F. Kohen

A direct non-competitive idiometric enzyme immunoassay for serum oestradiol

We report a novel non-competitive enzyme immunoassay for oestradiol based on the use of two types of anti-idiotypic antibody that recognize different epitopes within the hypervariable region of the primary anti-oestradiol idiotypic antibody (Ab1). The first anti-idiotype, the betatype, competes with the analyte for an epitope of the primary antibody at the binding site. On the other hand, the second anti-idiotype, the alphatype, binds to the Ab1 in the presence of analyte but does not bind to the betatype/Ab1 complex because of steric hindrance. In the present format the biotinylated alphatype was captured onto anti-biotin IgG which was adsorbed on the surface of microtitre wells. Reaction mixtures containing the Ab1 complexed sequentially with an enzyme labelled second antibody reagent, with oestradiol standards or serum samples and with the betatype anti-idiotypic antibody were then allowed to react with the immobilized alphatype anti-idiotypic antibody. The enzyme activity of the bound fraction measured at 405 nm increased with increasing oestradiol concentrations over the range 0.06-2.5 ng/ml. The detection limit of the assay was 28 pg/ml. The intra-assay variation ranged from 3.5 to 12.4%, and inter-assay variation from 6 to 13.4%. The results obtained by the colorimetric idiometric immunoassay correlated well with those obtained by a direct radioimmunoassay (n = 85, r = 0.97). This non-competitive immunoassay, termed idiometric assay, for haptens permits the development of sensitive immunoassays with a wide working range, and a variety of end-point determinations depending on the label used (e.g., enzyme, chemiluminescent or fluorogenic compound).

Chemluminescence immunoassay for estriol in saliva

Estriol is determined in whole saliva of pregnant women by a direct solid-phase chemiluminescence immunoassay. The assay uses a monclonal antibody raised against estriol-6-crboxymethyloxime/bovine serum albumin and the homologous chemiluminescent marker conjugate estriol- 6-carboxymethyloxime/aminopentylethylsiolominol (E3/APEI). The anti-estriol antibody is bound to the wells of a microtitration plate via a second antibody directed against the monoclonal antibody; 50 μl of saliva and 12.5 pg of E3/APEI per well are used. The incubation time is 10 min at room temperature. The calibration graph covers 5–750 pg of estriol and the detection limit is 4.5 pg (0.31 nmol l-1. Mean recovery of added estriol is 98%. Within-assay coefficient of variation is 11.8–5% for 0.21–6.5 nmol l-1 E3, and the between-assay value is 15.7–6.9% for 0.27-3.5 nmol l-1 E3. The correlation of E3 concentration in time-matched samples of saliva and sera from pregnant women was good (r = 0.934). Total assay time including calculation of results is 3 h for 40 saliva samples.

Development of a chemiluminescence immunoassay for salivary progesterone using microtitre plates as a solid phase

Abstract Progesterone is determined in whole saliva by a direct solid-phase chemiluminescence immunoassay (CIA), which uses a monoclonal antibody raised against progesterone-11α-hemisuccinate bovine serum albumin (BSA) and the homologous marker conjugate progesterone-11α-hemisuccinate aminobutylethylisoluminol. Rabbit anti-mouse immunoglobulins (second antibody) are coated on the wells of Nunc Maxisorb microtitre plates during an overnight incubation. Then monoclonal anti-estradiol antibody is bound overnight to the second antibody and residual “binding” places are blocked with BSA. Saliva is frozen, thawed and the clear supernate obtained after centrifugation is used. The calibration graph is linear over the range 3–200 pg of progesterone and the detection limit is 1.6 pg per well (0.096 nmol l −1 ). The mean recovery of added progesterone is 97%. The within-assay relative standard deviation (RSD) is 2.7–9.7% for 0.3–5.4 nmol l −1 progesterone and the between-assay RSD is 5.7–10.6% for 0.79–3.7 nmol l −1 progesterone. In pregnancy salivary progesterone rises from 1 nmol l −1 (6–15 weeks) to 4 nmol l −1 (36–40 weeks).

Recent advances in chemiluminescence-based immunoassays for steroid hormones

Several formats of solid-phase separation techniques for the measurement of steroids and urinary steroid conjugates using chemiluminescence as an end point are described. These formats include: (1) immunoadsorption of second antibodies directed against the first antibody on solid support; (2) specific immunoadsorbents consisting of primary antibodies covalently coupled to polymer beads; (3) second antibody coupled to a polymer containing magnetic particles. In these assays a steroid-chemiluminescent marker conjugate serves as the labelled ligand, and highly specific homologous monoclonal antibodies are used to provide optimal specificity and rigorous standardization. These techniques enabled the direct measurement of steroid glucuronides in diluted urine and of steroids in plasma.

[32] Surface chemiluminescent immunoassays of steroids

Publisher Summary This chapter describes the surface chemiluminescent immunoassays of steroids. Chemiluminescent conjugates of steroids or steroid glucuronides were prepared by conjugating carboxy derivatives of steroids or steroid glucuronides to primary amino derivatives of isoluminol. In the approach described here the following features are incorporated: (1) a steroid-chemiluminescent marker conjugate serves as the labeled ligand; (2) specific polyclonal or monoclonal antibodies are utilized either in the liquid phase or in the solid phase covalently coupled to polymer beads; (3) a solid-phase second antibody directed against the first antibody is utilized when the binding reaction is carried out in the liquid phase; (4) a centrifugation step after the binding reaction is used to remove interfering luminescent compounds along with unbound steroid; (5) dissociation of the label is achieved by incubation of the antibody bound fraction at pH 13 for 30 min at 60°; and (6) luminometry is performed by oxidation of the label with a H 2 O 2 -microperoxidase system. These assays have enabled the development of simple, reliable assays for the measurement of plasma steroids, for example, estradiol, testosterone, urinary steroid metabolites—for example, pregnanediol 3α-glucuronide and estrone 3-glucuronide, and therapeutic drugs such as digoxin.

Application of chemiluminescence immunoassays for steroid hormones in clinical endocrinological investigations in women

Abstract Immunoassays based on chemiluminescence for the measurement of serum and plasma steroids (estradiol, estriol, progesterone, testosterone, and cortisol), urinary steroid conjugates (estrone-3-glucuronide, estriol-16α-glucuronide and pregnanediol-3α-glucuronide) and peptide hormones (choriogonadotropin and luteinizing hormone) are surveyed briefly. These immunoassays are simple, robust and valid alternatives to radioimmunoassay. Homogeneous procedures and recent solid-phase assays based on purified specific antibodies, covalently coupled to polymer beads are discussed. Some new results are presented for solid-phase chemiluminescence immunoassays: estradiol is quantified in extracts of serum by using a monoclonal antibody to estradiol with estradiol-6-carboxymethyloxime-aminobutylethyl isoluminol as the marker ligand, and progesterone is quantified in unextracted serum by using a polyclonal antibody to progesterone, progesterone-11-hemisuccinyl-aminobutylethyl isoluminol as the marker ligand, and danazol (17α-pregna-2,4-dien-20-yno[2,3-d]-isoxazol-17-ol) to displace progesterone from serum binding-proteins. Their clinical utility is demonstrated.

Serum estradiol measurement by solid-phase chemiluminescence immunoassay and direct radioimmunoassay

Abstract Estradiol017β is determined in serum extracts by solid-phase chemiluminescence immunoassay. The results are compared with those obtained from unextracted serum in routine conditions with a commercial radioimmunoassay (r.i.a.) kit. For the chemiluminescence procedure, a purified monoclonal antibody to estradiol-6-carboxymethyloxime/bovine serum albumin and the homologous chemiluminescent marker conjugate estradiol-6-carboxymethyloxime aminobutylethylisoluminol are used. Bound and free ligand are separated by washing and simple centrifugation. Results obtained by the chemiluminescence assay ( y ) and by r.i.a. ( x ) on 170 serum specimens from women during ovulation induction showed good correlation ( y = 1.01 x − 16 with r = 0.95). The methods are similar in selectivity, detection limit (ca. 10 ng l −1 ) and precision (interassay relative standard deviation, 8–13%).

Improvement of assay sensitivity in immunoassay : chemiluminescence immunoassay for estradiol as a model

Abstract Variations of solid-phase format and ensuing assay sensitivity were compared. The assay, a chemiluminescence immunoassay for estradiol, uses a homologous combination of a monoclonal antibody to estradiol-6-carboxymethyl-oxime-bovine serum albumin and estradiol-6-carboxymethyloxime-aminobutylethylisoluminol (E 2 -ABEI) as chemiluminescent marker. When polyacrylamide beads were utilized as the solid phase for the monoclonal antibody, the detection limit (4.9 pg E 2 per cuvette) was moderate. When the monoclonal antibody was bound to microtitre plates, the detection limit decreased to 0.71 pg E 2 per well. Delayed addition of the E 2 -ABEI marker improved the detection limit to 0.37 pg E 2 per well. An alternative system was also evaluated: microtitre plates were coated with a second antibody and monoclonal antibody was incubated with the other reagents. This assay format, in combination with delayed addition of the marker, resulted in a detection limit of 0.25 pg E 2 per well.

Separation-Required Chemiluminescence Immunoassays for Steroids

Recent studies by Schroeder et al. (1978), from our laboratory (Kohen et al., 1985) and elsewhere (Pratt et al., 1978) have indicated that chemiluminescence immunoassay can be a feasible alternative to radioimmunoassay (RIA) of haptens. For our studies we chose as a chemiluminescent marker, derivatives of isoluminol possessing alkyl chains of an optional length of 2–6 atoms and terminating with a primary amino group (Schroeder and Yeager, 1978). The advantages of these compounds are that they can be measured at the level of pmol (Schroeder and Yeager, 1978), are stable (Pazzagli et al., 1983) and can be conjugated easily to carboxy derivatives of steroid or steroid glucuronides (Kohen et al., 1983) to yield the corresponding steroid-chemiluminescent tagged conjugates which can be utilized as markers in immunoassay procedures. Using these markers, we adopted two types of formats: (a) assays that do not require physical separation of bound and free hormone, so called “homogeneous”, and (b) assays that require a separation step, so called “heterogeneous”.