 diluted in water) was added to all the wells and was ing formula: ALD B0 2 SD, where B0 mean A at 0 incubated for 20 min at 37 °C. The enzyme reaction was dose; SD ϭ SD of 30-fold A at 0 dose, A ϭ absorbance ␮ LD stopped by adding 100 Lof0.5MH2SO4, and the color at lower detection limit). The lower detection limit was

intensity was measured at 450 nm. The logit-log transfor- obtained by interpolating the ALD value from the calibra- mation of the calibration curve of cortisol ELISA yielded tion curve. The lower detection limit of the assay with the following equations: F-21-HS-ADH-HRP conjugate was 0.05 ␮g/100 mL; as ␮ ϭ Ϫ ϩ compared with 0.28 g/100 mL, with F-21-HS-HRP con- Y 1.78X 0.83 jugate. We have assayed 42 random human samples by [using F-21-HS-ADH-HRP conjugate] ELISAs utilizing F-21-HS-ADH-HRP and F-21-HS-HRP as conjugates. There was a good correlation between the Y ϭ Ϫ1.62X ϩ 1.18 results obtained by ELISAs using the two above enzyme conjugates (r ϭ 0.97). [using F-21-HS-HRP conjugate] For the first time, the preparation of HRP-ADH reagent, 1412 Technical Briefs

using periodate-oxidation-mediated reaction and its po- 5. Boorsma DM, Streefekerk JG. Periodate or glutaraldehyde for preparing peroxidase conjugates? J Immunol Methods 1979;30:245–55. tential application in immunoassay for the preparation of 6. Jeanson A, Cloes JM, Bouchet M, Rentier B. Comparison of conjugation enzyme conjugate is described. This is mainly to over- procedure for the preparation of monoclonal antibody-enzyme conjugates. come the problem of direct conjugation of HRP to immu- J Immunol Methods 1988;111:261–70. 7. Basu A, Shrivastav TG. One step enzyme linked immunosorbent assay for noreactant with bifunctional coupling reagents. This ob- direct estimation of serum cortisol. J Immunoassay 2000;21:39–50. stacle is caused by the nonavailability of amino groups, 8. Shrivastav TG, Pandey PK, Kumari GL. Enzyme immunosorbant assay of because a majority of ␣ and ⑀ amino groups of most of the prolactin with penicillinase as label. Clin Chem 1988;34:2205–7. 9. O’Shannessy DJ, Wilchek M. Immobilization of glycoconjugates by their commercially purified HRPs are blocked by allylisothio- oligosaccharides: use of hydrazido-derivatized matrices. Anal Biochem cyanate (4). To introduce amino groups in HRP, we 1990;191:1–8. coupled ADH to it. ADH is a bifunctional cross-linking 10. Satoh A, Kojima K, Koyama T, Ogawa H, Matsumoto I. Immobilization of saccharides and peptides on 96-well microtiter plates coated with methyl reagent-containing hydrazide group at both ends. The vinyl ether-maleic anhydride copolymer. Anal Biochem 1998;260:96–102. reagent provides a 10-atom bridge length between cross- 11. Fleminger G, Hadas E, Wolf T, Solomon B. Oriented immobilization of linked molecules after conjugation. ADH has been cou- periodate–oxidized monoclonal antibodies on amino and hydrazide deriva- tives of Eupergit C. Appl Biochem Biotechnol 1990;23:123–37. pled to affinity matrices (9), 96-well microtiter plate, (10), 12. Bayer EA, Ben-Hur H, Wilchek M. Avidin and streptavidin-containing probes. beads (11), avidin and streptavidin (12), and enzymes Methods Enzymol 1990;184:174–87. (13) to produce a hydrazide derivative for coupling to 13. Keren Z, Berke G, Gershoni JM. Identification of cell surface glycoproteins by periodate-alkaline phosphatase hydrazide. Anal Biochem 1986;155:182–7. aldehyde ligands. For coupling ADH to HRP, we have 14. Dent AH, Aslam M. Enzymes. In: Aslam M, Dent A, eds. Bioconjugation: used carbohydrate moieties of HRP. HRP contains ϳ20% protein coupling techniques for the biomedical sciences. London: Macmillan of carbohydrate by weight. The carbohydrate of HRP Reference Ltd., 1998:121. 15. Schalkwijk J, van den Berg WB, van de Putte LBA, Joosten LAB, van den principally consists of mannose, N-acetyl glucosamine, Bresselaar L. Cationization of catalase, peroxidase and superoxide dis- xylose, and fucose (14). These carbohydrate residues are mutase. Effect of improved intraarticular retention on experimental arthritis good sites for derivatization with periodate-oxidation in mice. J Clin Invest 1985;76:198–205. 16. Hermanson GT. Bioconjugate techniques. San Diego: Academic Press, without any substantial loss in enzyme activity (15).We 1996:121. exploited these carbohydrate sites for generating HRP- aldehyde by periodate oxidation reaction and, subse- quently, 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 Novel Hemoglobin Variant [␤66(E10) Lys3Asn], with immunoreactant with bifunctional coupling reagents or Decreased Oxygen Affinity, Causes Falsely Low Hemo- aldehyde ligands. In the present work, HRP-ADH has 1* globin A1c Values by HPLC, Ulrich Friess, Alexander been conjugated to the carboxylic group of cortisol by the Beck,1 Elisabeth Kohne,2 Rainer Lehmann,1 Sylvia Koch,3 activated ester method for developing competitive direct Hans-Ulrich Haring,1 Reinhold-Michael Schmuelling,1 and ELISA. The hydrazide-containing reagents provide a Erwin Schleicher1 [1 Department of Medicine IV (Endocri- built-in spacer to accommodate greater steric accessibility nology, Metabolism, and Clinical Chemistry) and 3 De- (16). The present finding revealed that the use of a linking partment of Medicine II (Hematology), University of reagent in the enzyme conjugate increases the detection Tuebingen, Otfried-Mueller-Strasse 10, D-72076 Tuebin- limit of the competitive immunoassay. The improvement gen, Germany; 2 Department of Pediatrics, Hemoglobin in the detection limit may be caused by the linker group Facility, University of Ulm, Prittwitzstrasse 43, D-89075 preventing steric hindrance. The lower detection limit of Ulm, Germany; * author for correspondence: fax 49-7071- the competitive immunoassay with the use of a HRP- 295974, e-mail [email protected]] ADH reagent may be treated as an added advantage apart from overcoming the hurdles of direct conjugation with The lysine in position 66 of the ␤ chain of hemoglobin HRP. (Hb) is located within the heme-binding pocket and is The HRP-ADH reagent may also be used for coupling functionally important for oxygen binding (1). The lysine to nucleic acid for nucleic acid hybridization assays and to is exceptionally prone to glycation (2) and to modification proteins for preparing enzyme conjugates for immuno- by advanced glycation endproducts. In this journal, assay and immunochemistry, and may also be useful for Iwamoto et al. (3) recently proposed an automated im- staining glycoproteins and other glycoconjugates on pro- munologic assay for Hb that is specifically advanced tein blots after periodate oxidation. glycation endproduct-modified at Lys66. Here we report on a novel Hb variant at this position References [␤66(E10) Lys3Asn] found in a 73-year-old Caucasian 1. Dent AH, Aslam M. The preparation of protein-small molecule conjugate. In: Aslam M, Dent A, eds. Bioconjugation: protein coupling techniques for the patient with a 9-month history of type 1 diabetes mellitus biomedical sciences. London: Macmillan Reference Ltd., 1998:364–482. secondary to recurrent necrotizing pancreatitis and cho- 2. Dent AH, Aslam M. Enzymes. In: Aslam M, Dent A, eds. Bioconjugation: ledocholithiasis. He was referred to us because of insuf- protein coupling techniques for the biomedical sciences. London: Macmillan Reference Ltd., 1998:120. ficient metabolic control: in the previous 4-month period, 3. Ornstein L. Discussion on “Enzyme-labeled antibodies for light and electron his fasting blood glucose (FBG) had been in the range of microscopic localization of antigens” by PK Nakane et al. J Histochem 6.3–13.6 mmol/L and his postprandial blood glucose had Cytochem 1966;14:790. 4. Nakane PK, Kawaoi A. Peroxidase-labeled antibody: a new method of been in the range of 8.8–17.0 mmol/L. On admission, his conjugation. J Histochem Cytochem 1974;22:1084–91. FBG was 13.6 mmol/L. The blood smear results, complete