Geoff Barnard

An assay for urinary estriol-16α-glucuronide based on antibody-enhanced chemiluminescence

An immunoassay for estriol-16 alpha-glucuronide in pregnancy urine is described that utilizes antibody-enhanced chemiluminescence. The steroid glucuronide was covalently conjugated with the chemiluminescent marker aminobutyl-ethyl-isoluminol. The light yield of this conjugate upon oxidation was augmented by specific antibody, and this effect was inhibited by addition of the homologous steroid glucuronide (10-100 pg) in a dose-dependent manner. The assay does not require separation of bound and free ligand and proved satisfactory with respect to sensitivity, precision and accuracy. Assay results obtained by radioimmunoassay and chemiluminescence immunoassay were in good agreement (r = 0.98; n = 25).

The measurement of progesterone in serum by a non-competitive idiometric assay

A novel non-competitive idiometric time-resolved fluoroimmunoassay for the determination of serum progesterone was developed, based on the use of two types of anti-idiotypic antibody that recognize different epitopes within the hypervariable region of the primary antiprogesterone antibody. The first anti-idiotype, the betatype, competes with progesterone for an epitope of the primary antiprogesterone antibody at the binding site. The second anti-idiotype, the alphatype, binds to the antiprogesterone antibody in the presence of progesterone, but does not bind to the betatype antiprogesterone complex due to epitope proximity. In the present configuration, the biotinylated alphatype was captured onto anti-biotin IgG which was immobilized on microtiter wells. Reaction mixtures containing europium-labeled antiprogesterone antibody complexed sequentially with progesterone in standards or serum samples and with the betatype anti-idiotypic antibody were then reacted with the immobilized alphatype anti-idiotypic antibody. After 30 min of incubation, the fluorescence of europium is measured by time-resolved fluorescence and is proportional to the concentration of progesterone over the range 0-320 nmol/mL. The method demonstrates good sensitivity, precision, and comparability with a direct competitive radioimmunoassay. The idiometric assay for progesterone is suitable for dipstick technology and biosensors.

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).

The measurement of oestrone-3-glucuronide in urine by non-competitive idiometric assay

We report a novel non-competitive idiometric assay for the measurement of oestrone-3-glucuronide (EG) in diluted urine. The method is based on the use of two types of anti-idiotypic antibody, the beta-type and alpha-type, that recognize different epitopes within the hypervariable region of the primary anti-EG antibody (Ab1). The beta-type anti-idiotypic antibody is analyte sensitive and competes with the analyte for an epitope of the primary antibody at the binding site. On the other hand, the alpha-type is analyte insensitive, but does not bind the Ab1 in the presence of the beta-type due to epitope proximity. In the present format, reaction mixtures containing the europium labelled Ab1 are reacted sequentially with EG standards or diluted urine samples, with the beta-type anti-idiotypic antibody and biotinylated alpha-type anti-idiotypic antibody on immobilized streptavidin coated microtiter plates. After 1 h incubation, the fluorescence of europium is measured by a time-resolved fluorescence and is proportional to the concentration of EG over a range of 0-10 nmol/l. The method demonstrates good sensitivity, precision and comparability with an alternative competitive fluorescent immunoassay. The idiometric assay for EG may be applied for the monitoring of ovarian function in women and is suitable for dipstick technology.

[25] Amplified bioluminescence assay using avidin-biotin technology

Publisher Summary This chapter discusses the amplified bioluminescence assay using avidin-biotin technology. The high affinity of avidin for biotin (KD = 10–5 M) provides a powerful tool for studies in many areas such as (1) isolation of biotin-derivatized materials by affinity chromatography, (2) localization and visualization of various antigens, (3) drug delivery, (4) lymphocyte stimulation, and (5) immunoassays. In immunoassay, advantage is taken of the four biotinbinding sites of avidin to amplify the sensitivity of the assay. The chapter describes a specific example that illustrates this general approach. A monoclonal or polyclonal antibody to a peptide hormone—for example, hCG—is immobilized onto a solid matrix. The antigen is added, followed by a biotinylated preparation of an antibody directed against a second epitope on the antigen. After the immunological reaction, a secondary probe consisting of avidin and a biotinylated NAD+-dependent enzyme [for example, biotinylated glucose-6-phosphate dehydrogenase (G6PDH)] is added. The end point is determined by bioluminescence using glucose 6-phosphate and NAD+ as substrates and bacterial luciferase/FMN/ decanal for initiation of light output. The results of an immunobioluminometric assay (IBMA) for hCG is given. The chapter describes the procedures for the preparation of biotinyl-G6PDH, preparation of biotinylated monoclonal anti-β-hCG, preparation of acetyl N-hydroxysuccinimide ester, preparation of acetylated avidin, and preparation of antibody-coated microtiter plates.

Novel assay procedure for assessing ovarian function in women

Abstract Augmented urinary steroid excretion of ocstrone-3-glucuronide (E 1 -3-G) precedes the mid-cycle LH-surge and is followed by increased pregnanediol-3α-glucuronide (P-3α-G) excretion when ovulation has ensued. We developed a solid-phase chemiluminescence-monitored immunoassay (C.I.A.) for these two metabolites which incorporates the following features: (1) Monoclonal antibodies (a.b.) are utilized to provide optimal specificity and rigorous standardization. Selected stable hybridomas between P3-X63-Ag8 variants and immune mouse spleen cells yielded a.b. titres of 1:80,000–500,000 ( K a , 10 10 l/mol) in ascites fluid. (2) The steroid conjugates are assayed as such, obviating the need for time-consuming hydrolysis and extraction steps. (3) An aminoalkyi derivative of isoluminol attached covalently to the steroid conjugate via the sugar moiety affords the labelled ligand (detection limit 4 pg). eliminating the use of radioactive labels and attending health hazards, disposal problems and radio-chemical decomposition of steroid. (4) Immunoadsorption of the analytes to the a.b.-coated cuvettes eliminates interfering luminescent urine components. Concurrent urine analyses by C.I.A., R.I.A. and G.L.C. yielded concordant results (r > 0.95). The same principles were found applicable to the assay of urinary oestriol-16α-glucuronide and plasma progesterone. The C.I.A. described represents the first application of monoclonal a.b. to hormone assay and oners significant advantages over existing methods in speed, safety and economy.

PREPARATION AND PROPERTIES OF ANTI-BIOTIN ANTIBODIES

Publisher Summary This chapter discusses the preparation and properties of anti-biotin antibodies. The chapter produces a high-affinity monoclonal antibody to biotin and compares the binding properties of the monoclonal anti-biotin with that of streptavidin in the development of noncompetitive immunoassays for polypeptide hormones and haptens. The V H and the V L cDNA of this antibody are sequenced and cloned. A comparative analysis of the V H sequence of the anti-biotin antibody with those of avidin and streptavidin reveals a similarity in the CDR2 and CDR3 regions of the antibody with known biotin-binding motifs in two of the homologous stretches of avidin and streptavidin. The V L sequence shows no similarity to such stretches of avidin or streptavidin. The second homologous stretch of avidin and streptavidin contains the sequence XXXYXT(S)XX, and the CDR2 region of the heavy chain of the antibody contains RTNYNSGL. Similarly, the fourth homologous stretch of avidin and streptavidin contains the sequence XXTXF(W)XG and the CDR3 region of the antibody heavy chain contains HTNWDG. These results seem to indicate a similar pattern in the sequences that may dictate the quality of biotin binding.

Noncompetitive immunoassay for small molecules

This chapter provides an overview of the noncompetitive immunoassay for small molecules. All immunoassay procedures are divided into two basic types—that is, competitive and noncompetitive. In 1968, Miles and Hales introduced a noncompetitive, two-site sandwich assay for measuring compounds with more than one antigenic determinant. These excess reagent assays have been termed immunometric assays and have largely replaced the earlier competitive method for the measurement of protein hormones in biological fluids. The advantages of immunometric assays are greater sensitivity, precision, and working range of the analyte. The major disadvantage of the two-site assay is that it is not suitable for the measurement of small molecules. It has been suggested that the fundamental difference between competitive and noncompetitive methods is based solely on the detection of antibody occupancy. A way to detect antibody occupancy that is a noncompetitive method for the measurement of small molecules using two anti-idiotypic antibodies that recognize different epitopes within the hypervariable region of the primary antibody is devised.

Alternatives to Radioimmunoassay

The most significant developments in recent years in immunoassay technology have been: (1) the use of monoclonal antibodies; (2) the introduction of simpler techniques to separate the antibody-bound and free fractions; (3) the tagging of an antibody instead of the antigen in a move from limited to excess reagent systems; and (4) the emergence of several methods utilizing non-radioactive labels as an alternative to radioactive labeling. To date, at least ten labels have been advocated (bacteria, erythrocytes, latex particles, gold sols, enzymes, substrates and cofactors; dyes, fluorescent, phosphorescent and chemiluminescent compounds).