Establishing albumin levels in sheep serum by a specific fluoroimmunoassay.

A rapid and sensitive fluoroimmunoassay (FIA) was developed to measure albumin levels in sheep serum. The assay employed ovine albumin labelled with fluorescein as the tracer and a rabbit antiserum raised against sheep albumin. Separation of antibody-bound from free albumin was achieved using a second antiserum directed against the Fc component of rabbit IgG. Assay procedures were simplified by mixing the two antisera prior to use. Satisfactory assay validation parameters were demonstrated and the reagents were stable for at least 1 year at 4 degrees C. In contrast to the bromocresol green dye binding method, the accuracy of the FIA was unaffected by contaminating immunoglobulins. The FIA was used to establish reference ranges for serum albumin levels in lambs, in normal ewes and in ewes undergoing a prolonged course of immunisation. Mean serum albumin levels for these three groups were 38.8, 51.3 and 37.8 gL-1 respectively. The sensitivity of the assay makes it suitable to monitor albumin levels in therapeutic products derived from sheep blood or serum.

Development of a specific fluoroimmunoassay for ovine albumin.

A fluoroimmunoassay has been developed to measure serum levels of albumin in sheep. It employs ovine albumin labelled with fluorescein as the tracer and a rabbit antiserum raised against ovine albumin. Separation of the antibody bound and free fractions is achieved using a second antiserum directed against the Fc of rabbit immunoglobulin G and, to simplify the assay, the two antisera are premixed prior to use. Assay validation parameters are satisfactory and the reagents are predicted to be stable for at least one year at 4 degrees C. In contrast to the bromocresol green method, the assay is unaffected by immunoglobulins. A reference range for serum albumin levels has been established in lambs, normal ewes and ewes undergoing immunisation. Mean serum levels were 38.8, 51.3 and 37.8 g/l respectively. The sensitivity of the assay also enabled its use to monitor albumin levels at various stages during the production of specific antibody fragments from ovine antisera for therapeutic purposes.

Ovine desipramine antibody fragments reverse desipramine cardiovascular toxicity in the rat.

STUDY OBJECTIVE: To develop a model of severe desipramine cardiovascular toxicity and to determine whether partial neutralization of the antigen by desipramine-specific Fab antibody fragments ameliorates its cardiovascular effects. METHODS: We administered desipramine to rats until the QRS interval tripled in duration and mean arterial pressure (MAP) was less than 100 mm Hg. Animals were then assigned to one of six groups: (1) no treatment, (2) normal saline solution control treatment (.9% NaCl infusion equal to the volume of Fab infusion), (3) nonimmune Fab control treatment (infusion of Fab equal to that in the 9.6% neutralization treatment), (4) 9.6% desipramine Fab (infusion of ovine desipramine Fab equal to 9.6% of an equimolar neutralization), (5) 19.2% desipramine Fab, and (6) 30.0% desipramine Fab. RESULTS: QRS-interval duration, heart rate, and MAP were recorded for 60 minutes. Animals in groups 1 through 3 demonstrated slow and incomplete improvement. Animals in groups 4 through 6 showed improvement in QRS-interval duration and heart rate within 4 minutes (P<.05) compared with untreated animals. A dose-response relationship was evident; animals given the highest dose of desipramine-specific Fab showed the greatest improvement. CONCLUSION: Partial neutralization of desipramine by specific Fab fragments produces rapid improvement of QRS-interval duration and heart rate in a rat model of severe desipramine toxicity.

Comparison of a new ovine antigen binding fragment (Fab) antivenin for United States Crotalidae with the commercial antivenin for protection against venom-induced lethality in mice.

Snake venom poisoning is a medical emergency requiring immediate attention and the exercise of considerable judgment. Of the estimated 8,000 bites inflicted by venomous snakes in the United States each year, approximately 6,000 are treated with commercial antivenin. The only commercially available antivenin for North American Crotalidae envenomation is Antivenin (Crotalidae) Polyvalent (equine origin) (ACP; Wyeth Laboratories, Philadelphia, PA). A common complication is the high incidence of hypersensitivity reactions, occurring in more than 75% of patients treated with ACP. To minimize these side effects, a novel, affinity-purified, antigen binding fragment (Fab) antivenom (FabAV) for Crotalidae venom poisoning has been produced from the sera of sheep. The new product is Antivenin Polyvalent Crotalid (Ovine) Fab (Crotab; Therapeutic Antibodies, Inc., Nashville, TN). The current report compares the potencies in mice of FabAV and ACP against venom-induced lethality. The results indicate that FabAV is 3.1-9.6 times more potent than ACP for the prevention of lethality of the nine United States venoms tested. For one of the venoms, Crotalus viridis helleri, FabAV was efficacious while ACP was not.

Reduction of desipramine cardiotoxicity and prolongation of survival in rats with the use of polyclonal drug-specific antibody Fab fragments.

STUDY OBJECTIVE: Tricyclic antidepressants (TCAs) are a leading cause of death from intentional drug overdose. Available therapies are often unsatisfactory. In this study we evaluated the use of a high-affinity drug-specific polyclonal Fab fragment (TFab) as an antidote to desipramine toxicity. DESIGN: We gave anaesthetized rats under mechanical ventilation IV desipramine so that we might study the effect of TFab on survival or IP desipramine to facilitate study of the interaction of TFab and hypertonic sodium bicarbonate (NaHCO3), the standard clinical treatment for TCA overdose. INTERVENTIONS: For the study of the effects of TFab and NaHCO3 on survival, each rat was given a constant IV infusion of desipramine until it died, together with TFab 2 g/kg, bovine serum albumin, or .9% NaCl starting 5 minutes after the desipramine infusion. In the study of the interaction of TFab and NaHCO3, each rat received 30 mg/kg IP desipramine followed by TFab (molar TFab:desipramine ratio, .11), NaHCO3, TFab+NaHCO3, or NaCl at the time of maximal toxicity (15 minutes). RESULTS: In the survival protocol, QRS-interval duration, systolic blood pressure, and heart rate were significantly improved by TFab, and survival was prolonged by 58% compared with that in the albumin and NaCl groups (P < .001). The molar ratio of TFab to administered desipramine was .21. The unbound fraction of desipramine in serum at the time of death was reduced by TFab, but the unbound desipramine concentration was not, suggesting that TFab prolonged survival by delaying the increase in the unbound serum desipramine concentration. In the interaction protocol, neither TFab nor NaHCO3 was effective alone, but the combination significantly reduced QRS-interval prolongation (P = .001). CONCLUSION: These data demonstrate the efficacy of TFab in reducing desipramine-induced cardiovascular toxicity and prolonging survival. The pharmacokinetic effects of TFab in rats with severe desipramine toxicity were similar to those observed in sublethal desipramine toxicity. Therapeutic benefit is enhanced by the concurrent use of NaHCO3 and may be achieved despite binding only a fraction of the desipramine dose.

Toxicity of high doses of polyclonal drug-specific antibody Fab fragments.

Drug-specific antibody Fab fragments have been used as a treatment for acute drug overdose. For some drugs, the required Fab dose may be very high (up to several g/kg) and may have adverse effects of its own. The current study evaluated the potential toxicity of an affinity purified sheep polyclonal Fab (TFab) directed at the two antidepressants desipramine (DMI) and nortriptyline. TFab 4 g/kg was administered to anesthetized rats i.v. over 10, 25 or 60 min, with or without a toxic dose of DMI. This high dose of TFab, which is in excess of that needed to reduce DMI toxicity, was used in order to exaggerate any adverse effects. In the absence of DMI, TFab produced minimal changes in the electrocardiographic QRS duration, systolic blood pressure and heart rate compared with control animals and was well tolerated. In the presence of DMI, groups receiving TFab as a 10 or 25 min infusion showed a therapeutic effect (lessening of DMI toxicity) over the first 60 min compared with the control group, but one of six animals in each of the TFab groups died prior to the end of the 180 min experiment. No control animals died, but progressive QRS prolongation and decreasing blood pressure toward the end of the experiment suggested that DMI toxicity was increasing over time. These data suggest that, when administered alone, very high doses of rapidly infused TFab are well tolerated. When administered with DMI, TFab is effective in initially reducing DMI toxicity. However, this dose of TFab may later aggravate DMI toxicity and/or the effects of prolonged anesthesia.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversal of desipramine toxicity in rats with polyclonal drug-specific antibody Fab fragments.

Drug-specific antibodies, or fragments containing their binding site(s), are a potential means of treating drug overdose. Affinity purified polyclonal ovine Fab (TFab) with a high average affinity constant (Ka = 1.4 x 10(10) M-1) for the common tricyclic antidepressants was evaluated as a possible treatment for tricyclic antidepressant toxicity. Groups of eight anesthetized rats received 30 mg/kg body weight of desipramine (DMI) intraperitoneally, followed after 15 minutes by a 10-minute intravenous injection of 3 ml normal saline solution, 2 gm/kg nonspecific Fab as a control (CFab), or 1 or 2 gm/kg TFab (representing a molar ratio of TFab to DMI of 0.11 and 0.22, respectively). The animals were observed for 3 hours. During the initial 15 minutes, serum DMI levels in the four groups reached 2.3 to 2.9 micrograms/ml, the QRS duration increased by 67.5% to 77.9%, and the systolic pressure fell to between 65% and 85% of its initial value. The group given saline solution showed a gradual return of all these parameters toward normal, whereas CFab caused a transient further QRS prolongation. CFab also caused an initial rise in blood pressure, which then fell progressively, and two of the rats died 2 to 3 hours later with hypotension and bradycardia. Serum DMI concentration did not change significantly in either the saline or CFab groups.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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)

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.

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.