An indirect haemolytic assay for assessing antivenoms.

Dilutions of antivenom, venom, human erythrocytes and a phosphatidylcholine suspension, were incubated for 30 min at 37 degrees C. After centrifugation, the liberated haemoglobin was measured spectrophotometrically. The assay was used to assess an ovine antivenom against the venom from the South American rattlesnake, Crotalus durissus terrificus, and an equine Wyeth antivenin (Crotalidae, polyvalent). The ovine antivenom was more than five times as effective as the equine product. It also neutralized venoms from the Western diamondback rattlesnake, Crotalus atrox, and the fer-de-lance, Bothrops atrox. However, antivenoms raised against venoms from other Crotalus and Bothrops species provided little protection against the haemolytic activity of C. d. terrificus venom.

Comparison of three different activation methods for coupling antibodies to magnetisable cellulose particles.

The periodate and 1,1'-carbonyldiimidazole activation methods were compared with the cyanogen bromide procedure for coupling antibodies to magnetisable cellulose/iron oxide solid-phase particles. Fluoroimmunoassays for quinine, primaquine, normetanephrine and cannabinoids were employed to assess the binding properties of such coupled solid phases. The cyanogen bromide and 1,1'-carbonyldiimidazole methods gave similar products in most cases, while the specific binding capacity of periodate coupled particles was between two and five times lower. Nevertheless, comparable standard curves could be obtained with solid phase coupled by each method. The periodate and 1,1'-carbonyldiimidazole methods are acceptable alternatives, notably for laboratories lacking the facility to handle the toxic cyanogen bromide.

Development of a magnetisable solid-phase fluoroimmunoassay for primaquine and carboxyprimaquine.

Primaquine coupled to keyhole limpet hemocyanin was used as an immunogen to produce antiprimaquine antibodies in three sheep. The antisera obtained were characterised by the increase in fluorescence polarisation found upon binding to fluorescein-labelled primaquine prepared via same route. All sheep showed a good antibody response and one antiserum was coupled to magnetisable solid-phase particles to facilitate the separation of the antibody bound from free labelled antigen and the removal of interfering components which may be present in the sample. The fluoroimmunoassay requires addition of 100 microliters of standard or sample (urine or serum) to 100 microliters tracer (150 nmol/l) followed by 100 microliters of magnetisable solid-phase particles (12.5 g/l). After one hour incubation followed by the usual washing and eluting procedures, using a magnetic rack, the fluorescence of the supernatant was measured directly in a fluorimeter. Sodium salicylate was incorporated in the tracer solution to block the non-specific binding of tracer to the protein in serum samples. Cross-reactivity studies showed that the antibodies have high specificity for the 8-aminoquinoline nucleus but not to the 8-N-aminobutyl side chain. Thus carboxyprimaquine cross-reacted equally with primaquine and the assay can be used to measure their combined level. After extraction of primaquine from a basified sample with methylene chloride, the assay may be applied for the quantitation of either primaquine (in the organic phase) or its acidic metabolites including carboxyprimaquine (in the aqueous phase) separately. This approach was applied for the determination of total primaquine (primaquine and its metabolites) and extracted primaquine in urine samples following a single oral dose of 45 mg primaquine.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct single-reagent fluorescence polarisation immunoassay for valproic acid in serum.

A fluorescence polarisation immunoassay for quantitating serum concentrations of valproic acid was developed and validated. Its low molecular weight and lack of structural features caused difficulties in producing suitable antibodies. However, success was achieved using 2-propyl-6-aminohexanoic acid to make the fluorescein-labelled drug and two immunogens, the first using glutaraldehyde to link the drug derivative to keyhole limpet haemocyanin, and the second by carbodiimide activation of cellulose hydroxyl groups and coupling them to the drug derivative and killed Mycobacteria. It was found that both immunogens produced a good antibody response in sheep. The antibodies were highly specific and the assay results correlated well with an in-house gas-liquid chromatographic method.

A specific ELISA method for determining chloroquine in urine or dried blood spots.

Reported is an enzyme-linked immunosorbent assay (ELISA) that has been optimized and validated for the determination of chloroquine in urine or dried blood spots. The assay employs antisera raised in sheep to a chloroquine derivative conjugated to keyhole limpet haemocyanin and chloroquine conjugated to porcine thyroglobulin adsorbed onto the wells of a microtitration plate. The competitive binding of the antiserum to the wells was monitored using an alkaline-phosphatase-conjugated second antibody and a specific substrate. The assay exhibits no cross-reactivity with known chloroquine metabolites, other antimalarials, and commonly used drugs. The method was used to determine chloroquine in dried blood spot extracts and urine from a patient who was receiving a prescribed prophylactic chloroquine regimen. The drug was detected in the urine for 17 weeks and in the dried blood spots for 4 weeks after termination of the therapy.

Development of a single reagent polarisation fluoroimmunoassay for the detection of opiates in urine.

The design, development, and optimisation of a simple polarisation fluoroimmunoassay to detect the opiate group of drugs, in urine, is described. Urine (10 microL) is added to 1.5 mL of a single reagent, prepared by mixing ovine anti-opiate serum with fluorescein-labelled morphine. After incubation for 30 min at 30 degrees C, or 60 min at room temperature, fluorescence polarisation is measured. The assay can be used as a "stat" test or automated for large batch screening and detects the commonly abused opiates, heroin (through its metabolite morphine), codeine, and dihydrocodeine.

Development of fluoroimmunoassays for flecainide.

Antibodies raised in sheep against a flecainide:protein conjugate and fluorescein-labeled drug were used to develop simple fluoroimmunoassays for the measurement of flecainide acetate in serum or plasma. A rapid, nonseparation assay, based on fluorescence polarization was optimized for therapeutic drug monitoring. A separation fluoroimmunoassay, using antibodies covalently linked to magnetizable particles to avoid the need for centrifugation, was also optimized and validated for monitoring flecainide therapy. It is applicable to lipemic, hemolyzed or icteric samples unsuitable for the nonseparation approach and for laboratories with access only to a simple fluorimeter. Finally, the separation fluoroimmunoassay was modified slightly to improve markedly sensitivity for use in pharmacokinetic studies.

Polarisation fluoroimmunoassay for quinine in serum and urine.

The development and validation of a polarisation fluoroimmunoassay for the antimalarial drug quinine is described. The assay is performed either by sequential addition of the reagents or by a single-reagent technique whereby the tracer and antibodies are premixed. Serum samples require pepsin digestion prior to assay while urine specimens are assayed directly. The reliability criteria of the assay are satisfactory and no cross-reaction is detected with quinidine (the optical isomer of quinine) or with common antimalarial drugs. The assay was applied to the measurement of quinine in urine specimens obtained from a single-dose pharmacokinetic study and the results correlated with those of the benzene extraction fluorescence method for quinine measurement.

Quinine directly determined in serum or urine by separation fluoroimmunoassay.

We developed and validated a magnetizable solid-phase fluoroimmunoassay for directly determining quinine in serum or urine. We prepared the immunogen by coupling quinine hemisuccinate to keyhole-limpet hemocyanine, using this to immunize three sheep, and coupling the antisera to magnetizable solid-phase particles to facilitate separation of bound antigen from interfering components of serum or urine. We tested the stability of two fluorescein-labeled derivatives of quinine--one prepared by direct conjugation of quinine to dichlorotriazenyl aminofluorescein, the other by conjugating quinine hemisuccinate to fluorescein thiocarbamyl ethylenediamine. The latter was unstable. Using the former label and an 30-min incubation, we saw no interference by quinidine (an enantiomer of quinine) or other antimalarial drugs at their therapeutic concentrations.

Single-reagent polarisation fluoroimmunoassay for cotinine (a nicotine metabolite) in urine.

A rapid, single-reagent, non-separation, non-isotopic immunoassay was developed for determining levels of the nicotine metabolite, cotinine, in urine. The single reagent was prepared by pre-mixing an appropriate dilution of sheep anti-cotinine serum with a fluorescein-labelled cotinine tracer. All normal reliability criteria were satisfied. The assay was found to be specific for cotinine and there was no cross reactivity with other available nicotine metabolites and structurally-related compounds. The results obtained correlated closely with those of an established radioimmunoassay. The assay was well-suited to application in the discrimination of active smokers from non-smokers (and passive smokers).

Direct homogeneous phosphoroimmunoassay for carbamazepine in serum.

In this "phosphoroimmunoassay," a phosphorescent label is used: erythrosin (tetraiodofluorescein). Its long-lived phosphorescence was detected after pulsed excitation in a time-resolved luminescence spectrometer. To achieve convenient open-atmosphere phosphorimetry in liquid solution at room temperature, we used sodium sulfite as an "in situ" chemical deoxygenator, to eliminate oxygen quenching of the excited triplet state. Time-resolved detection allows for complete rejection of short-lived background signals, including those from fluorescent or light-scattering components of biological fluids that can interfere in fluoroimmunoassays. We chose the antiepileptic drug carbamazepine (CBZ) and its active metabolite CBZ-10, 11-epoxide (CBZE) to demonstrate the development and validation of nonseparation assays based on quenching the phosphorescence of erythrosin-labeled drug upon binding to antibody. These two compounds cross reacted equally with the antiserum used; hence, we measured the activity of both in patients' sera. Alternatively, simple pre-treatment of the sample with acid destroys CBZE immunoreactivity and enables specific assay of CBZ.

Single-reagent polarisation fluoroimmunoassay for the cocaine metabolite, benzoylecgonine, in urine.

A polarisation fluoroimmunoassay is described for the detection in urine of benzoylecgonine, the major metabolite of cocaine. The method uses a single reagent comprising sheep anti-benzoylecgonine serum pre-mixed with a fluorescein-labelled benzoylecgonine derivative as tracer. Under the assay conditions the antiserum has equal cross-reactivity with cocaine and the metabolite. The test is performed by the addition of 10 microL of urine to an aliquot of the single reagent, incubation for a few minutes and measurement of fluorescence polarisation. The assay has a positive/negative cut-off level of 1 mg/L benzoylecgonine and results compare favourably with the EMIT-d.a.u.TM (Syva) system.

Single-reagent polarization fluoroimmunoassay for theophylline in serum.

A polarization fluoroimmunoassay for theophylline was developed employing fluorescein-labeled drug and antiserum precombined in a single reagent. Assay was performed simply by addition of sample to an aliquot of the single reagent, incubation, and determination of fluorescence polarization. Because of the relatively rapid dissociation kinetics of the labeled drug from antibody binding, added unlabeled theophylline caused displacement within a practical time period. The precision, accuracy, and specificity of the simplified single-reagent assay were similar to those obtained by a conventional immunoassay procedure using the same reagents. Results for the assay of patients' serum specimens correlated well with those by an established enzymoimmunoassay.

Direct determination of theophylline in serum by fluoroimmunoassay using highly specific antibodies.

Described is the development of a fluoroimmunoassay for theophylline using a fluorescein labelled derivative of theophylline as tracer and antibodies coupled to magnetizable solid-phase particles. Three approaches are described for the preparation of antibodies for theophylline, of which one produced highly specific, high titre antibodies. The fluoroimmunoassay using these antibodies required a 10 microL sample, reached equilibrium within 5 min, and the results correlated closely with those of an established enzymoimmunoassay method. Potentially interfering endogenous fluorophores from the serum sample were reliably removed at the separation step of the bound and free fractions. There was no significant cross-reactivity with all other structurally related compounds.

Dual-label fluoroimmunoassay for simultaneous determination of primidone and phenobarbital.

A dual-label magnetizable solid-phase fluoroimmunoassay for direct determination of serum levels of primidone and its main metabolite, phenobarbital, in a single tube was developed and optimized. Fluorescein isothiocyanate was used to label phenobarbital and a rhodamine 101 derivative (XRITC) to label primidone; these could be independently quantitated with no fluorimetric "cross-talk." The dual-label assay employs mixed immunochemical reagents but is otherwise similar in performance to conventional, single-drug, magnetizable solid-phase fluoroimmunoassays. The ability to measure simultaneously two related analytes, such as a drug and its metabolite, represents a useful extension of immunoassay. This was illustrated in the present work by assay of sera from patients on primidone therapy. Dual-label assay results correlated well with those by a conventional enzymoimmunoassay (for primidone) and by a polarization fluoroimmunoassay (for phenobarbital).

Single-reagent polarization fluoroimmunoassay for barbiturates in urine.

We describe a rapid polarization fluoroimmunoassay for screening for the presence of barbiturates in urine. The single reagent is prepared by pre-mixing antiserum with a fluorescein-labeled barbiturate derivative as tracer. Use of a mixture of two sheep antisera, raised against different barbiturate immunogens, results in an assay with a broad cross-reactivity spectrum for most common barbiturates. One adds urine to the pre-mixed reagent, incubates the solution for a few minutes at room temperature, and measures the fluorescence polarization. The tracer/antiserum reagent is stable for at least six months at 4 degrees C. Results compare favorably with two other commonly used screening techniques for barbiturates, thin-layer chromatography and the EMIT-stTM (Syva) system. Although designed as a qualitative screen, some drugs can be quantified by means of a standard curve of the relevant barbiturate.

Influence of the hapten-fluorophore bridge on binding parameters in a fluoroimmunoassay for carbamazepine.

Raising antibodies to a hapten (drug or steroid) requires that it be coupled to a carrier protein through a bridge such that the hapten has maximum exposure with minimum changes in its configuration. In raising antisera in six sheep against carbamazepine (CBZ) coupled to bovine albumin, we found that some subpopulations of the antibodies recognized the bridge linking the drug to the carrier protein. To study the influence of the bridge on a fluoroimmunoassay for CBZ, we prepared four tracers by linking the carbamyl nitrogen of CBZ to fluorescein via four alkyl bridges of different lengths and structures. We calculated various binding parameters--including antibody affinity, binding capacity, and heterogeneity index--for each tracer and chose for the final fluoroimmunoassay the tracer that gave the best displacement with CBZ. We then optimized and validated the assay for direct measurement of CBZ in serum or plasma. The antibodies are coupled to magnetizable particles, which greatly facilitates separation and ensures removal of endogenous interferents.

Direct determination of primidone in serum or plasma by a magnetisable solid-phase fluoroimmunoassay.

A solid-phase separation fluoroimmunoassay for primidone in plasma or serum was developed using antibodies coupled to magnetisable cellulose/iron oxide particles and a fluorescein-labelled primidone derivative as tracer. Endogenous fluorophores and other potentially interfering components in serum samples were reliably and completely removed at the time the antibody-bound and free fractions were being separated. The separation and wash steps were greatly facilitated by use of magnetic sedimentation. The assay reached equilibrium within 30 minutes; all normal reliability criteria were satisfied; and the results correlated closely with those of an established enzymoimmunoassay method. The assay is specific for primidone, and no detectable interference by all other commonly employed anticonvulsants was found at serum levels of 1 g/l.