188Re DD-3B6/22 Fab' for use in therapy of ovarian cancer: labelling and animal studies.

A fast and high yielding method of 188Re radiolabelling DD-3B6/22 Fab' is described. An inert atmosphere [N2(g)] and ascorbic acid was essential for preparation and storage of therapeutic levels (< or =2 GBq/mg) for up to 24 h. Immunoreactivity was greater than 75%. Pharmacokinetic studies in nu/nu mice demonstrated localisation of 188Re DD-3B6/22 Fab' was equivalent and correlated well with the behaviour observed for 99mTc DD-3B6/22 Fab' used to image ovarian cancer. Excellent stability at the target site in vivo supports the potential use of 188Re DD-3B6/22 Fab' in the therapy of ovarian cancer.

Serological cross-reactivity between Brucella abortus and yersinia enterocolitica 0:9: IV. Evaluation of the M- and C-epitope antibody response for the specific detection of B. abortus infections.

Smooth lipopolysaccharides (SLPS) from Brucella abortus contain A-epitopes against which the majority of serum antibodies are directed during infections. SLPS from Yersinia enterocolitica 0:9 possesses identical epitopes, which are the cause for serological cross-reactivity. All Brucella spp. possess M- and C-epitopes which are not present in Y. enterocolitica 0:9. In order to examine the usefulness of these M- and C-epitopes for discriminatory serological testing, a panel of sera were used in this study, comprising sera from Y. enterocolitica 0:9-infected heifers, sera from B. abortus-infected cattle of comparable strength in the serological brucellosis tests to the sera from Y. enterocolitica 0:9-infected heifers, sera from B. abortus-infected bovines with strong serological reactions and sera from animals free from B. abortus or Y. enterocolitica infections. These sera were tested in blocking ELISAs with seven M- and one C-epitope-specific monoclonal antibodies in combination with SLPS from B. melitensis M16 high in M-epitopes as antigen. Strong B. abortus sera inhibited most strongly, while negative sera showed no or little inhibition. Sera with weak or intermediate titres blocked to a lower extent. Unexpectedly, the sera from Y. enterocolitica 0:9-infected heifers showed inhibition behaviour virtually identical to the comparable sera from B. abortus infected animals. Absorbing out of the A-epitope specific serum antibodies with either Y. enterocolitica 0:9 SLPS or with Y. enterocolitica 0:9 bacteria, indicated the presence of M- or C-epitope-specific serum antibodies in some sera from B. abortus-infected cattle but not in the sera from Y. enterocolitica 0:9-infected animals. These results demonstrate that the M- or C-epitope-specific antibody response in sera from B. abortus infected cattle is only of limited value for the serological discrimination between B. abortus and Y. enterocolitica 0:9 infections.

Rapid detection of D-dimer using a fiber optic biosensor.

We describe a rapid and sensitive method for detection and quantification of D-dimer and other crosslinked fibrin degradation products (XL-FDPs), which are present in elevated concentrations in patients with sepsis and thrombotic disorders. The method utilizes a sandwich fluoroimmunoassay immobilized in the sensing region of an evanescent wave biosensor. Physiological concentrations of D-dimer and high molecular weight XL-FDP could be determined in buffer and plasma samples on calibrated fibers in 11 min. Samples from septic patients were assayed using ELISA and the fiber optic method; concentrations determined by fiber optic assay were strongly correlated with those determined by ELISA (r = 0.918); intra- and inter-assay errors were comparable to those from ELISAs. Given its accuracy and rapid response time, this fiber optic biosensor shows great potential for development as a diagnostic tool.

Detection of malignant tumors using Tc-99m labeled Fab' fragments from a monoclonal antibody with specificity for D-dimer of cross-linked fibrin.

The authors present the case studies of two patients whose malignant tumors were detected with a Tc-99m labeled antifibrin monoclonal antibody (DD-3B6/22), which is specific for cross-linked fibrin. The first case was a malignant fibrous histiocytoma involving the proximal aspect of the left thigh, whereas in the second case, the patient was receiving treatment for a squamous cell carcinoma of the right mainstem bronchus. The results highlight the potential of this anti-D-dimer radiopharmaceutical for noninvasive detection of malignant tumors.

Autologous red cell agglutination test for antibodies to feline immunodeficiency virus.

The T-lymphotropic lentivirus, feline immunodeficiency virus (FIV) is now recognised as a major viral pathogen affecting domestic cat populations worldwide. A rapid, autologous red cell agglutination test for antibodies to FIV has been developed. A synthetic peptide analog corresponding to the immunodominant epitope within the FIV transmembrane glycoprotein gp40 residues (680-715) KVEAMEKFLYTAFAMQELGC (Acm)NQNQFFK(BrAc)KIPLELWTR was conjugated to an anti-feline erythrocyte antibody using a thio-ether linkage. Within 3 min of adding this reagent to 20 microliters of whole blood, circulating antibody to the peptide epitope caused agglutination of the red blood cells. The performance of this simple test is comparable with the two commercially available enzyme immunoassay (EIA) kits and an EIA based on this peptide. A variant of the gp40 (680-715) peptide corresponding to the FIV, PPR strain gp40 (678-716) sequence was also synthesised and no difference in reactivity was observed in an EIA on 211 seropositive samples, indicating that the peptide-based test may be applicable to other known strains of the virus.

Technetium-99 m labelling of DD-3B6/22 antifibrin monoclonal antibody fragment Fab' for thrombus imaging.

The antifibrin DD-3B6/22 monoclonal antibody Fab' fragment, a murine immunoglobulin, IgG3, has been labelled with technetium-99 m (99mTc) via a transchelation reaction, to specific activity in excess of 30 mCi/mg protein. The radiolabelling of Fab' was dependent on time, temperature, pH, antibody concentrations and nature of intermediary transchelation complex used. The resultant radioconjugate was stable in vitro and in vivo. Blood clearance of 99mTc-Fab' in rat followed two compartment kinetics with the half time of the fast phase being 0.5 h. The main route of excretion was via the kidneys with little uptake indicated by other tissues. The results suggest that the inherent specificity of the antibody, small molecular size, rapid plasma clearance, high specific radioactivity, together with the physical properties of the 99mTc label, combine to make this labelled monoclonal antibody (MoAb), potentially suitable as a radiopharmaceutical for the scintigraphic detection of thrombi in humans.

Automated determination of cross-linked fibrin derivatives in plasma.

Automated assays for the measurement of cross-linked fibrin derivatives in plasma (XL-FbDP) have been developed utilizing latex beads coated with anti-D dimer monoclonal antibody (DD-3B6/22) for both the Cobas Fara Chemistry Centrifugal and the Cobas Mira analysers (Roche, Basle, Switzerland). The analysers were programmed to mix plasma and latex reagent simultaneously and analyse absorbance changes over a 10-15 min period. Results were interpolated by the analyser from a standard curve derived from a polymer of D-dimer. Both assays had high precision (< 5% CV) for values between 100 and 1000 ng/ml and provided clear discrimination between normal samples and samples from patients suffering from the thrombotic diseases, DVT/PE and DIC. The results obtained for XL-FbDP determination with both methods compared well with established methods: a high correlation was obtained with a semi-quantitative manual latex method for both the Fara (r = 0.92) and Mira (r = 0.83) and correlations (r) of 0.81 (Fara) and 0.84 (Mira) were obtained with an enzyme immunoassay (EIA). Correlation between the two automated procedures was high (r = 0.96). The automated method will enable laboratories to provide a rapid and accurate quantitation of XL-FbDP.

Evaluation of thrombus detection in a rabbit model using a technetium-99m-labelled anti-fibrin monoclonal antibody.

Technetium-99 m (99mTc)-labelled conjugates of an anti-fibrin monoclonal antibody, DD-3B6/22, have been assessed for their detection of vascular thrombi in a rabbit model. DD-3B6/22 binds to a D-dimer epitope present on cross-linked fibrin but absent from the fibrin monomer or fibrinogen. Injection of a 99mTc-labelled Fab' fragment of DD-3B6/22 allowed delineation of model thrombi as early as 30 min postinjection (p.i.) with optimal localization at 4-5 h. Thrombus label uptake at 4 h p.i. was 0.304 +/- 0.106% injected dose/g (% ID/g) compared with 0.022 +/- 0.001% ID/g after the injection of a control Fab' fragment. These results suggest that the 99mTc-labelled Fab' fragment of DD-3B6/22 has excellent potential for scintigraphic detection of vascular thrombi in humans.

Preclinical evaluation of 99m technetium-labeled DD-3B6/22 Fab' for thrombus detection.

Injection of 99mTc-labeled Fab' fragments of the anti-fibrin antibody DD-3B6/22, in the baboon, resulted in clear visualisation of both fresh and aged autologous thrombi by gamma scintigraphy. Whole body scintigraphy, pharmacokinetics and urine analysis showed rapid renal excretion of the conjugate with little accumulation of label in other organs. 99mTc-DD-3B6/22 Fab' appears a suitable candidate for further investigation as a radioimaging agent for thrombi in humans.

Rapid whole blood assay for HIV-1 seropositivity using an Fab-peptide conjugate.

A rapid whole blood test has been developed for circulating antibodies to human immunodeficiency virus type 1 (HIV-1), based on agglutination of autologous red blood cells. Evaluation of the test revealed that 100% of seropositive HIV-1 patients (both asymptomatic and AIDS cases) were detected (n = 94) with a specificity of 99.5% in healthy blood donors (n = 596). The assay uses an Fab fragment of a monoclonal antibody specifically directed against glycophorin (a transmembrane glycoprotein present on the surface of human red blood cells). This anti-red blood cell Fab is conjugated via the inter-heavy chain cysteines to a synthetic peptide corresponding to the immunodominant epitope of the HIV-1 viral coat protein gp41 (579-613). Addition of this reagent to 10 microliters of whole blood results in the Fab-peptide conjugate coating the red blood cells with peptide. In the presence of circulating antibodies to the HIV-1 peptide, red cell agglutination occurs within 2 min. The sensitivity and specificity of this reagent indicate that it is appropriate for use as a rapid diagnostic test for HIV-1 seropositivity.

The simpliRED D dimer test: a novel assay for the detection of crosslinked fibrin degradation products in whole blood.

A new system for the detection of fibrin degradation products in whole blood has been developed. The test provides a clearly visible agglutination of the patient's red blood cells in the presence of elevated levels of the crosslinked fibrin derivative, D dimer. The test, which uses a bispecific reagent prepared from Fab' fragments of monoclonal antibodies, gives a positive result in 1-2 minutes. One monoclonal antibody (RAT-1C3/86) was raised against human red blood cells, and the second (DD-3B6/22) was specific to the crosslinked fibrin derivative, D dimer. Addition of the bispecific reagent to a drop of patient's whole blood resulted in red blood cell agglutination when elevated levels of D dimer were present in the sample. Clinical trials showed sensitivity equivalent to that of current commercial tests. Samples from patients with thrombotic disease states as well as normals were examined. The test was compared with commercial latex agglutination and enzyme immunoassay systems and showed good correlation with the presence of elevated levels of crosslinked fibrin degradation products. This technology represents an advance which allows rapid "on the spot" whole blood analysis, for the diagnosis of thrombotic disorders.

Detection of experimental thrombi in rabbits with an 131I-labelled fibrin-specific monoclonal antibody.

The detection of thrombi in rabbits has been investigated with 131I-labelled DD-3B6/22, a monoclonal antibody (Mab) reactive at high affinity (Kd = 2.68 x 10(-10) M) with human D Dimer (DD). DD-3B6/22 bound well to both "fresh" and "aged" human clots in an in vitro assay but showed poor binding to rabbit clots. However, reactivity was restored to rabbit blood if it was seeded, before clotting, with human DD covalently coupled to Sepharose beads. Thus, a rabbit model was developed in which blood was allowed to clot around DD-Sepharose beads introduced into the jugular vein. Gamma camera imaging showed that intact 131I-labelled DD-3B6/22 localised to these clots within 24 h. Uptake at this time was 0.202 +/- 0.012% injected dose per gram (%ID/g) compared with 0.086 +/- 0.018%ID/g after injection of control antibody. 131I-labelled F(ab')2 fragments of DD-3B6/22 allowed earlier scintigraphic detection of the clot which was evident 4 h after injection. Uptake in the clot at 24 h was 0.154 +/- 0.038 %ID/g compared with 0.109 +/- 0.027 %ID/g for a control F(ab')2. As antigen levels in the clot are estimated to be less than 300 micrograms DD, thus representing a very small human clot, the DD-3B6/22 Mab would appear to have a good potential for the sensitive detection of thrombi in a clinical setting.

A rapid whole-blood immunoassay system.

A new immunoassay system has been developed which allows the detection of circulating antigens, antibodies or drugs in whole blood without specialized personnel or equipment. This is achieved by the use of bispecific reagents, which comprise specific antibodies or antigens that are coupled to a non-agglutinating antierythrocyte antibody. Within two minutes, these reagents cause specific agglutination of a patient's own red cells in samples that contain the relevant analyte. Levels of low molecular weight haptens also can be measured by the use of an indirect, agglutination-inhibition assay. This simple immunoassay method would fulfil the needs of many physicians and Third-World countries and also has mass-screening and veterinary applications.

Similarities of Giardia antigens derived from human and animal sources.

A total of 37 Giardia stocks isolated from humans and 14 stocks derived from animal sources have been analysed for antigenic differences. Separation of the proteins of the stocks by polyacrylamide gel electrophoresis showed no major differences among the stocks. Immunoblotting of these antigens demonstrated some minor differences which were not correlated with geographic location, allozyme type, virulence or any other distinguishing characteristic of the stocks. Immunofluorescence tests using monoclonal antibodies revealed some differences between stocks but the monoclonal antibodies did not significantly inhibit growth in inhibition assays.

Anti-fibrin monoclonal antibodies for radioimmunodetection: preliminary assessment in a rat model system.

The D Dimer (DD) site formed by linkage of D domains from adjacent fibrin (FN) molecules is unique to cross-linked FN and its degradation products and is not found in FN monomer or fibrinogen (FB). Thus monoclonal antibodies (MAb) reactive to DD should have a very suitable specificity for in vivo thrombus detection. Two anti-DD MAbs have been labelled with 131-I and assessed as scintigraphic agents in a normal rat model system. Each rat received 3 sc implants of antigen covalently coupled to Sepharose beads: 1) Human DD 2) Human FB 3) Glycine (GL) (control). Scintigraphic images taken 7 days after injection of 131-I anti DD MAb showed clear localisation of both anti-DD MAbs to the DD implant rather than to the FB or GL implants with no localisation in normal tissues. This was confirmed in biodistribution studies. Injection of anti-DD MAbs DD-3B6/22 and DD-IC3/108 resulted in DD: blood ratios of 10.4 +/- 0.6 and 4.9 +/- 0.3 respectively. These results suggest that anti-DD MAbs will have potential for thrombus radioimmunodetection.

Identification of Mycobacterium bovis isolates using a monoclonal antibody.

A rapid immunoperoxidase slide assay for the identification of Mycobacterium bovis culture isolates is described. The monoclonal antibody used in this assay is specific for the M. tuberculosis complex of organisms. All M. bovis isolates tested, including 151 separate field isolates of M. bovis were positive as were 11 out of 12 M. tuberculosis strains and 4 out of 6 Bacillus Calmette Guerin (BCG) strains. One strain each of M. africanum and M. microti was negative. This assay provides a considerable improvement in both time and expense over the conventional methods of biochemical typing of M. bovis.

Autologous red cell agglutination assay for HIV-1 antibodies: simplified test with whole blood.

An antibody detection procedure based on agglutination of autologous red cells has been developed for samples of whole blood. A nonagglutinating monoclonal antibody to human red blood cells conjugated to a synthetic peptide antigen (in this case residues 579 to 601 of the HIV-1 envelope precursor, Arg-Ile-Leu-Ala-Val-Glu-Arg-Tyr-Leu-Lys-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp- Gly-Cys - Ser-Gly-Lys) permitted the detection of antibodies to the human immunodeficiency virus type 1 (HIV-1) in 10 microliters of whole blood within 2 minutes. Agglutination was specifically inhibited by addition of synthetic peptide antigen but not by unrelated peptides. The frequency of false positive results was 0.1% with HIV-1 seronegative blood donors (n = 874). The false negative results were approximately 1% (n = 81). The autologous red cell agglutination test is potentially suitable for simple, rapid, qualitative screening for antibodies to a variety of antigens of medical and veterinary diagnostic significance.

Production and characterization of monoclonal antibodies specific for Mycobacterium bovis.

A series of monoclonal antibodies (MAbs), specific for Mycobacterium bovis and BCG strains, were tested extensively for cross-reactivity to a wide range of mycobacterial species using ELISA, Western blotting and dot-blot analysis. The MAbs bound specifically to M. bovis and BCG and showed limited cross-reactivity with some strains of M. tuberculosis. All these MAbs recognized a 22 kDa protein previously termed MPB70, and by competitive ELISA analysis appeared to detect at least three M. bovis-specific determinants on the MPB70 molecule.

A latex agglutination assay for D dimer: evaluation and application to the diagnosis of thrombotic disease.

Although latex agglutination assays have been used for some years to diagnose thrombotic disorders, only recently has it been possible to measure specifically the products of fibrin breakdown in the presence of fibrinogen degradation products, by using monoclonal antibodies. We have evaluated a preparation of latex particles coupled to the monoclonal antibody DD-3B6/22, which is specific for cross-linked fibrin degradation products (XDP) and allows accurate discrimination between normal and pathological conditions. Of samples from 515 apparently healthy volunteers, 97.7% failed to agglutinate the latex; the normal reference interval for XDP determined by enzyme immunoassay was less than 78-320 micrograms/L. The use of different anticoagulants with or without the addition of a protease inhibitor had no significant effects on the results of the latex assay. The latex preparation provides a useful, rapid diagnostic tool for assaying small numbers of samples or as an emergency test.

Monoclonal antibody-based enzyme immunoassay for trypsinogen in neonatal screening for cystic fibrosis.

An enzyme immunoassay (EIA) with monoclonal antibodies against human trypsinogen in neonatal blood-spots has been evaluated for screening for neonatal cystic fibrosis (CF). In a retrospective study, 36 of 39 CF samples were distinguished from controls matched for age and storage time. 7 infants with CF were detected in 16,500 infants screened in a prospective study. The EIA is quicker and less labour intensive than conventional assays for the detection of immunoreactive trypsin and may have further advantages of specificity and sensitivity for monitoring the release of pancreatic zymogens in CF.