Detection and differentiation of active and inactive isoforms of coagulation factors II, VII, IX, and X in prothrombin complex concentrate by mass spectrometry.

PURPOSE: Prothrombin complex concentrates (PCCs) are plasma products containing a mixture of four inactive/proactive coagulation factors. The activated forms of human coagulation factors, like Thrombin (FIIa), Convertin (FVIIa), activated Christmas factor (FIXa) and the activated Stuart-Prower factor (FXa), are impurities in PCCs. Until now no valid assay exists to differentiate the non activated proform (inactive) from active coagulation factor isoforms in PCCs in one measurement. Therefore, the aim of this study was to establish a mass spectrometry (LC-MS/MS)-based assay to address this issue in the ready to use medicinal product. METHODS: Bottom-up proteomics combining double digestion (Glu-C & Lys-C) and LC-MS/MS, was used to differentiate the inactive and active forms of the coagulation factors Prothrombin (FII), Proconvertin (FVII), Christmas factor (FIX) and the Stuart-Prower-factor (FX) in PCCs. RESULTS AND CONCLUSIONS: A targeted pseudo-multiple reaction monitoring (pMRM-LC-MS/MS)-assay was developed for the specific detection of four different coagulation factors in PCCs. Proteotypic peptides for the inactive/active isoforms (zymogen) of the four coagulation factors were identified and validated by the investigation of six investigational and one commercially available PCCs. In conclusion, the semi-quantitative determination and the distinction between the active and the inactive isoform of the respective coagulation factors were possible in one liquid chromatography tandem mass spectrometry (LC-MS/MS) run.

Application of capillary zone electrophoresis and reversed-phase high-performance liquid chromatography in the biopharmaceutical industry for the quantitative analysis of the monosaccharides released from a highly glycosylated therapeutic protein.

Two assays for the quantitative determination of the neutral and amino-monosaccharides attached to a therapeutic glycoprotein were developed using capillary zone electrophoresis (CZE) and RP-HPLC. These assays meet the strict batch release requirements of the quality control in biopharmaceutical industry. The monosaccharides were released from the glycoprotein by hydrolysis with 2N trifluoroacetic acid. In the CZE assay the monosaccharides were reacetylated prior to derivatization with 8-aminopyrenesulfonic acid (APTS), reacetylation in the glycoprotein matrix was investigated in detail. The RP-HPLC method used pre-column derivatization with anthranilic acid in methanol-acetate-borate reaction medium; reacetylation was not necessary. However, epimerization of the different monosaccharides was observed and studied in detail. For the quantitative assay, separation of the amino-monosaccharide epimers had to be developed. The HPLC assay was validated.

Structural base of the interaction of a monoclonal antibody against p24 of HIV-1 with its peptide epitope.

The interaction of a murine monoclonal antibody (CB 4-1) against the core protein p24 of HIV-1 with its peptide antigen was studied in detail. The amino acid sequence of the variable regions of the heavy and light chain as derived from DNA sequencing was used to model the structure of the antigen binding region on the basis of reported Fab structures from the Brookhaven Protein Data Base. A linear peptide epitope responsible for the p24 binding to the antibody was determined by peptide scan. Subsequent N- and C-terminal truncation of the corresponding sequence region as well as amino acid substitutions were performed to recognize the epitope and the amino acid residues critical for antibody binding. These data were used to derive a structural model of the peptide-antibody interaction.

Enzyme immunoassay techniques. An overview.

In spite of the great variety of enzyme immunoassays (EIA) they can be classified into two groups 'analyte-observed' and 'reagent-observed' assays, depending on their reaction principle. The latter are favored by use of monoclonal antibodies and are characterized by a greater sensitivity, a larger measuring range, a lower susceptibility to disturbing influences. They can be used only for detection of macromolecules. For heterogeneous EIAs to be used on laboratory scale, simple adsorption of antigens and antibodies is still recommendable though affinity constants decrease by at least one order of magnitude and antibody density at the solid phase and analyte binding capacity are not parallel due to increasing steric hindrance. For this reason, the antibody with the higher affinity constant should therefore always be used as solid-phase antibody. Microparticles used as solid phase for heterogeneous assays, due to their very high binding capacity for the analyte and extremely short diffusion distances, guarantee 'one step' assays of only a few minutes. Of the limited number of enzymes suitable as markers in immunoassays, horseradish peroxidase is the enzyme of choice followed by alkaline phosphatase. Although enzyme and enzyme-labelled reagents are detectable by fluorogenic product measuring with a sensitivity, which is 10-1000 times higher than using chromogenic substrates, the sensitivity of the assays can be increased only by factor 2-10. Labelling enzymes cannot only be covalently bound to the antibody, but also via anti-enzyme antibodies. Pros and cons of the different methods of coupling the enzyme/anti-enzyme complex to analyte-containing immune complexes are discussed. Different EIA variants to detect specific antibodies are reviewed. Among them only capture EIAs permit precise isotype analysis of antibodies of a distinct idiotype. Homogeneous EIAs are widely spread for hapten determination but even variants based on proximal linkage are no alternatives to heterogeneous EIAs for determination of macromolecules. Different parameters are defined which permit to assess the quality of an immunoassay and which should be used in routine assays as internal controls in the laboratory.

Purification and immunochemical characterization of a natural human polyreactive monoclonal IgM antibody.

In vitro and in vivo experiments to explain the function of natural polyreactive antibodies, usually of the IgM isotype, require large amounts of purified antibodies. We have developed a two-step purification procedure using a human natural polyreactive monoclonal IgM antibody (CB03). This combines hydrophobic interaction chromatography on phenyl-Superose and gel filtration over Superose 12 and readily permits scaling-up to isolate mg to g amounts of antibody. Retention of the CB03 antibody during gel filtration by precipitation and interaction with the gel matrix was overcome by the addition of 10 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate. The yield of purified antibody was 34% and Fab fragments were obtained from the purified CB03 antibody by hot tryptic digestion (yield, 68% of theoretical amount). In an enzyme-linked immunosorbent assay, Fab and complete antibody had similar reaction patterns with different antigens.

Establishment of human Ig producing heterohybridomas by fusion of mouse myeloma cells with human lymphocytes derived from peripheral blood, bone marrow, spleen, lymph node, and synovial fluid. Effect of polyclonal prestimulation and cryopreservation.

50 fusion experiments were carried out to analyse heterohybridization efficiencies on mouse myeloma cells of the P3 X63 Ag8/653 line with human lymphocytes derived from peripheral blood, bone marrow, lymph node, spleen or synovial fluid. We found higher yields of growing and human Ig-producing hybridoma lines when lymphocytes from spleen or lymph node were fused. Although primary hybridomas could be established from fusions with bone marrow-derived cells, only in nine out of 1616 initially seeded wells was Ig production registered. Four fusions using immune cells from synovial fluid were made without success. Independently of the source of lymphocytes pokeweed mitogen (PWM) prestimulation had no enhancing effect on the percentage of wells with cell growth and this did not alter the IgM:IgG ratio in primary hybridomas (9:1), although cells from all compartments used here (with the exception of bone marrow cells) could be stimulated with PWM to produce both IgG and IgM in cultures. Cryopreserved lymphocytes from different sources could be used for fusions with comparable results registered for the fresh material.

The high efficiency, human B cell immortalizing heteromyeloma CB-F7. Production of human monoclonal antibodies to human immunodeficiency virus.

This paper describes the construction of a new heteromyeloma cell line designated CB-F7. The cell line was derived from xenogeneic somatic cell hybridization between normal human B lymphocytes and the murine HAT-sensitive P3X63Ag8/653 cell line. CB-F7 cells were characterized by rapid cell growth (doubling time about 16 h) and high cloning efficiencies in culture medium supplemented with 10% or 5% fetal calf serum, respectively. The karyotype of the cells consists of about 75-78 chromosomes as well as two chromosomal fragments. Fusions of the cells with human peripheral blood cells resulted in approximately 2-6 clones per 10(5) seeded lymphocytes. Furthermore, the cells are ouabain resistant and therefore suitable for fusions with EBV-transformed lymphoblastoid cell lines. Using CB-F7 as the parental cell line a number of specific human mAb producing hybrids were established. For the first time, we describe here the generation of hybrids secreting human monoclonal antibodies to human immunodeficiency virus (HIV). Two monoclonal antibodies of IgG type and one of IgM type reacted with the major core protein p25 and one IgG antibody reacted with the transmembrane protein gp41.

Immune restoration in children after partial splenectomy.

Splenectomy (SE) is recognized to be a therapeutical approach in treating children with severe autoimmune diseases (chronic idiopathic thrombocytopenia; hemolytic anemia) or hypersplenism because of portal hypertension. Nevertheless, removal of a main immune organ results in elevated infection risk for these patients. Partial splenectomy (PSE) was developed as a therapeutical compromise to retain immunologically active spleen tissue. Here, we document the analysis of immune parameters obtained from children after both partial and total splenectomy, which have been followed up for a period of more than 6 years: (i) Lymphocytes from both groups of patients failed to produce IgG in response to pokeweed mitogen in vitro. This was observed in 11/20 splenectomized patients even 10 years after operation, whereas in PSE patients a restoration of this parameter after 1-2 years was seen. (ii) In patients after PSE, but not in splenectomized persons, an elevated number of HLA-class II positive cells had been detected suggesting a different situation of immune regulation following this operation. However, in parallel with an improvement of B cell in vitro activity this parameter was found to achieve normal values. Our findings indicate that partial splenectomy may be a therapeutical alternative, if the therapeutic goal can be achieved by this procedure.

Interaction of cyclophilin and cyclosporins monitored by affinity capillary electrophoresis.

The affinity capillary electrophoretic separation of the complex of the enzyme cyclophilin (Cyp) with the immunosuppressive drug cyclosporin A (CsA) from uncomplexed Cyp and CsA in phosphate buffer (pH 8) under non-denaturing conditions by equilibrium-mixture analysis is reported. Using a new approach combining mobility-shift analysis and electrophoretically mediated microanalysis the binding constant of rhCyp18 to CsA and derivatives was estimated.

Lymphocytotoxic autoantibodies in progressive systemic sclerosis.

In 53 patients with progressive systemic sclerosis (PSS) the lymphocytotoxic activity of their serum was measured in a microlymphocytotoxicity assay. In 21 of the 53 patients the test reacted distinctly positively in the heterologous system, and in 9 of these 21 also in the autologous system. After preparation of the immunoglobulins from these positive sera, whole cytotoxic activity was detected only in the IgM fraction but not in the IgG fraction. When using prepared T lymphocytes as target cells in the microlymphocytotoxicity test, the cytotoxic activity of the positive PSS sera showed itself to be directed against this lymphocyte population. Further analysis using the Western-blot technique showed that the IgM autoantibody in PSS sera reacted with the cell surface of CD4+ lymphocytes. The cross reactivity with extractable nuclear antigens was rather improbable. These results suggest that lymphocytotoxic autoantibodies may play a role in immunological disturbances in PSS.

Influence of hydroxyurea on lectin-induced early and late events in peripheral blood lymphocyte cultures.

Activation events induced by lectins in human lymphocyte cultures differed not only in time kinetics of their appearance but were also in a different manner inhibited by hydroxyurea (HU). 4F2 and Tac expression, thermostable sheep erythrocyte rosette formation, cell volume distribution changes and the enhancement of the purine metabolic rate [( 3H]adenine incorporation) induced by PHA, Con A or PWM were not influenced by HU treatment, suggesting G1-phase dependency of these markers. The decrease in the mitogen-induced marker expression after 48 h of incubation with HU can be explained by the unability of activated cells to process the cell cycle, to divide and to become newly positive cells. Mitogen-induced RNA synthesis was partially, DNA synthesis, PWM-induced Ig synthesis and lectin-mediated HLA class II antigen expression were totally inhibited by HU. It holds especially for the DR antigen that its appearance in a polyclonal model of lymphocyte activation occurs in a later (postmitotic G1) phase of the cell cycle. The inhibitory effect of HU on late activation parameters could be removed after washing the cells. Mitogen-activated, HU-treated PBL cultures after removing HU continued cell cycle progression without requirement for further addition of lectin.

Development of specific IgG-producing human hybridomas by fusions of lymphocytes from actively immunized persons.

More than 13,500 initial hybridoma lines derived from fusions of lymphocytes from non-boosted persons were tested for IgG production against Tetanus Toxoid. However, only 2 were found to produce IgG monoclonal antibodies of the desired specificity. Peripheral blood lymphocytes were then taken from actively immunized donors 3, 7, 14 or 60 days after boosting and fused to the HAT-sensitive heteromyeloma cell line CB-F7. A strong enhancement of both IgG-producing hybridomas and specific IgG-producers was detected in fusions of lymphocyte material derived 7 days after booster injection (239 of 731 IgG-producing lines showed anti-TT-specificity). Two months after boosting no more specific IgG-producing hybridomas could be established from the peripheral blood of the donors. Data were discussed regarding an optimized human monoclonal antibody production against bacterial antigens.

Characterization of neutralizing monoclonal antibodies directed against Staphylococcus aureus alpha-toxin.

A panel of neutralizing murine monoclonal antibodies (MAbs) against Staphylococcus aureus alpha-toxin has been established, using formaline-inactivated alpha-toxin as an immunogen. Five independent groups of neutralizing epitopes have been identified representing five functionally important structures in the toxin molecule. Because none of the antibodies binds to overlapping decapeptides representing the toxin sequence or to bromocyanogen cleavage products of alpha-toxin, they may all bind to conformational epitopes. Nevertheless, they all bind to monomeric alpha-toxin in a Western blot. Three of the antibodies bind to the toxin monomer in an enzyme-linked immunosorbent assay (ELISA) in the presence, but not in the absence, of detergent. These epitopes are not accessible in hexameric toxin; two of them may represent the contact sites of the toxin monomers upon hexamerization and one is related to a structurally important glycine-rich central hinge region. Two different antibodies bind to monomeric toxin in an ELISA in the presence and absence of detergent and their epitopes are present more than once on oligomeric toxin; they bind strongly to hexameric toxin in a Western blot. The binding properties of the antibodies to alpha-toxin in different assay systems are summarized in an epitope model, which describes the presence of neutralizing domains in the different conformational steps required for pore formation.

Cell biology of human IgM-producing hybridomas derived from a fusion of human spleen lymphocytes with mouse myeloma cells.

Hybrids were derived from the fusion of mouse myeloma cells with human spleen cells from a patient with active idiopathic thrombocytopenia. Of 288 initially seeded cultures, 186 were found to produce human Ig. The growth and Ig production rates, cloning efficiencies using different feeder layers and the karyotype were determined for 9 clones that stably produced human monoclonal IgM (2-100 micrograms/ml) for at least 9 months. All cells of the Ig-producing hybridoma clones were positive for cytoplasmic-Ig, whereas only 20-65% of cells expressed surface Ig (mu and chains). Human monoclonal antibodies in mass cultures were derived in serum-free PRMI 1640 medium. Two clones produced human IgM (nearly 2 mg/ml) in the ascitic fluid of nude mice. Feeder cells of peritoneal macrophages from Balb/c mice enabled more efficient recloning of human x mouse hybrids than did thymocytes. Nearly all subclones derived from 2 clones were found to produce the same monoclonal antibodies as the parental lines. Information on the individual parameters of a hybridoma cell line may be helpful in the large-scale production of human monoclonal antibodies.

[Adjuvant autologous tumour cell vaccination in patients with renal cell carcinoma. Overall survival analysis with a follow-up period in excess of more than 10 years]. / Adjuvante autologe Tumorvakzine beim Nierenzellkarzinom. Gesamtüberlebensanalyse mit einem Nachbeobachtungszeitraum von mehr als 10 Jahren.

BACKGROUND: Organ-confined renal cell carcinoma (RCC) is associated with tumour progression after surgical therapy in approximately 30% of cases. However, of all recently available adjuvant treatment options, only the autologous tumour cell lysate vaccination therapy (Reniale) has been able to demonstrate a significant positive impact on progression-free survival in a phase III trial. Nevertheless, this therapeutic option has not yet been established as a standard adjuvant treatment. MATERIALS AND METHODS: Between August 1993 and December 1996, a total of 1,267 patients who underwent radical tumour nephrectomy at 84 German centres received Reniale outside a controlled trial. Of these patients, 692 presented at stage pT2-3, pNx-2, M0 (based on the 4th version of TNM classification). These patients were matched with a cohort of 861 patients not receiving any adjuvant treatment who underwent surgical therapy for RCC in a 15-year period in the Carl-Thiem-Klinikum in Cottbus, Germany. Matching criteria included age, gender, pT stage, pN stage, grading, histological cell type, and UICC stage. This resulted in 495 matched pairs (study group n=990) that were comparable regarding demographic and tumour-specific criteria. Statistical analyses included univariate and multivariate analyses of overall survival (OS). Median follow-up time of all patients still alive at the end of the trial (n=667) was 11 years. RESULTS: In the vaccine group, OS after 5 and 10 years was 80.6% and 68.9%, respectively, whereas control patients had an OS of 79.2% and 62.1%, respectively (p=0.066). The 5-year OS of patients with pT3 RCC was 71.3% after vaccination therapy and 65.4% for control patients. After 10 years, 53.6% of the patients in the vaccine group and 36.2% in the control group were still alive (p=0.022). Median survival of patients with pT3 RCC was 81 months (SD 7.8) in the control group. This period was not achieved in the vaccine group. Multivariate Cox analysis revealed a significant positive impact of Reniale on OS among the whole study group [hazard ratio (HR) 1.28, p=0.030]. The analysis of patient subgroups showed a significant positive influence of Reniale for patients presenting with pT3 tumours (HR 1.67, p=0.001). CONCLUSION: Adjuvant postsurgical treatment with Reniale in patients presenting with stage pT3 RCC results in a significant enhancement of OS and should be considered especially in this group of patients. Further clinical trials integrating the recent TNM classification and comprising different risk constellations should follow in order to ultimately assess the value of adjuvant treatment with vaccination immunotherapy.

A prospective multicentre study on the safety of long-term intensive plasmapheresis in donors (SIPLA).

BACKGROUND AND OBJECTIVES: The safety of chronic intensive donor plasmapheresis has not been determined in large prospective studies examining dropout rates, dropout reasons and predictors of withdrawals. MATERIALS AND METHODS: Twenty-one plasma centres recruited 3783 donors who were switched from a moderate to an intensive plasmapheresis programme and observed over a 3-year period. Individuals weighing < 70 kg and > or = 70 kg donated 750 ml and 850 ml of plasma per session, respectively. The maximum of annual donations was limited to 60. Total serum protein (TSP) and haemoglobin (Hb) or haematocrit (Hct) were determined at each donation, and immunoglobulin G (IgG) at every fifth donation. Dropout rates, dropout reasons and potential predictors of withdrawal were analysed. RESULTS: Dropouts were predominantly due to socioeconomic (49.2% of all donors) or medical reasons not related to plasma donations (10.4% of all donors). Sixteen per cent of donors dropped out when IgG, TSP or Hb levels fell below threshold values. Severe clinical adverse events related to plasmapheresis were observed in five subjects. The incidence in severe cardiovascular diseases was lower in donors than in the general population. The risk factors that led to dropping out as a result of low IgG, TSP or Hb levels included younger age, female gender, low initial IgG levels and a high donation frequency. Neither body weight nor the amounts of plasma donated per kilogram of body weight per session were associated with ceasing due to medical reasons, whether related or unrelated to plasma donations. Females and males within the respective lowest body weight category were not at higher risk of dropping out. CONCLUSION: Long-term intensive donor plasmapheresis under conditions investigated in this study is safe. All donors weighing > or = 70 kg are safely able to donate 850 ml of plasma in each session up to 60 times per year, provided that they are carefully monitored.

Evaluation of type D retroviruses as diagnostic tools in HIV infections.

A type D retrovirus isolated from a permanent human cell line (PMFV) was employed as diagnostic reagent both in Southern transfer hybridization experiments using the cloned genome as a probe and in immunoblot analysis using SDS disrupted virus particles. Hybridization experiments performed under conditions of different stringencies revealed a close homology of PMFV to SAIDS type D retroviruses of serotype 1 (SRV-1, SAIDS retrovirus D/NE), a related homology to the prototype type D virus (MPMV) and to viruses of serotype 2 (SRV-2), but no homology to the endogenous type D retrovirus of squirrel monkeys (SMRV) and the human AIDS virus (HIV-1). Antigens of PMFV showed cross-reactivity only to antibodies of a SAIDS infected macaque, but no reaction to anti HIV-antibodies of seropositive patients. Thus, the type D virus isolated from a human cell line and closely related to SAIDS type D viruses of macaques is not related to the AIDS virus in humans.

[Model calculations for HIV screening of blood and plasma donors with a combination of 2 screening tests: test strategies, validity, costs and effectiveness]. / Modellrechnungen zum HIV-Screening bei Blut- und Plasmaspendern mit der Kombination zweier Suchtests: Testrategien, Validität, Kosten und Nutzen.

OBJECTIVE: The strategies for combining two screening tests for HIV infections in blood or plasma donors are formulated in biometric terms and analyzed with respect to their value, i.e. their validity, cost and effectiveness. DESIGN: Biometrical modeling using assumptions on the validity of the single tests, the conditional correlations between them, as well as on the cost of testing and the consequences of false-negative or false-positive test results. RESULTS: If the test combination is defined as positive whenever at least one of the single tests is positive, then this rule (the 'believe the positive' rule, BTP), due to its lower specificity, has extremely low positive predictive values. In case of high prevalence rates of the infection (e.g. 1:1,000), the BTP rule leads to lower total cost than single testing, unless the latter has very high sensitivity (e.g. 99%). For smaller prevalence rates (< 1:50,000), which are more typical of the selected group of blood or plasma donors, combination testing is of little value because the extra cost of detecting one additional infection (compared with single testing) may reach several 100 million DM. CONCLUSION: The cost for detecting additional cases of HIV infection by using combination instead of single testing in HIV screening is so high that this decision requires a public consensus.

Serological investigations of red foxes (Vulpes vulpes L.) for determination of the spread of tick-borne encephalitis in Northrhine-Westphalia.

Serum samples from 786 red foxes shot between January 1995 and August 1996 in the southern half of Northrhine-Westphalia, located in western Germany, were tested for the presence of antibodies against tick-borne encephalitis (TBE) virus using the Immunozym FSME IgG All Species-ELISA (Immuno, Heidelberg, Germany) as a screening test: 759 sera were negative, 23 (2.9%) were borderline, and four (0.5%) were positive. Nine of the 27 ELISA reactive sera were confirmed by the TBE Western-Blot (Immuno, Heidelberg, Germany). Furthermore these 27 sera were tested for neutralizing antibodies by means of a plaque reduction neutralization test (PRNT) against TBE and West Nile viruses. Only one single serum was found to have a neutralization titre (+1:800 PRNT80) against TBE virus. All other 26 sera were negative for neutralizing antibodies against TBE or West Nile virus. Since the titre of the single serum is low, it can be interpreted that if TBE virus is present, its prevalence is extremely low. Northrhine-Westphalia is not classified as a TBE-endemic area. Further calculated serological testing of game and virological investigation of collected ticks in the affected area seem to be meaningful and necessary.