Enhanced immunoassay sensitivity using chemiluminescent acridinium esters with increased light output

Chemiluminescent acridinium ester labels are widely used in clinical diagnostics especially in automated immunochemistry analyzers such as Siemens Healthcare Diagnostics’ ADVIA Centaur® systems. Although, chemiluminescence from acridinium compounds was discovered more than 50 years ago, details regarding the excitation process are still not well understood particularly in relation to acridinium structure and overall light output. Herein, we report an empirical study that correlates the presence of electron-donating methoxy groups at C-2 and/or C-7 in the acridinium ring with increased light output. We further demonstrate that these high light output labels can be combined with hydrophilic functional groups such as hexa(ethylene)glycol to generate unique acridinium esters that are stable and are useful in improving immunoassay sensitivity for both competitive and sandwich automated immunoassays.     

Future applications of magic lite technology

The features that Magic Lite products offer as an immunoassay delivery system are discussed. The use of paramagnetic particles and acridinium ester labels confers advantages of speed, sensitivity, and stability. The technology has been used to measure analytes of widely varying molecular weights and serum concentrations, indicating its potential to detect the full range of clinically relevant analytes. Initial development efforts have indicated that the advantages of the system can be effectively exploited in an automated instrument.     

Acridinium ester-labelled DNA oligonucleotide probes

Chemiluminescent acridinium ester derivatives have been synthesized and covalently attached to suitably modified synthetic DNA oligonucleotides. Attachment of acridinium ester label to primary aliphatic amine group(s) present in the synthetic DNA probe molecule is rapid and efficient. Methods have been developed for efficient separation of acridinium ester-labelled DNA from unincorporated labelling reagent and underivatized DNA. The basic hydrogen peroxide detection reaction and photon counting conditions for measurement of chemiluminescence emission from acridinium ester-labelled DNA probes have been optimized. Under optimal conditions, the observed detection limit for the labelled DNA (1:1 mole ratio) is the same as for the free acridinium ester label, which is 2 attomole sensitivity in the best case studied.     

Nucleophilic addition to the 9 position of 9-phenylcarboxylate-10-methylacridinium protects against hydrolysis of the ester

The chemiluminescent reaction of an acridinium ester (AE) requires addition of peroxide to the 9 position of the acridinium ring. The addition of a hydroxide ion to the 9 position of an acridinium ester to form the carbinol adduct has also been well documented. We have observed a similar addition of other nucleophiles to the acridinium ring to form an acridan adduct. The adduct formed with bisulphite has been particularly well-characterized for rate of formation, rate of reversion, and reaction equilibrium. The formation of an adduct (other than H₂O₂) has been demonstrated to decrease significantly the reactivity of the adjacent ester bond to alkaline hydrolysis. The resulting, more stable adduct is very useful when the acridinium ester is used as a label in DNA probe-based assays. The adduct is highly resistant to hydrolysis under the conditions often desired for DNA probe-based assays (high temperature, elevated pH, extended storage).     

Immune complex transfer two-site chemiluminescent immunoassay for serum growth hormone in alevin chum salmon

An immune complex transfer two-site chemiluminescent immunoassay (CLIA) for salmon growth hormone (GH) was developed to measure serum GH in alevin chum salmon (Oncorhynchus keta) using a chemiluminescent acridinium ester as a label. The immune complex transfer method dramatically reduced non-specifically bound of acridinium ester-labelled antibody without a decrease in the specific binding. Consequently, we could detect lower levels of GH than achieved previously in a two-site CLIA for salmon GH. The detection limit of the assay was 7.8 fg/mL and the standard curve was linear up to 250 fg/mL. Coefficients of variation were 2.2–7.7% within-assay and 5.3–9.1% between-assay. We have developed a highly sensitive and reproducible GH method and applied it to measurement of GH in alevin chum salmon. © 1998 John Wiley & Sons, Ltd.     

Tracermer signal generators: An arborescent approach to the incorporation of multiple chemiluminescent labels

The synthesis, conjugation, and chemiluminescent evaluation of zero, first, and second order acridinium-based Tracermer^TM signal generators are described. Members of this family of labels have potential use as tracers in diagnostic assays and are structurally similar to arborol dendrimers. Tracermer^TM-BSA conjugates showed up to a sixfold increase in light emission compared to the normal acridinium label.     

On the use of acridinium indicators for the chemiluminescent determination of the total antioxidant capacity of dietary supplements

Acridinium salts, due to their chemiluminogenic properties, have found several applications in biomedical analysis as labels and indicators, where the assessment of emission intensity is used for the end‐point detection. This work presents the use of chemiluminescent indicators in the form of selected acridinium esters in order to determine the antioxidant properties of exemplary formulations, namely quercetin, vitamin C and the dietary supplement, Apiextract. The principle of measurements is based on a change in the kinetics of emission decay derived from the acridinium cations in alkaline solutions of hydrogen peroxide in the presence of an antioxidant (the analyte). The proposed system makes a beneficial alternative to related methods, which mostly rely on the assessment of emission efficiency and use the luminometric standard luminol – due to superior parameters of acridinium chemiluminescence, among others ‐ high temporary emission efficiency. The features of the proposed method are manifested by a shorter time period of analysis and lower background signals associated with the environmental influences, as compared to typical approaches. The chromatographic (RP‐HPLC) analyses of the substrates and products generated during chemiluminogenic oxidation of acridinium cations under assay conditions are also presented.     

Direct chemiluminescence immunoassay (CLIA) for muramyl tripeptide phosphatidyl-ethanolamine in plasma

A competitive chemiluminescent immunoassay for quantitation of muramyl tripeptide phosphatidyl-ethanolamine (MTP-PE) in plasma has been developed. The assay is based on the use of an acridinium ester-labelled analogue of muramyl tripeptide and a rabbit antiserum. It includes an overnight incubation and a separation with a second antibody covalently coupled to paramagnetic particles. The sensitivity of detection is 0.012 nmol/l, the assay working range is 0.1-5 nmol/l, and the inter-assay CVs are ? 10%. Using up to 6000-fold sample dilutions, a wide working range (0.1-30 000 nmol/l) is obtained. Rat plasma samples were collected during and one day after intravenous infusion of MTP-PE. Following infusion, the concentrations in plasma declined multiphasically. Half-life time was 0.37 h ± 0.03 (mean ± SD, alpha phase) and 1.76 h ± 0.08 (mean ± SD, beta phase), clearance and volume of distribution were 0.09 ± 0.02 l/h × kg (mean ± SD) and 0.06 ± 0.01 l/kg (mean ± SD) respectively. The use of an acridinium ester as a chemiluminescent (CL) label overcomes the problems associated with reagents of limited shelf-life.     

Method for reducing the rate of light emission from chemiluminescent and bioluminescent reactions using frozen reagents

Frozen assay reagents have been used to reduce the rate of light emission from the rapid chemiluminescent acridinium ester and the bioluminescent firefly luciferase reactions. Melting of the assay reagent delays the initiation of the light emission, thus eliminating the need to initiate these rapid reactions by injection of the assay reagents in front of the photodetector.     

Chemiluminescent immunoassay (CLIA) for urinary albumin

A simple chemiluminescent immunoassay (CLIA) for urinary albumin has been developed based on the use of a chemiluminescent acridinium ester-labelled human albumin and a commercially available antiserum. It includes two incubation steps and a second polyethylene glycol-assisted antibody separation. The sensitivity of detection is 0.016 mg/l, the assay working range is 0.1-5 mg/l, and the inter-assay CVs are ≤ 15%. Using 10? and 50-fold sample dilutions in assay buffer, a wide working range (1-250 mg/l) is obtained covering normal and pathological conditions. Timed overnight urine samples (bed rest conditions) were collected on three consecutive days for each patient. Albumin excretion rate (AER) was 4.7 ± 2.7 μg/min (x ± SD), range 1-15.9 μg/min in 36 healthy subjects (17♂, 19♀, ages 4-56 years), with day-to-day variations of 28.5 ± 20% (x ± SD), range 3.3-76.1%. The use of an acridinium ester as a chemiluminescent (CL) label overcomes the disadvantages of short shelf-life and health and safety hazards associated with radioisotopes. Results compare favourably with those obtained using a commercially available RIA kit.     

Effect of surfactants on the intensity of chemiluminescence emission from acridinium ester labelled proteins

In order to establish optimum conditions for the chemiluminescent (CL) reaction of two acridinium ester labelled proteins (human albumin and rabbit anti-human albumin IgG), we investigated the effects of the following factors known to influence the CL emission: pH, presence of proteins, relative concentrations of components of CL reaction and presence of surfactants. Under optimal conditions of pH and hydrogen peroxide concentration, hexadecyl trimethyl ammonium chloride (CTAC) increased the intensity of the CL reaction of the acridinium ester labelled albumin by 42-fold. Triton X-100, Tween-20, 23 lauryl ether (Brij 35) and sodium dodecyl sulphate (SDS) exerted a much smaller effect. In the case of the acridinium ester labelled antibody, the greatest increase was obtained with Triton X-100 (15-fold) followed by CTAC, Brij 35 and Tween 20 (SDS decreased the emission intensity).     

Electrochemical immunoassays

Immunoassays are routinely used to detect, specifically, low levels of many antigens. The trend away from the use of radioisotopic labels has resulted in a proliferation of alternative labels, many of which have electrochemical activity. The more successful of these assays have used enzyme labels, coupled with amperometric or potentiometric methods of detection of the products. A number of assays have also been designed which are specifically electrochemical in origin, not simply adaptations of currently used spectrophotometric methods. Much effort has been expended in developing a potentiometric immunoassay that measures the change in potential that should occur when an antibody binds to its antigen. The use of electroactive labels has resulted in a number of assays for drugs. The advantages of an enzyme-linked mediated assay for lidocaine, an antiarrhythmic drug, are discussed.     

Synthesis and chemiluminescent characteristics of two new acridinium esters

Two new acridinium esters with a 2-(succinimidyloxycarbonyl)ethyl side arm, namely, 9-(2,6-dibromophenoxycarbonyl)-10-methyl-2-(2-(succinimidyloxycarbonyl)ethyl)acridinium trifluoromethanesulfonate and 9-(4-(2-(succinimidyloxycarbonyl)ethyl)phenoxycarbonyl)-2,7-dimethoxy-10-methylacridinium triflate, have been produced and characterized. The chemiluminescent properties and hydrolytic stabilities of the new acridinium esters have been investigated.     

Photocleavage of DNA by the p-nitrobenzoyl group covalently linked to proflavine.

We have synthesized two novel DNA photocleaving agents,3,6-diamino-10-[6-(4-nitrobenzoyloxy)hexyl]acridinium chloride and 3,6-diamino-10-[6-(4-nitrobenzamido)-hexyl]acridinium chloride, and studied their DNA binding mode and cleavage properties. These compounds contain the photoactive p-nitrobenzoyl group attached to proflavine via an amide or ester linker group and a polymethylene chain. Spectroscopic and viscometric studies have shown that the compounds bind DNA by an intercalative mode. The presence of covalently-bonded intercalator is essential for the UV (310 nm) induced DNA scission. Above a critical ratio, an increase in the relative concentration of compound to DNA did not induce further cleavage. The cleavage efficiency was dependent on the type of linker group. These results are discussed in regard to possible mechanisms for photoinduced DNA breakage.     

Flow injection chemiluminescence study of acridinium ester stability and kinetics of decomposition

Decomposition of phenyl acridinium-9-carboxylate is monitored using electrogenerated chemiluminescence in a flow system. The formation of the pseudobase from the acridinium ester [AE] is described by rate = k′₁[AE] + k″₁[AE][OH^?]^0.5, where k′₁ = 0.020 ± 0.006 s^?1 and k″₁ = 2.1 ± 0.8 (L/mol)^?0.5 s^?1. Irreversible decomposition of the pseudobase is described by rate = k′₂[AE][OH^?], where k′₂ = 20.1 ± 3.8 (L/mol s). These kinetic equations, plus measurement of variation in emission intensity for constant acridinium ester concentration, are used to predict the resulting emission intensity v. pH behaviour given various contact times (in the 0.25 to 25 s range) for the acridinium ester to be in an alkaline solution prior to initiation of the chemiluminescence reaction.     

Homogeneous enzyme immunoassay of estradiol using estradiol-3-O-carboxymethyl ether as hapten

A homogeneous enzyme immunoassay for estradiol estimation has been developed, which can be extended to other steroids. A new procedure for the preparation of estradiol -3-0- carboxymethyl ether by a simple one step reaction in high yield (90%) has been described. This hapten has been used for raising highly specific anti-estradiol antibody in rabbits and for preparation of enzyme conjugates. Two different enzymes, lysozyme and glucose -6- phosphate dehydrogenase have been studied for their suitability as enzyme labels. Our results indicate that lysozyme-conjugate meets the essential requirement for a practical enzyme immunoassay. The advantage of the present nonradioactive procedure is the overall simplicity, low cost and high stability of the reagents.     

Synthesis,structure elucidation,and chemiluminescent activity of new 9-substituted 10-(ω-(succinimidyloxycarbonyl)alkyl)acridinium esters

Several new acridinium esters 2 – 9 having their central acridinium ring bearing a 9-(2,5-dimethylphenoxycarbonyl), 9-(2,6-bis(trifluoromethyl)phenoxycarbonyl) or 9-(2,6-dinitrophenoxycarbonyl) group, and a 10-methyl, 10-(3-(succinimidyloxycarbonyl)propyl), 10-(5-(succinimidyloxycarbonyl)pentyl), or 10-(10-(succinimidyloxycarbonyl)decyl) group, have been synthesized and their chemiluminescent properties have been tested. The 2,5-dimethylphenyl acridinium esters emit light slowly (glow) when treated with alkaline hydrogen peroxide, while the 2,6-dinitrophenyl and 2,6-bis(trifluoromethyl)phenyl esters emit light rapidly (flash). The substituent at the 10 position affects the hydrolytic stabilities of the compounds.     

Development of enantioselective chemiluminescence flow- and sequential-injection immunoassays for alpha-amino acids

The development of an enantioselective flow-through chemiluminescence immunosensor for amino acids is described. The approach is based on a competitive assay using enantioselective antibodies. Two different instrumental approaches, a flow-injection (FIA) and a sequential-injection system (SIA), are used. Compared to the flow-injection technique, the sequential injection-mode showed better repeatability. Both systems use an immunoreactor consisting of a flow cell packed with immobilized haptens. The haptens (4-amino-L- or D-phenylalanine) are immobilized onto a hydroxysuccinimide-activated polymer (Affi-prep 10) via a tyramine spacer. Stereoselective antibodies, raised against 4-amino-L- or D-phenylalanine, are labeled with an acridinium ester. Stereoselective inhibition of binding of the acridinum-labeled antibodies to the immobilized hapten by amino acids takes place. Chiral recognition was observed not only for the hapten molecule but also for a series of different amino acids. One assay cycle including regeneration takes 6:30 min in the FIA mode and 4:40 min in the SIA mode. Using D-phenylalanine as a sample, the detection limit was found to be 6.13 pmol/ml (1.01 ng/ml) for the flow-injection immunoassay (FIIA) and 1.76 pmol/ml (0.29 ng/ml ) for the sequential-injection immunoassay (SIIA) which can be lowered to 0.22 pmol/ml (0.036 ng/ml) or 0.064 pmol/ml (0.01 ng/ml) by using a stopped flow system. The intra-assay repeatability was found to be about 5% RSD and the inter-assay repeatability below 6% (within 3 days).     

Evidence for the localization of chlorophyll in lipid vesicles: a spin label study.

Fatty acid spin labels containing nitroxide groups at different positions in the fatty acid chain have been incorporated into lipid vesicles. Changes in esr parameters of the spin labels in the presence in the membrane of phytol, propionic acid phytol ester or chlorophyll $\text{a}$ and the kinetics of chlorophyll $\text{a}$ mediated photodestruction of the spin labels suggest a localization of the macrocyclic ring of the chlorophyll molecule in the polar head group region of the membrane.     

Osmium-labeled polynucleotides. Incorporation of additional heavy atoms (mercury) via ligand substitution reactions

The reaction of osmium tetroxide with biological materials, particularly nucleic acids, has considerable utility in electron microscopy and X-ray crystallography. This heavymetal label introduces an electrophilic center which may serve as a means of attachment of nucleophiles. Nucleophilic ligand substitution reactions have been exploited as a means of adding more heavy metals (mercury) onto the osmium label. Furthermore, the technique is quite general and could be used to modify the osmium label with, for example, fluorescent groups. The nucleophilic exchange reactions have also been studied using ¹H nmr spectroscopy with a representative heterocyclic osmate ester derivative, the bis(pyridine) osmate ester derivative of TMP. These studies have defined the nature of the ligands which lead to stable osmium labels.