One-Step Monoclonal Antibody Based Enzyme-LinkedImmunosorbent Assay for Direct Determination

of Cortisol in Serum

Rong Liu,1,2 Faju Chen,2 Kun Zou,2 and Tangbin Yang1

Monoclonal antibodies to cortisol have obvious potential advantages as starting materials for assay systems todetect their levels in body fluids. This is very important for monitoring pituitary gland and adrenal functions. Todevelop a one-step competitive heterogeneous enzyme-linked immunosorbent assay (ELISA), a monoclonal anti-cortisol antibody was generated using a reasonably designed haptenic derivative. Cortisol-3-O-carboxy-methyloxime was coupled to carrier protein bovine serum albumin (BSA) to enhance its immunogenicity. Spleencells were prepared from a BALB/c mouse, which had repeatedly been immunized with a conjugate of cortisol-3-O-carboxymethyloxime-bovine serum albumin (cortisol-3-O-CMO-BSA), to be fused with SP2/0 myelomacells. After one fusion experiment, four hybridoma clones secreting a practical antibody were established. One ofthe resulting monoclonal antibodies, 2C9D11B5, showed an affinity constant (Ka) of 1.4�1010 M–1 for cortisoland provided a practical calibration curve (limit of detection [LOD], 0.26 ng per assay) in this ELISA systememploying cortisol-21-hemisuccinate-horseradish peroxidase (cortisol-21-HS-HRP) as a tracer. Cross-reactivitieswith related C-21 steroids were acceptably low: 11-deoxycortisol (3.5%), cortisone (0.47%), corticosterone(<0.01%), progesterone (<0.01%), 17-hydroxyprogesterone (1.2%), 6-hydroxycortisol (7.6%), and tetrahydro-cortisol (<0.01%). The intra-assay and inter-assay coefficient of variations (CVs) ranged from 4.3% to 9.2% and3.8% to 10.4 %, respectively. The analytical recoveries were 92.3% to 116.3%. Serum cortisol levels of healthyvolunteers were determined after chilled acetone, stripped to be 292.76 ± 201.38 ng/mL (n¼ 5), which are in thereference range.

Introduction

C ortisol is an important hormone in the body, se-creted by the adrenal glands and involved in the fol-

lowing functions: proper glucose metabolism, regulation ofblood pressure, insulin release for blood sugar maintenance,immune function, and inflammatory response. Because it issecreted at higher levels during the body’s ‘‘fight or flight’’response to stress, and is responsible for several stress-relatedchanges in the body, cortisol is also termed the ‘‘stress hor-mone.’’(1,2) Clinically, measurement of serum cortisol is auseful index for monitoring pituitary gland and adrenalfunctions(3,4) and diagnosing adrenocortical diseases includ-ing Cushing’s syndrome,(5–7) Addison’s disease,(8) congenitaladrenal hyperplasia,(9) and stress-related illness.(10) Thus,development of practical immunoassays of this steroid hasbeen an important subject in biomedical analytical chemis-try.(11,12) The production of monoclonal antibodies (MAbs) tocortisol has been reported by a number of groups.(12–15) Suchreagents have obvious potential advantages as starting ma-

terials for assay systems since monoclonal antibodies offergreat scope for the development of new assay techniques,particularly those that use labeled antibody. Several MAb--based enzyme-linked immunosorbent assays (ELISAs)have been reported for estimating the cortisol in urine,(14)

saliva,(16–18) and serum.(12,13,19–22) However, none of the re-sulting antibodies afforded a truly excellent immunoassaysystem showing high sensitivity and specificity, and all theseELISAs required extra extraction steps with many organicsolvents, which not only needed correction of recovery butalso increased the cost of the assay by negatively affecting thetime, labor, and material.

In this present study, the C-3 position on cortisol was usedto conjugate with bovine serum albumin (BSA) for immuno-gen preparation and to generate MAb. The resulting MAbwas equipped with suitable binding properties to develop anELISA system to measure human serum cortisol levels, inwhich cortisol-21 hemisuccinate-horseradish peroxidase(cortisol-21-HS-HRP) was used as a tracer and no extractionsteps were required for estimation of cortisol from serum

1Medical Science College, China Three Gorges University, Yichang, Hubei, China.2 Hubei Province Key Laboratory of Natural Products Research and Development, China Three Gorges University, Yichang, Hubei, China.

HYBRIDOMAVolume 30, Number 3, 2011ª Mary Ann Liebert, Inc.DOI: 10.1089/hyb.2010.0115

279

sample. To the best of our knowledge, no similar method,based on both MAb and site heterologous competitive ELISAfor direct estimation of serum cortisol, has been reporteduntil now.

Materials and Methods

Reagents and materials

Cortisol, 11-deoxycortisol, cortisone, corticosterone, pro-gesterone, 17-hydroxyprogesterone, 6-hydroxycortisol,tetrahydrocortisol, cortisol-3-O-carboxymethyl-oxime (cortisol-3-O-CMO), cortisol-21-hemisuccinate (cortisol-21-HS), HRPtype VI (EC 1.1 1.1.7), BSA, Ovalbumin (OVA), Freund’scomplete adjuvent (FCA), 1-ethyl-3-(3-dimethyl-amino-propyl)-carbodiimide-HCl (EDAC), N-hydroxysuccinimide(NHS), dimethylformamide (DMF), hypoxanthine-thymidine(HT), hypoxanthine-aminopterin-thymidine (HAT), Dulbecco’smodified Eagle’s medium-high glucose (DMEM), and 3,3’,5,5’tetramethyl benzidine (TMB) were all purchased from SigmaChemical Company (St. Louis, MO).

Two-month-old BALB/c mice were purchased from BeijingVital River Laboratory Animal Technology Co. (Bejing, China).The myeloma cell line of SP2/0 origin was obtained fromWuhan University Type Culture Collection (Wuhan, China).ImmunoPure� Monoclonal Antibody Isotyping Kit (HRP/ABTS) and BCA Protein Assay Kit were purchased from Pierce(Rockford, IL). ELISA plates (96 wells) were purchased fromCanada JET Bio-chemicals (Toronto, Canada). Other cell cultureplastic wares were obtained from Costar (Cambridge, MA).

Absorbance values were read in dual-wavelength mode(450–630 nm) with a microplate reader (model 680, Bio-RadLaboratories, Hercules, CA). All other reagents used in thisstudy were of analytical grade and obtained from standardsources.

Preparation of immunogen and coating conjugate

Cortisol-3-O-CMO was covalently linked to BSA or OVAby activated ester method with little modification.(13,23–27)

Briefly, 25 mg of cortisol-3-CMO, 20 mg of NHS, and 30 mg ofEDAC were mixed and dissolved in 250 mL of DMF and left atroom temperature for 1 h. After completion of the conversion,the reactant was extracted twice with 10 mL of ethyl acetate,and the pooled solvent was washed twice with 10 mL water.The ethyl acetate fraction was dehydrated with anhydrousNa2SO4, and evaporated under a gentle stream of nitrogen.

The active ester of cortisol-3-CMO was conjugated to car-rier proteins using a proper molar ratio of steroid to protein.BSA or OVA solutions were mixed with a solution of cortisol-3-CMO ester (25 mg dissolved in 0.5 mL of DMF). After 30 minof stirring, the reactant was dialyzed against cold runningwater overnight; the solution was brought to pH 4.5 with 1 NHCl and centrifuged at 4000 g for 15 min, and the dialysis wasrepeated until the free steroid and NHS were removed com-pletely. The precipitate was dissolved in water adjusted to pH7.0 with 1 N NaOH, diluted to a concentration of 10 mgprotein/mL and stored at �808C. The concentration of pro-tein was assessed by Pierce BCA Protein Assay Kit. The twoconjugates are shown in Figure 1.

To determine effectiveness of conjugation, ultravioletspectral measurements were carried out on a UV-2102PCspectrophotometer (UNICO Instruments, Shanghai, China).

The solvent used was 0.05 mol/L phosphate buffer (pH 7.4) or0.1 mol/L KOH. Spectral analysis and calculations were car-ried out as described by Hosoda and associates(27) by com-paring the absorbance (molar extinction coefficient of 23560)of the conjugate cortisol-3-CMO ester with BSA or OVA withthose of BSA or OVA, cortisol, and cortisol-3-CMO ester alonein both solvents.

Preparation and dilutionof cortisol-21-HS-HRP conjugate

According to the method of Basu and associates,(28) theprocedure that cortisol-21-HS was coupled to HRP, whichwas employed as a tracer in one-step competitive heteroge-neous ELISA system, is shown in Figure 2. The optimal di-lution of cortisol-HRP conjugate was found by thecheckerboard assay. The diluted conjugate was stored in theconjugate dilution buffer at 48C for future use. This was stablefor more than one year at 2–88C.

Immunization

Female BALB/c mice (each 6–8 weeks of age; each 5 heads)were immunized bi-weekly with cortisol-3-O-CMO-BSA. Theconjugate (50 mg) was subcutaneously injected with anemulsion of FCA (primary immunization) or FIA (boosterimmunizations) and sterile saline (1:1; 0.2 mL) at multiple siteson the back. Ten days after the third booster injection, bloodwas collected and checked for the titer of the serum antibodiesto cortisol. The conjugate (50 mg) in a sterile saline (0.2 mL)was intraperitoneally injected into the two BALB/c mice thatshowed a relatively high immune response, from whichspleen cells were prepared 3 days later.

Monoclonal antibody production

The cell fusion experiment was performed according to ourprevious work.(29,30) In brief, the immune spleen cells (1�108

cells) and 1/5 numbers of SP2/0 myeloma cells(31) were fused

FIG. 1. Conjugation of carrier proteins with cortisol-3-O-CMO to prepare immunogen and coating antigen.

280 LIU ET AL.

with 50% PEG 4000 in incomplete DMEM’s medium con-taining 10% DMSO and 0.001% poly-L-arginine–HCl solution(1 mL). The fused cells were suspended in HAT mediumsupplemented with 20% FCS and cultured in 96-well micro-titer plates (200mL/well) overnight in a humidified incubatorin an atmosphere of 5% CO2 at 378C. After further culture forabout 1 week in HAT medium, the hybridoma supernatantswere submitted to screening by ELISA described below. Theantibody-secreting hybridomas were expanded in HT me-dium, subcloned by limiting dilution, and grown until con-fluence was reached. The resulting supernatant was used forcharacterization of the MAbs contained therein.

ELISA for MAb detection and characterization

The checkerboard procedure was used to optimize thecoating antigen concentrations. To screen antibody in thesupernatant of cell culture medium, cortisol-3-O-CMO-OVAwas coated onto the wells of microtiter plates. Supernatantsof the wells containing a monoclonal cell growth were char-acterized for titer, specificity, and affinity.(29) Selectedantibody-producing clones were cultured in 150 mL flasks oralternatively cells were injected i.p. into pristine-pretreatedmice. Supernatants or ascitic fluids were collected and puri-

fied using ammonium sulfate precipitation and protein Acoupled agarose column. Purified immunoglobulin wasused to establish a calibration curve in culture supernatant.For the cross-reactivity study, seven related C-21 steroids (11-deoxycortisol, cortisone, corticosterone, progesterone, 17-hydroxyprogesterone, 6-hydroxycortisol, and tetrahydrocortisol)were added in place of cortisol. Supernatant of cell culturemedium with proper dilution reacted with cortisol or theother derivatives in another container and transferred intothe cortisol-3-O-CMO-OVA conjugate precoated microtiterwells, washed, and added to HRP-IgG, and incubated for30 min. The rest was performed following the procedure shownin Figure 3. Class and subclass determination was performedusing HRP/ABTS as instructed by the manufacturer.

Preparation of cortisol standards in stripped serum

Pooled human serum was treated with chilled acetonefollowed by centrifugation for 30 min at�208C. The pellet waslyophilized. The original volume of the serum was recon-stituted by dissolving the lyophilized material in normal sa-line. Five cortisol working standards such as 2.0, 6.0, 18.0,54.0, and 162.0 ng/mL, respectively, were prepared in theabove serum matrix.

FIG. 2. Conjugation of cortisol-21-HS with ADH-HRP as an enzyme conjugate. (A) Oxidation of vicinal hydroxyl groups ofcarbohydrate moieties of HRP by meta-periodate and, subsequently, formation of HRP aldehyde. (B) Formation of a hy-drazone bond between HRP-aldehyde and ADH. (C) Conjugation of cortisol-21-HS with ADH-HRP to form amide bond.

DIRECT DETERMINATION CORTISOL IN SERUM 281

One-step heterogeneous competitive ELISAprocedure for cortisol evaluation

Wells of the microtiter plate were coated with 100mL ofdiluted cortisol-3-O-CMO MAb by second antibody tech-nique, as described elsewhere. The optimal antibody dilution(1:20,000) of cortisol MAb was obtained by checkerboardassay. After antibody coating, the wells were blocked byblocking and stabilizing buffer. The plates were stored underdesiccation at 48C for future use. The binding capacity ofcoated microwells remained unchanged for more than oneyear.

To the cortisol MAb coated wells, 50mL of cortisol stan-dards or serum samples were added in duplicate. The 50 mL ofcortisol-HRP conjugate was added to all the wells and incu-bated for 2 h at 378C. After incubation, the contents of thewells were decanted and washed in PBST four times by filling,decanting, and flicking. Finally, for measuring the boundenzyme activity, 100mL of substrate solution was added to allthe wells and incubated for 20 min at 378C. The reaction wasstopped by adding 50 mL of 6 M H2SO4 and the color wasmeasured at 450 nm in ELISA plate reader.

Results and Discussion

Production of MAbs

It is well established that cortisol is a useful index formonitoring pituitary gland and adrenal functions and diag-nosing adrenocortical diseases.(5–10,12) Thus, development ofpractical immunoassays of this steroid has been an importantsubject in biomedical analytical chemistry. The specificity ofantibodies against haptens such as steroids is significantlyinfluenced by the position on the hapten molecule used forconjugation with a carrier protein to yield an immunogen.Various kinds of antisera against cortisol have been generatedso far using the C-3, C-6, C-7, or C-21 position on the steroidnucleus for the conjugation.(33) Some of these antisera pro-vided practical radioimmunoassay (RIA) systems but werenot always available for developing enzyme immunoassay(EIA) systems. Indeed, reasonably specific polyclonal anti-bodies have been prepared against cortisol(33) and 11-deoxy-cortisol(12) using corresponding haptenic derivatives having achemical bridge at their C-4 position. These antibodies were

also useful for developing EIA systems providing practicalsensitivity and specificity.(12,34) Basu and colleagues foundthat the C-3 on cortisol is suitable for conjugation to producespecific antisera and that C-21 is useful for conjugation toprepare a tracer. Making use of two components, they es-tablished a one step competitive enzyme-linked immunosor-bent assay to determine the serum cortisol level directly,(13)

but it was limited by the supply of polyclonal antibodies.Since MAbs, which are obtainable by hybridoma technol-

ogy,(35) can be supplied constantly maintaining well-definedand unique binding properties and consequently allow us tostandardize assay performance, generating an MAb that isavailable for use in modern immunoassay systems is veryimportant. From these points of view, a few studies have beendone so far to generate monoclonal anti-cortisol anti-bodies.(12,15,36) Crichton’s group produced several antibodiesshowing low cross-reaction with 11-deoxycortisol after varioustrials for immunization using cortisol 3-(O-carboxymethyl)oxime (3-CMO) as the haptenic derivative.(15) In the study ofLewis and colleagues, MAbs were produced after immuni-zation with 21-acetate of 3-CMO conjugated with BSA andshowed exceptionally high cross-reactivity to prednisone(>1200%) and 11-deoxycortisol (>100%). One of the anti-bodies was applied to ELISA for determining the plasmacortisol level but exhibited somewhat poor sensitivity as wellas significant cross-reaction with 11-deoxycortisol (19%).(36)

In both studies, the resulting antibodies did not afford trulyexcellent immunoassay systems, showing high sensitivityand specificity using the same strategy to prepare coatingantigen.(15,36) Kobayashi et al. generated an MAb showingimproved affinity and specificity for cortisol using a cortisol-

100 µl of Cortisol-3-O-CMO-OVA (1 µg/ml) was coated on each well of microtiter platesAt 4 °C overnight

Added 120 µl of 0.1% gelatin each well to block microtiter plates at 37 °C for 1 h Washed with pH 7.4 PBST (3×30")

Added each well 100 µl of mixture of a series of cortisol or related compounds andmonoclonal antibody at 37 °C for 30 min

Washed with pH 7.4 PBST (3×30") Added each well 100 µl of goat anti-mouse IgG-HRP conjugate at 37 °C for 1h

Washed with pH 7.4 PBST (3×30") Added to 100 µl of TMB / substrate solution at 37 °C for 15 min

Added to 50 µl of H2SO4 (2 M) at room temperature

Detection absorbance at 450 nm

FIG. 3. Flow chart of competitive immunoassay for the MAb screening.

Table 1. Identification and Characterization

of Anti-cortisol Monoclonal Antibodies

HybridomaClass andsubclass Type

Titer in culturemedium

Affinity(M�1)

2C9D11B5 IgG1 k 1:6400 1.40�1010

2E7C3B2 IgG1 k 1:4000 4.53�1010

1E11B5D11 IgG1 k 1:8000 6.78�109

3D1E9D9 IgG1 k 1:3200 1.07�1010

282 LIU ET AL.

[C-4]-BSA conjugate for immunization of several murinestrains.(12) One of the resulting antibodies was equippedwith suitable binding properties to develop an ELISA sys-tem to determine human urinary and serum cortisol levels,which showed relatively high cross-reactivities with 17a-hydroxyprogesterone (12%) and 6b-hydroxycortisol (8.4%).

In the present study, a designed haptenic derivative, whichexposed characteristic functional groups on the target haptenoutside of the carrier protein, was proven to be easy to gen-erate specific antibodies against haptens. Then we immunizedBALB/c murine strains with the cortisol-[C-3]-BSA conjugate.

Two BALB/c mice that showed relatively high immuneresponses were used as spleen donors. After one fusion ex-periment and the HAT selection, hybridomas were observedin almost all the microcultures (a total of 490 wells were used),among which four hybridoma cultures were found to be se-creting an antibody that strongly binds to cortisol-3-O-CMO-OVA in the ELISA. These hybridomas were cloned andgrown, and four corresponding kinds of MAbs were obtainedas the culture supernatant and ascites, but only one clonesecreting the anti-cortisol antibody 2C9D11B5 showed prac-tical binding properties in the heterogeneous ELISA system.

Characterization of MAbs

Table 1 summarizes the characterization of selected clonesin terms of titer, affinity, class, and subclass, demonstratingthat the antibodies were of G1 subclass with k light chain. Thebinding characteristics of the antibody were investigated bythe ELISA using cortisol-3-O-CMO-OVA as a labeled antigen.The optimum dilution of the antibody (culture supernatant)was arbitrarily determined to be 1:4000, at which the enzymeactivity of B0 corresponds to approximately 1.5 absorptionunit (15 min incubation). The absorption due to nonspecificbinding in the absence of antibody was below 5% of the B0value. These were of high-affinity type antibodies with af-finity constant ranging from 109 to 1010 M�1. The specificity of(2C9D11B5) was examined by a cross-reaction study withseven related compounds, taking the reactivity of cortisolas 100% (Table 2); this showed that 2C9D11B5 had a highspecificity for free cortisol, acceptably low reactivity with11-deoxycortisol, cortisone, corticosterone, progesterone,17-hydroxyprogesterone, 6-hydroxycortisol, and tetrahydro-cortisol, which showed obviously better specificity than theother antibodies from the viewpoint of clinical application.Namely, this antibody afforded much lower cross-reactivitieswith 11-deoxycortisol and cortisone, both of which have avery similar chemical structure to cortisol and could poten-tially interfere with determination of serum/plasma cortisollevels in some cases. Cross-reactivities with the other ana-logs were similar or lower than those of an EIA system that

Hosoda and colleagues previously established using apolyclonal antibody elicited with the same immunogen.(27)

However, cross-reactivity with 6-hydroxycortisol, which isone of the major urinary metabolites of cortisol, was signifi-cantly higher.

Calibration curve and assay evaluation

Effects and optimization of pH value. According to ourprevious work,(32) the value of pH is one of the key factorsinfluencing the characteristics of assay, and extremely higheror lower pH value affected the IC50 and ODmax of ELISAgreatly, so we investigated the effect of medium pH (7.2 to 8.0)on ELISA performance. It seems that the reaction betweenantibody and analyte may be prone to change at lower cortisollevels. The physiological pH (7.2) was selected as the opti-mum for the assay, based on the favorable IC50 value andODmax of the calibration curve (Fig. 4).

Calibration curve. Among the four MAbs, 2C9D11B5showed the lowest assay values for serum specimens, con-sistent with the cross-reactivity, and consequently, this anti-body was determined to possess the most suitable propertiesfor practical use.

2C9D11B5 provided dose-response curves for cortisol witha practical measurable range in one-step heterogeneouscompetitive ELISA, which was separately obtained taking thestandard variation (SD) at each dose (Fig. 5). Each standardconcentration was used in triplicate in each assay. The coef-ficient of variations (CVs) of the standard(s) ranged from 4.1

Table 2. Percent Cross-reaction on Monoclonal Antibody 2C9D11B5 Raised

Against Cortisol-3-O-CMO-BSA in ELISA

Steroid Cross-reactivity (%) Steroid Cross-reactivity (%)

Cortisol 100.0 17-Hydroxyprogesterone 1.211-Deoxycortisol 3.5 6-Hydroxycortisol 7.6Cortisone 0.47 Tetrahydrocortisol <0.01Corticosterone <0.01 Progesterone <0.01

FIG. 4. Influence of pH on performance of assay. Resultsare the means of two independent experiments.

DIRECT DETERMINATION CORTISOL IN SERUM 283

to 8.3%. The lower detection limit of the assay (i.e., concen-tration equivalent to B0-2SD) was 0.26 ng per assay.

Analytical recoveries. The ability of the assay to accu-rately quantify cortisol in serum samples was tested. Knownamounts of standard cortisol (6.0–100 ng/mL) were addedexogenously to chilled acetone extracted serum specimens.After this, the concentration of cortisol was determined andrecovery was calculated in each fraction of serum. Table 3indicates the precision profile of the assay. The analysis of fivespiked serum samples for inter- and intra-assay (n¼ 8, repli-cate of each pool) gave CVs of 4.3–9.2% and 5.1–10.4% at alllevels. The analytical recoveries of inter- and intra-assay are inthe ranges of 92.8–110.2% and 92.3–116.3%, respectively.

Clinical application of ELISA

Since serum/plasma-free cortisol levels are a useful indexin clinical diagnosis, we next examined the specificity fromthe viewpoint of applicability to serum samples. However, itsmeasurement is often accompanied by overestimation due tovarious kinds of metabolites having similar structures andcoexisting in excess amounts. Considering this, we collected

sera from five healthy men and submitted the ELISA systemsto measure the serum levels and found that a single extractionwith chilled acetone permits reasonable assay values. Al-though direct measurement afforded obviously over-estimated assay values (data not shown), we could obtainacceptable assay values by employing chilled acetone ex-traction as a pretreatment. The mean of SD concentrationmeasured is shown in Table 4. The recoveries ranged between92.8% and 110.2% (Table 3), which are all in the referencerange. The serum cortisol levels obtained from healthy vol-unteers were in the reference range, which strongly suggestedthat the present ELISA is valid for clinical application.

Of greatest concern in this direct ELISA is the matrix in-terference during the sample preparation procedure. Themulticentric evaluation of enzyme immunoassay of plasmacortisol showed that the precision of the assay was adequate(<12%), whereas recovery often differed markedly, whichmay be due to a binding protein in the direct assays.(13) Fewnumbers of immunoassays other than ELISA have been re-ported in the literature for direct estimation of cortisol inserum. These assays employed danazol in time-resolvedfluoroimmunoassay(37) and sodium salicylate in radioimmu-noassay(38) as the cortisol displacing agent. We have usedsodium salicylate as a cortisol-displacing and cortisol-bindingglobulin (CBG) blocking agent for the estimation of cortisoldirectly from serum without compromising sensitivity of theassay.

FIG. 5. Calibration curve of heterogenous competitive ELISA for cortisol.

Table 3. Recovery of Cortisol from Exogenously

Spiked Pooled Serum by ELISA

Inter-assay (n¼ 5) Intra-assay (n¼ 5)Spiked serumconcentration(ng/mL)

Averagerecovery (%) CVa (%)

Averagerecovery (%)

CV(%)

6.0 95.7 6.7 96.6 5.120.0 105.4 8.5 116.3 3.840.0 92.8 4.3 109.6 4.780.0 110.2 7.1 92.3 9.3100.0 100.6 9.2 95.5 10.4

aCoefficient of variation; the measure basal cortisol level forpooled serum is 65 ng/mL.

Table 4. Measurement of Human

Pooled Serum Cortisol

Subject Mean (SD) ng/mL CV%

A 72.3 (0.74) 10.57B 185.7 (0.95) 5.42C 321.2 (1.16) 4.71D 527.5 (0.89) 1.73E 357.1 (2.10) 6.73

284 LIU ET AL.

In the present procedure, the chilled acetone strippedpooled human serum (matrix) was also used for preparingstandards with improved accuracy. This matrix resembledserum whereas matrix prepared by conventional procedure,like stripping by charcoal, did not resemble serum because,along with low molecular weight compound, charcoal alsoadsorbed the proteins. The serum matrix prepared by thisnew method can be employed for the standard preparation ofdifferent steroid ELISAs, which may possibly eliminate thematrix effect. But before considering this procedure as theuniversal method of matrix preparation for other steroids, itshould be tested for accuracy of the steroid assays.

The analytical variables of direct ELISA, such as sensitivity,accuracy, precision, and correlation coefficient, are in agree-ment with the standardization of a method. One reason for thisis that we used acetone stripped pooled human serum toprepare standards. Another reason is that in our system cor-tisol-21-HS-HRP conjugate was diluted in HRP conjugatebuffer that contains 0.1% sodium salicylate, which can displacecortisol from CBG and block the binding of cortisol to CBG.

Conclusion

We have developed a one-step heterologous competitiveELISA for cortisol based on a specific MAb (2C9D11B5)produced from a conjugate of cortisol-3-O-carboxymethyloxime-bovine serum albumin and cortisol-21-hemisuccinate-horseradish peroxidase as a tracer. At optimal experimentalconditions, LOD of this assay was 0.26 ng/mL. This ELISAwas finally applied to analyze cortisol levels in healthy vol-unteers’ serum samples. The analysis of five spiked serumsamples for inter-assay and intra-assay (n¼ 8, replicate ofeach pool) gave CVs of 4.3–9.2% and 5.1–10.4% at all levels.The acceptable recoveries ranged from 92.8 to 110.2% and 92.3to 116.3%, respectively. To eliminate the matrix effect, thechilled acetone stripped pooled human serum (matrix) wasemployed for the preparation of standard for different ste-roids ELISAs, which was valid for clinical application.

Acknowledgment

This work was supported by a scientific grant (KJ2009B025)from China Three Georges University.

Author Disclosure Statement

The authors have no financial interests to disclose.

References

1. Caroprese M, Albenzio M, Marzano A, Schena L, Annic-chiarico, and Sevi A: Relationship between cortisol responseto stress and behavior, immune profile, and productionperformance of dairy ewes. J Dairy Sci 2010;93(6):2395–2403.

2. Hankin BL, Badanes LS, Abela JR, and Watamura SE: Hy-pothalamic-pituitary-adrenal axis dysregulation in dys-phoric children and adolescents: cortisol reactivity topsychosocial stress from preschool through middle adoles-cence. Biol Psychiatry 2010;68(5):484–490.

3. Duplessis C, Rascona D, Cullum M, and Yeung E: Salivary andfree serum cortisol evaluation. Mil Med 2010;175(5):340–346.

4. Levine A, Zagoory-Sharon O, Feldman R, Lewis JG, andWeller A: Measuring cortisol in human psychobiologicalstudies. Physiol Behav 2007;90(1):43–53.

5. Tiryakioglu O, Ugurlu S, Yalin S, Yirmibescik S, Caglar E,Yetkin DO, and Oglu P: Screening for Cushing’s syndromein obese patients. Clinics (Sao Paulo) 2010;65(1):9–13.

6. Bertagna X, Guignat L, Groussin L, and Bertherat J: Cush-ing’s disease. Best Pract Res Clin Endocrinol Metab2009;23(5):607–623.

7. Raff H: Utility of salivary cortisol measurements in Cush-ing’s syndrome and adrenal insufficiency. J Clin EndocrinolMetab 2009;94(10):3647–3655.

8. Nieman LK, and Chanco Turner ML: Addison’s disease.Clin Dermatol 2006:24(4):276–280.

9. Demirci C, and Witchel SF: Congenital adrenal hyperplasia.Dermatol Ther 2008;21(5):340–353.

10. Schelling G: Post-traumatic stress disorder in somatic dis-ease: lessons from critically ill patients. Prog Brain Res2008;167:229–237.

11. Gatti R, Antonelli G, Prearo M, Spinella P, Cappellin E, andDe Palo EF: Cortisol assays and diagnostic laboratory pro-cedures in human biological fluids. Clin Biochem 2009;42(12):1205–1217.

12. Kobayashi N, Sun P, Fujimaki Y, Niwa T, Nishio T, Goto J,and Hosoda H: Generation of a novel monoclonal antibodyagainst cortisol-[C-4]-bovine serum albumin conjugate: ap-plication to enzyme-linked immunosorbent assay for uri-nary and serum cortisol. Anal Sci 2002;18(12):1309–1314.

13. Basu A, and Shrivastav TG: One step enzyme linked im-munosorbent assay for direct estimation of serum cortisol. JImmunoassay 2000;21(1):39–50.

14. Lewis JG, Manley L, Townsend LC, and Elder PA: An en-zyme linked immunosorbent assay (ELISA) for urinary freecortisol. Clin Chim Acta 1986;159(2):205–209.

15. Crichton D, Grattage L, McDonald A, Corrie JE, Steel CM,Hubbard AL, Al-Dujaili EA, and Edwards CR: The pro-duction and assessment of monoclonal antibodies to cortisol.Steroids 1985;45(6):03–517.

16. Hubl W, Taubert H, Freymann E, Meissner D, Stahl F, andDomer G: A sensitive direct enzyme immunoassay for cor-tisol in plasma and saliva. Exp Clin Endocrinol 1984;84(1):63–70.

17. Cooper TR, Trunkfield HR, Zanella AJ, and Booth WD: Anenzyme-linked immunosorbent assay for cortisol in the sa-liva of man and domestic farm animals. J Endocrinol1989;123(2):R13–16.

18. Dressendorfer RA, Kirschbaum C, Rohde W, Stahl F, andStrasburger CJ: Synthesis of cortisol-biotin conjugate andevaluation as a tracer in an immunoassay for salivary cor-tisol measurement. J Steroid Biochem Mol Biol 1992;43(7):683–692.

19. Comoglio S, and Celada F: An immunoenzymatic assay ofcortisol using E. coli P-galactosidase as label. J ImmunolMethods 1976;10(2-3):161–170.

20. Ogihara T, Miyai F, Nishi K, Ishibashi K, and Kumahara Y:Enzyme labelled immunoassay for plasma cortisol. J ClinEndocrinol Metab 1977;44(1):91–95.

21. Lewis JG, and Elder PA: An enzyme linked immunosorbentassay (ELISA) for plasma cortisol. J Steroid Biochem1985;22(5):673–676.

22. Shrivastav TG, Kumari GL, and Rao PN: Enzyme immu-noassay of cortisol in human plasma using penicillinase aslabel. Clin Chim Acta 1988;174(1):83–91.

23. Mattox VR, Litwiller RD, and Nelson AN: A comparison ofprocedures for attaching steroidal glucosiduronic acids tobovine serum albumin. J Steroid Biochem 1979;10(2):167–172.

DIRECT DETERMINATION CORTISOL IN SERUM 285

24. Pandey PK, Shrivastav TG, Kumari GL, Rao PN, Grover PK,and Murthy HG: Enzyme immunosorbant assay of oestra-diol in unextracted plasma using penicillinase as label. ClinChim Acta 1990;190(3):175–184.

25. Asahina K, Kambegawa A, and Higashi T: Development of amicrotiter plate enzyme-linked immunosorbent assay for17a, 20b–21-trihydroxy-4-pregnen-3-one, a teleost gonadalsteroid. Fish Sci 1995;61:491.

26. Tintos A, Mıguez JM, Mancera JM, and Soengas JL: Devel-opment of a microtitre plate indirect ELISA for measuringcortisol in teleosts, and evaluation of stress responses inrainbow trout and gilthead sea bream. J Fish Bio 2006;68(1):251–263.

27. Hosoda H, Sakai Y, Yoshida H, Miyairi S, Ishii K, andNambara T: The preparation of steroid N-hydroxysuccinimideesters and their reactivities with bovine serum albumin.Chem Pharm Bull 1979;27(3):742–746.

28. Basu A, Shrivastav TG, and Kariya KP: Preparation of enzymeconjugate through adipic acid dihydrazide as linker and itsuse in immunoassays. Clin Chem 2003;49(8):1410–1412.

29. Yang TB, Zhong P, Qu LN, Wang CY, and Yuan YH: Pre-paration and identification of anti-2, 4-dinitrophenylmonoclonal antibodies. J Immunol Method 2006a;313(1-2):20–28.

30. Yang TB, Wang JH, Qu LN, Zhong P, and Yuan YH: Pre-paration and identification of anti-melatonin monoclonalantibodies. J Pineal Res 2006b;40(4):350–354.

31. Ren XF, Zhang FM, Chen FJ, and Yang TB: Development ofa sensitive monoclonal antibody-based ELISA for the de-tection of clenbuterol in animal tissues. Food Agri Immunol2009;20(4):333–344.

32. Zeng K, Yang TB, Zhong P, Qu LN, Zhou SY, He J, and JiangZS: Development of an indirect competitive immunoassayfor parathion in vegetable samples. Food Chem 2007;102(4):1076–1082.

33. Hosoda H, Miyairi S, Kobayashi N, and Nambara T: Spe-cificity of antisera raised against cortisol-4-bovine serumalbumin conjugates in radioimmunoassay. Chem PharmBull 1980;28(11):3369–3374.

34. Hosoda H, Kobayashi N, Miyairi S, and Nambara T: Thespecificity in enzyme immunoassay for plasma and urinecortisol. Chem Pharm Bull 1981;29(12):3606–3610.

35. Kohler G, and Milstein C: Continuous cultures of fused cellssecreting antibody of predefined specificity. Nature 1975;256(5517):495–497.

36. Lewis JG, Manley L, Whitlow JC, and Elder PA: Productionof a monoclonal antibody to cortisol: application to a directenzyme-linked immunosorbent assay of plasma. Steroids1992;57(2):82–85.

37. Mikola H, and Miettinen P: Preparation of europium-labeledderivatives of cortisol for time-resolved fluoroimmunoassays.Steroids 1991;56(1):21.

38. Kane JW: Use of sodium salicylate as a blocking agent forcortisol-binding-globulin in a radioimmunoassay for corti-sol on unextracted plasma. Ann Clin Biochem 1979;16(4):209–212.

Address correspondence to:Dr. Tangbin Yang

Medical Science CollegeChina Three Gorges University

8 University AvenueYichang 443002

HubeiChina

E-mail: y_t_b@hotmail.com

Received: December 9, 2010Accepted: February 1, 2011

286 LIU ET AL.