Digoxin Specific Antibody (Fab) Fragments in 34 Cases of Severe Digitalis Intoxication

Abstract

34 patients aged between 2 and 80 years were treated with digoxin specific antibody (Fab) fragments for severe digitalis poisoning. In 27 cases, the glycoside was taken with suicidal intent; in 3 cases accidentally and 4 were iatrogenic. The following criteria were considered to be indications for use of Fab fragments: the appearance of life-threatening arrhythmias such as high-grade atrioventricular conduction disorders (grade 2 and 3 A-V block), multifocal ectopic beats, ventricular tachycardia, and relapsing ventricular fibrillation. Serum digoxin concentrations were between 3.4 and 29ng/ml before the start of treatment. Between 240 and 800mg of Fab were administered; the majority of patients received 480mg. Regression of arrhythmias was seen between 0.5 and 8 hours after Fab infusion. There was a rapid fall in the free digoxin in the serum to concentrations that were no longer measurable and a marked rise in bound digoxin with a simultaneous increase of excretion of bound digoxin in the urine. Fab therapy is considered to be a major advance in the treatment of severe, previously fatal, glycoside poisoning. No notable side effects or allergic reactions were observed.     

Digoxin toxicity: pediatric survival after asystolic arrest

We report the first case of a child with known cardiac disease who presented in full cardiac arrest secondary to digoxin poisoning and was successfully resuscitated. A 12-week-old female presented 1-week status post surgical repair of a congenital heart anomaly in asystolic cardiac arrest. The patient was successfully resuscitated with standard Advanced Pediatric Life Support. A toxic digoxin level returned, Digoxin-specific antibody fragments (Digibind, Fab) were administered, and all signs and symptoms of toxicity resolved. The patient was discharged 6 days after presentation with full neurological recovery.     

Reversal of Advanced Digitoxin Toxicity and Modification of Pharmacokinetics by Specific Antibodies and Fab Fragments

The effects of Fab fragments of high-affinity specific antibodies have been studied in a canine experimental model of lethal digitoxin toxicity. Selected antiserum from sheep immunized and boosted with a digoxin-serum albumin conjugate contained antibodies that cross-reacted with digitoxin with an average intrinsic association constant of 1.4 × 10¹⁰ M⁻¹ as determined by equilibrium dialysis. Rapid second-order association kinetics (k_f = 3.7 × 10⁶ M⁻¹ per s) and slow dissociation kinetics (k_r = 1.9 × 10⁻⁴ per s) were documented for the antibody-digitoxin complex. Eight dogs given 0.5 mg/kg digitoxin intravenously developed ventricular tachycardia after 23±4 (SEM) min. Control nonspecific Fab fragments were then given. All animals died an average of 101±36 min after digitoxin administration. Another eight dogs given the same digitoxin dose similarly developed ventricular tachycardia after 28±3 min. This group then received a molar equivalent dose of specific Fab fragments intravenously over 3 min, followed by a 30-min infusion of one-third of the initial dose. All dogs survived. Conducted sinus beats reappeared 18±4 min after initial Fab infusion, and stable normal sinus rhythm was present at 54±16 min. Plasma total digitoxin concentrations increased threefold during the hour after initial Fab infusion, while plasma free digitoxin concentration decreased to less than 0.1 ng/ml. Effects on digitoxin pharmacokinetics of these Fab fragments and the antibody population from which they were derived were further investigated in a primate species. Unlike common laboratory animals previously studied, the rhesus monkey was found to have a prolonged elimination half-life, estimated at 135 and 118 h by radioimmunoassay and [³H]digitoxin measurements, respectively, similar to man and thus providing a clinically relevant experimental model. Intravenous administration of 2 mol of specific Fab fragments per mole of digitoxin 6 h after 0.2 mg of digitoxin produced a rapid 4.3-fold increase in plasma total digitoxin concentration followed by a rapid fall (t_½ 4 h) accompanied by a 14-fold enhancement of urinary digitoxin excretion over control values during the 6-h period after Fab was given. Analytical studies were consistent with increased excretion of native digitoxin rather than metabolites, and the glycoside was found in equilibrium dialysis studies to be excreted in the urine in Fab-bound form. Administration of 2 mol of specific antibody binding sites per mole of digitoxin as intact IgG caused a greater and more prolonged increase in plasma total digitoxin concentration, peaking 13-fold above control levels. In contrast to the effects of Fab, however, specific IgG reduced the rate of urinary digitoxin excretion substantially below control values. We conclude that Fab fragments of antibodies with high affinity for digitoxin are capable of rapid reversal of advanced, otherwise lethal digitoxin toxicity, and are capable of reducing the plasma half-life and accelerating urinary excretion of digitoxin.     

Electrocardiographic Response of Digoxin-Toxic Fascicular Tachycardia to Fab Fragments: Implications for Tachycardia Mechanism

The electrocardiographic response of digoxin-induced fascicular tachycardia to Fab fragments was evaluated in two patients. In addition, we documented the response of the fascicular tachycardia to spontaneous premature ventricular depolarizations during different tachycardia rates, the response to a nonsustained episode of ventricular tachycardia, and the mode of spontaneous initiation and termination of short-lived episodes of the tachycardia during the treatment process. The following findings were noted: slowing of the tachycardia in response to Fab administration; change in the morphologic characteristics of the tachycardia from multiform to uniform; resetting of the tachycardia by spontaneous premature ventricular depolarization with the return cycle equal to the observed tachycardia cycle length; acceleration of the tachycardia in response to five beats of a faster nonsustained ventricular tachycardia; and initiation and termination of the tachycardia, both by spontaneously occurring premature ventricular depolarizations and in the absence of premature ventricular depolarizations. Both tachycardias resolved completely within 20 and 40 minutes, respectively, of Fab administration. We conclude that Fab administration can promptly resolve fascicular tachycardias precipitated by digoxin toxicity and that the observed electrocardiographic phenomena strongly suggest triggered activity as the electrophysiologic mechanism of fascicular tachycardia in man.     

Recurrent digoxin overdose and treatment with digoxin-specific Fab antibody fragments

A case of overdose with digoxin on three separate occasions is presented. The patient received digoxin-specific Fab antibody fragments on each presentation without adverse effects. A discussion of digoxin toxicity and specific treatment follows. Immunologic aspects of therapy are emphasized.     

Utilization of recombinant Fab fragments in a cTnI immunoassay conducted in spot wells

OBJECTIVES: To evaluate the performance of a new cTnI immunoassay utilizing site-specifically biotinylated recombinant Fab fragments on recently established spot wells. DESIGN AND METHODS: Two different cTnI-specific recombinant site-specifically biotinylated Fab fragments were produced. The performance of the new sandwich-type cTnI immunoassay in spot wells was evaluated in terms of binding capacity, assay kinetics and assay sensitivity and compared with a cTnI immunoassay carried out in conventional microtitration wells. Furthermore, the functionality of the recombinant Fab fragments was compared to the corresponding monoclonal antibodies in assay with one, two or three capture antibodies. RESULTS: The signal-to-background level was improved, providing an analytical detection limit of 0.002 microg/l with a surface of two capture Fab fragments. The spot wells increased the signal levels 2-fold and a further 4-fold improvement was detected with the Fab fragments already after 5 min assay time. CONCLUSIONS: The spot-concept in combination with site-oriented capture Fab fragments carries great promise as a very useful approach to improve the immunoassay performance of future point-of-care cTnI assays.     

Fatal cardiac glycoside poisoning due to mistaking foxglove for comfrey

Context: Accidental ingestion of foxglove (Digitalis purpurea) can cause significant cardiac toxicity. We report a patient who ingested foxglove mistaking it for comfrey and developed refractory ventricular arrhythmias. The patient died despite treatment with digoxin-specific antibody fragments (DSFab) and veno-arterial extracorporeal membrane oxygenation (VA-ECMO).

Case details: A 55-year-old woman presented to the emergency department with nausea, vomiting and generalized weakness eight hours after drinking “comfrey” tea. She had bradycardia (54 beats/min) and hyperkalemia (7.6?mEq/L). Electrocardiogram revealed a first-degree atrioventricular conduction block with premature atrial contractions, followed by polymorphic ventricular tachycardia three hours after arrival. A serum digoxin level was 151.2?ng/mL. The patient developed ventricular fibrillation while waiting for Digibind infusion. Resuscitation was performed and an emergent VA-ECMO was set up. A total of eight vials of Digibind were given over the next 16?hours. She temporarily regained consciousness, but remained hemodynamically unstable and subsequently developed lower limb ischemia and multiple organ failure, and she expired on hospital day seven. A botanist confirmed that the plant was foxglove.

Conclusions: The diagnosis of cardiac glycoside plant poisoning can be difficult in the absence of an accurate exposure history. In facilities where DSFab is unavailable or insufficient, early VA-ECMO might be considered in severely cardiotoxic patients unresponsive to conventional therapy.     

Acute massive digoxin overdose: survival without use of digitalis-specific antibodies

Acute massive digoxin overdose may result in life-threatening arrhythmias, with reported mortality of up to 20% prior to the introduction of digitalis-specific antibodies. Digitalis-specific Fab antibody fragments remain under experimental protocol and are not widely available. Interpretation of serum digoxin levels and indications for the use of Fab are not clearly established. The authors report a case of massive digoxin overdose in an 18-month-old child with the highest reported digoxin level (48 ng/ml) with which a victim survived without the need for Fab administration. She developed only mild manifestations of digitalis intoxication, and her serum potassium never exceeded 5.2 mEq/l. Her course may be explained by the distribution kinetics of digoxin, which follows a two-compartment model, and the relative resistance of children to digitalis intoxication. This case emphasizes the need for better criteria than the digoxin level for the administration of Fab. The serum potassium concentration, which is usually elevated in acute type digitalis intoxication, may be a better predictor of the need for Fab in acute massive digitalis ingestion.     

Recognition by recombinant autoimmune thyroid disease-derived Fab fragments of a dominant conformational epitope on human thyroid peroxidase.

To characterize the nature of thyroid peroxidase (TPO) autoantibodies present in the sera of patients with autoimmune thyroid disease, we cloned three IgG1/kappa Fab fragments which bind 125I-TPO. This was accomplished by the molecular cloning and expression in bacteria of IgG gene fragments from B cells infiltrating the thyroid of a patient with Graves' disease. The three Fab fragments (SP2, SP4, and SP5) are coded for by a common heavy chain (VH1, D, JH3) and three related, but different, light chains (VK1, JK2). The SP Fab fragments bind specifically to TPO with high affinities (6 x 10(-11)-2 x 10(-10) M) comparable to those of serum TPO autoantibodies. TPO autoantibodies represented by the SP Fab fragments are present in all 11 patients studied, constitute a high proportion (36-72%) of serum TPO autoantibodies in individual patients and interact with a conformational epitope on TPO.     

Reversal of lethal digoxin toxicity in guinea pigs using monoclonal antibodies and Fab fragments

To extend the potential application of digoxin-specific immunoglobulin (Ig) (Fab) fragments for the reversal of advanced digitalis intoxication, monoclonal digoxin-specific antibodies were obtained by fusion of myeloma cell lines with spleen cells from mice immunized with a digoxin-serum albumin conjugate. The monoclonal antibody from the cell line designated Dig 26-10 had high affinity (KA = 5 X 10(9) M-1) and specificity for digoxin and was tested for its efficacy in the reversal of advanced, otherwise lethal digoxin toxicity in guinea pigs given a loading dose of 500 micrograms of digoxin per kg b.wt. i.v. followed by continuous infusion of digoxin at 10 (IgG-treated group) or 50 (Fab-treated group) microgram/kg/min. Control animals given nonspecific rabbit or mouse Igs after the onset of digoxin-toxic ventricular arrhythmias all died. Administration of monoclonal digoxin-specific antibody as intact IgG in doses stoichiometrically equivalent to the digoxin dose fully reversed digoxin toxicity in six of eight animals and prolonged survival somewhat in the remaining two animals. Fab fragments from the Dig 26-10 monoclonal antibody were even more effective, with rapid reversal (mean time 7 min) of all arrhythmias and survival of all animals so treated. We conclude that murine monoclonal antibodies and their Fab fragments are capable of reversing advanced and otherwise lethal digoxin-induced arrhythmias in a guinea-pig experimental model and offer potential advantages over polyclonal antibodies in the management of this clinically important problem.     

Electrocardiographic changes with segmental akinesia after chloral hydrate overdose

We report a case of deliberate ingestion of 12.5 g chloral hydrate in a 25-year-old psychiatric patient. Coma and life-threatening ventricular dysrhythmias were observed soon after ingestion. Repeated electrocardiographic examination was consistent with ischemic changes appearing on day 3. They were associated with segmental abnormal left ventricular wall motion by echocardiography. A coronary angiogram was performed and was normal. Toxic metabolites of chloral hydrate, trichloroethanol and trichloroacetic acid were found in the urine until day 7. This case illustrates that with halogenated aliphatic hydrocarbons, sustained changes in cardiac contractility may occur in addition to early life-threatening ventricular dysrhythmias.     

Assessment of digoxin antibody use in patients with elevated serum digoxin following chronic or acute exposure

OBJECTIVE: To evaluate the use of antidotal therapy in patients with an elevated digitalis concentration following chronic or acute exposure. DESIGN AND SETTING: Retrospective review of patient records over 2 years in 20 city hospitals in France. PATIENTS: Overall 838 patients with an elevated serum digitalis concentration (digoxin[Symbol: see text]>[Symbol: see text]1.95[Symbol: see text]ng/ml or digitoxin[Symbol: see text]>[Symbol: see text]23[Symbol: see text]ng/ml) were included in the study. Of these, 67 (8%) had received antidotal therapy with Fab fragments. MEASUREMENTS AND RESULTS: The relationships between previously reported prognostic criteria and use of antidotal therapy were investigated. We identified five independent factors that were associated with the use of antidotal therapy: acute overdose (OR 15.74), Fab fragment availability in the hospital (11.06), serum potassium (1.81), and heart rate (0.96). Mortality was significantly lower in Fab-treated (6%, 4/67) than untreated patients (15%, 117/770). CONCLUSIONS: Antidotal therapy is underused in patients with an elevated digitalis concentration especially in patients with chronic digitalis exposure. These patients in our series presented a higher mortality rate than patients with acute poisoning. Although they were older and tended to have a history of cardiac disease, they did not differ from patients with acute poisoning with regard to the main severity criteria and prognostic factors. The use of identical criteria for antidotal treatment after acute and chronic poisoning should help optimize outcomes. Fab fragment availability is insufficient in France but ranks only second after type of poisoning (acute or chronic) in the multivariate association with Fab treatment.     

Alloantiserum-mediated suppression of histocompatibility-linked Ir-gene-controlled immune responses. Suppressive effects of IgG fragments derived from alloantisera

Fab, Fc, and F(ab)''₂ fragments were prepared by enzymatic hydrolysis of the IgG fraction of strain 13 antistrain 2 alloantisera. These fragments were not cytotoxic to lymphocytes bearing strain 2 histocompatibility antigens, but the Fab and F(ab)''₂ fragments retained functional combining sites as indicated by their ability to suppress the cytotoxicity mediated by the intact antistrain 2 antibodies. The F(ab)''₂ fragments were much more efficient as inhibitors in this system than the Fab fragments. F(ab)''₂ at 0.06 mg/ml and 0.45 mg/ml Fab produced comparable degrees of suppression. The F(ab)''₂ at 0.06 mg/ml completely suppressed DNP copolymer of L-glutamic acid and L-lysine (GL)-stimulated tritiated thymidine incorporation. The monovalent Fab at 0.45 mg/ml, however, had no significant effect on the in vitro responses to DNP-GL. Addition of the intact alloantisera can be delayed 3 h after initiation of the antigen-stimulated cultures with no loss of suppression. After a delay of 6 h 45% suppression was observed. The requirement for the divalent molecule and the observation that effective suppression of the in vitro responses is still obtained when the alloantiserum is added several hours after initiation of the cultures both suggest that the immunosuppression results from an active process affecting the lymphocyte membrane that renders the cell refractory to the antigenic stimulus.     

Extracorporeal life support and digoxin-specific Fab fragments for successful management of Taxus baccata intoxication with low output and ventricular arrhythmia

Introduction

Yew plants are evergreen shrubs which are widely spread throughout the northern hemisphere. Taxane alkaloid derivatives, mainly taxine B, represent the main toxins of Taxus baccata and are highly cardiotoxic. Due to the lack of randomized clinical trials, case reports on accidental or suicidal yew intoxications build the only source of knowledge of clinical treatment options.

A molecular approach for isolating high-affinity Fab fragments that are useful in blood group serology

BACKGROUND: Multiple mouse hybridoma antibodies recognize the antigens of the MNS blood group system. The Fab fragments of several of these antibodies were expressed on bacteriophage and as soluble proteins. The parental N92 anti-N IgG monoclonal antibody (parental N92 MoAb), but not its monovalent, soluble Fab fragment (N92 Fab fragment), agglutinated antigen-positive red cells by an antiglobulin method. Light-chain shuffling was used to isolate mutant N92 Fab fragments with higher affinity that would function by agglutination. STUDY DESIGN AND METHODS: Light-chain cDNA libraries, constructed from mice immunized with N-type glycophorin A, were inserted into a recombinant pComb3H vector containing the N92 Fd fragment. The N92 Fd fragment:light-chain libraries were panned on N-type glycophorin A or NN red cells, and antigen-binding clones were isolated. Purified parental N92 MoAb and the Fab fragments were evaluated by enzyme-linked immunosorbent assay and agglutination. RESULTS: The novel NNA7, C1, and G11 Fab fragments all bound to N-type glycophorin A with higher affinity than did the N92 Fab fragment. The affinity of the library-derived clones was equivalent to that of the parental N92 MoAb. Although their fine specificity differed slightly from the parental N92 MoAb, the clones functioned equivalently by agglutination using an antiglobulin method. CONCLUSIONS: Light-chain shuffling allowed the isolation of bacterially produced, high-affinity, soluble, monovalent recombinant anti-N Fab fragments that functioned well by agglutination. This approach is useful in obtaining inexpensive serologic reagents that may replace conventional MoAbs produced by tissue culture methods.     

Acute Digitalis Intoxication — Is Pacing Still Appropriate?

Abstract

Over a six year period, 92 patients intoxicated with either digitoxin or digoxin were admitted to our ICU. Fifty-one patients were treated with cardiac pacing and/or Fab fragments, and the mortality rate was 13% (14 were intoxications with digoxin, 36 with digitoxin, 1 was mixed). Forty-five cases were suicide attempts; six were accidental overdosages. Since cardiac pacing may trigger fatal arrhythmia or delay the administration of Fab fragments, we conducted a retrospective study to determine whether fatal outcomes could be related either to cardiac pacing or to unsatisfactory use of immunotherapy. In our study, prevention of life-threatening arrhythmia failed in 8% of cases with Fab and in 23% with pacing. Though Fab tended to be more effective, this difference was not significant. In our study, the main obstacles to the success of Fab were pacing-induced arrhythmias and delayed or insufficient administration of Fab. Iatrogenic accidents of cardiac pacing were frequent (14/39, 36%) and often fatal (5/39, 13%). In contrast, immunotherapy was not associated with any serious adverse effects (0/28, 0%) and was safer than cardiac pacing (p < 0.05). In conclusion, during digitalis intoxication, the pacemaker has limited preventive and curative effects, is difficult to handle, and exposes patients to severe iatrogenic accidents. Fab fragments act as a powerful antidote and are safer and much easier to use than pacing. These results encourage us to prescribe Fab fragments as first-line therapy during acute digitalis intoxication.     

Intradermal Anti-Loxosceles Fab Fragments Attenuate Dermonecrotic Arachnidism

OBJECTIVE: Bites from the brown recluse spider and other arachnids from the genus Loxosceles frequently induce necrotic skin lesions that can be recalcitrant to treatment and disfiguring. The authors used a rabbit model of dermonecrotic arachnidism to address the therapeutic efficacy of intradermal (id) polyclonal anti-Loxosceles Fab fragments (alphaLoxd Fab) raised against Loxosceles deserta spider venom. METHODS: Fab fragments were prepared by papain digestion and affinity chromatography from the IgG fraction of L. deserta antivenom raised in rabbits. Eighteen inbred New Zealand white rabbits were assigned to six groups of three. The rabbits received L. deserta venom (3 microg, id) injections into each flank. Cohorts of rabbits received single id injections (at one venom site/rabbit) of 30 microg alphaLoxd Fab at different times (T = 0, 1, 2, 4, 8, and 12 hours) after venom injection. In each rabbit the opposite flank was left untreated. As an additional control, one group of rabbits (T = 0) received nonspecific Fab (30 microg, id) in the opposite flank. Dermal lesions were quantified as a function of time through the use of a series of digital photographs and imaging software. In addition, myeloperoxidase (MPO) activity, a measure ofneutrophil accumulation, was determined in lesion biopsies. Lesion areas and MPO activities were analyzed by repeated-measures analysis of variance (ANOVA). RESULTS: Lesion areas and MPO activity were markedly reduced when alphaLoxd Fab was administered very early after venom injections. As the interval between venom inoculation and antivenom treatment increased, the therapeutic benefit of alphaLoxd Fab decreased. The final time tested that demonstrated therapeutic efficacy of alphaLoxd Fab was T = 4 hours. Lesion attenuation was no longer apparent when alphaLoxd Fab was given 8 hours post inoculation. CONCLUSIONS: Intradermal administration of alphaLoxd Fab attenuates Loxosceles-induced dermonecrotic lesion formation when given up to 4 hours after venom inoculation in this rabbit model.     

Detection of colorectal carcinoma by emission-computerized tomography after injection of 123I-labeled Fab or F(ab'')2 fragments from monoclonal anti-carcinoembryonic antigen antibodies

This clinical study was based on experimental results obtained in nude mice grafted with human colon carcinoma, showing that injected 131I-labeled F(ab')2 and Fab fragments from high affinity anti-carcinoembryonic antigen (CEA) monoclonal antibodies (MAb) gave markedly higher ratios of tumor to normal tissue localization than intact MAb. 31 patients with known colorectal carcinoma, including 10 primary tumors, 13 local tumor recurrences, and 21 metastatic involvements, were injected with 123I-labeled F(ab')2 (n = 14) or Fab (n = 17) fragments from MAb anti-CEA. The patients were examined by emission-computerized tomography (ECT) at 6, 24, and sometimes 48 h after injection using a rotating dual head scintillation camera. All 23 primary tumors and local recurrences except one were clearly visualized on at least two sections of different tomographic planes. Interestingly, nine of these patients had almost normal circulating CEA levels, and three of the visualized tumors weighed only 3-5 g. Among 19 known metastatic tumor involvements, 14 were correctly localized by ECT. Two additional liver and several bone metastases were discovered by immunoscintigraphy. Altogether, 86% of the tumor sites were detected, 82% with F(ab')2 and 89% with Fab fragments. The contrast of the tumor images obtained with Fab fragments suggests that this improved method of immunoscintigraphy has the potential to detect early tumor recurrences and thus to increase the survival of patients. The results of this retrospective study, however, should be confirmed in a prospective study before this method can be recommended for the routine diagnosis of cancer.     

Determination of free digoxin concentrations in serum for monitoring Fab treatment of digoxin overdose

A rapid method for assessing the free digoxin concentration in the serum of digoxin-overdosed patients receiving treatment with digoxin-specific Fab fragments has been developed. For this method, a protein-free ultrafiltrate is prepared from the patient's serum, and the digoxin in the ultrafiltrate (free digoxin) is measured by fluorescence polarization immunoassay. Both the inaccuracies associated with measurements of total digoxin by immunoassay in the presence of Fab and the long turnaround time associated with measurements of free digoxin by equilibrium dialysis were avoided. Good correlation was observed between measurements of free digoxin by this ultrafiltration technique and by equilibrium dialysis. The ultrafiltration method was used to evaluate the concentrations of free digoxin in a digoxin-overdosed patient treated with Fab at our hospital. In retrospect, the results suggest that her hospital stay could have been shortened by a timely appreciation of her increased concentration of free digoxin. Using the ultrafiltration method, one can determine free digoxin concentrations quickly, conveniently, and accurately in the clinical laboratory. This procedure therefore should be a valuable aid in monitoring the efficacy and adequacy of Fab treatment.     

Dual-label time-resolved immunofluorometric assay of free and total prostate-specific antigen based on recombinant Fab fragments

BACKGROUND: Recombinant Fab fragments are attractive as reagents for novel sandwich immunoassays, but no such assays have been described. We developed a dual-label two-site immunoassay based entirely on recombinant Fab fragments and compared it to the same assay with intact monoclonal antibodies. METHODS: The capture Fab fragment, which binds free prostate-specific antigen (PSA) and PSA in complex with alpha(1)-antichymotrypsin on an equimolar basis, is site-specifically biotinylated and attached to the solid phase in streptavidin-coated microtitration wells. The Fab fragment that detects only free PSA is site-specifically labeled with a fluorescent europium chelate, and the Fab fragment that detects both free and serpin-complexed PSA in an equimolar fashion is labeled with a fluorescent terbium chelate. Time-resolved fluorescence is used to measure both europium and terbium signals in one well. RESULTS: The detection limits of the assay (mean + 3 SD of zero calibrator) were 0.043 and 0.28 microgram/L, respectively, for free and total PSA. The within-run and day-to-day CVs were 2-11% and 4-10%, respectively. Mean recoveries were 93% and 98% in female and male sera, respectively. Compared with the commercial ProStatus PSA Free/Total Assay, the intercepts of the regression equations (r >0. 99) were not significantly different from zero, and the slopes were 0.95-1.01. In one female serum sample, PSA was falsely increased with the monoclonal assay but was undetectable with the recombinant assay. CONCLUSIONS: The performance of this novel assay based on recombinant components is comparable to a conventional assay based on monoclonal antibodies. The more complete control of the essential characteristics of site-specifically derivatized recombinant Fab fragments will be valuable for the design of miniaturized and multianalyte assay concepts where correct antibody orientation, density, and capacity as well as uncompromised binding affinity are required.