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.

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.

An increased serum level of free Apo(a) in renal patients is more striking than that of Lp(a) and is influenced by homocysteine.

Lipoprotein(a) [Lp(a)] excess combined with hyperhomocysteinaemia and hyperfibrinogenaemia may contribute to the high incidence of vascular diseases in dialysis patients. This study is aimed at investigating the role of free apolipoprotein(a) [fapo(a)] in renal patients. We have been able to show that, as compared with controls (0.53 mg/l), the median serum concentrations of fapo(a) in patients with nephrotic syndrome (2.58 mg/l) and with peritoneal dialysis (3. 40 mg/l) were strongly elevated (5- to 7-fold), while the fapo(a) levels in patients undergoing haemodialyis (1.02 mg/l) and after renal transplantation (0.90 mg/l) were about doubled. The observed differences in fapo(a) levels indicate that several mechanisms may increase the level of fapo(a), i.e., reduced renal clearance, enhanced hepatic synthesis, or homocysteine releasing apolipoprotein(a) from Lp(a). In the study collective, the median total homocysteine levels were significantly elevated in all patient groups, stronger in patients on haemodialysis (31.4 micromol/l) and peritoneal dialysis (31.2 micromol/l) than in patients with nephrotic syndrome (19.7 micromol/l) and after renal transplantation (19.5 micromol/l). In transplant patients with adequate renal function and without other apolipoprotein(a)-increasing factors, fapo(a) was significantly increased when total homocysteine exceeded 22 micromol/l. In conclusion, our findings let us presume that an increased fapo(a) level in renal patients possibly could be one of the reasons contributing to the high incidence of vascular diseases in these patients, because fapo(a) not covalently linked with Lp(a) is even more easily able to inhibit the fibrinolytic system than the complete Lp(a). These preliminary results have to be confirmed by further investigations.

The influence of citrate concentration on the quality of plasma obtained by automated plasmapheresis: a prospective study.

BACKGROUND: There is a need for more comprehensive work dealing with the quality of plasma collected by automated plasmapheresis using different final concentrations of citrate anticoagulant. A prospective study was performed to examine the influence of three concentrations of sodium citrate on the levels of clotting factors and markers of activated hemostasis and fibrinolysis. STUDY DESIGN AND METHODS: Fifty-one experienced plasma donors were recruited for subsequent 750-mL plasmapheresis procedures using 4-percent (wt/vol) sodium citrate. Anticoagulant-to-blood ratios of 1:16.6, 1:14.2, and 1:12.5 were used, corresponding to sodium citrate concentrations of 6 percent, 7 percent, and 8 percent (vol/vol), respectively. Between two plasmapheresis procedures, there was a washout period of 7 days. Determinations were made of the plasma levels of fibrinogen and factors V, VII, VIII, and IX, as well as antithrombin, tissue-type plasminogen activator, and several markers of activated hemostasis and fibrinolysis: activated factor VII, prothrombin splits products, D-dimers, and beta-thromboglobulin. RESULTS: The plasma samples anticoagulated with 6-percent citrate contained significantly higher levels of factors V, VIII, and IX than the samples anticoagulated with 8-percent citrate (p<0.0001, p< or =0.0001 and p = 0.009, respectively). The citrate concentration had no influence on the levels of fibrinogen, factor VII, antithrombin, or tissue-type plasminogen activator. There was no evidence that the plasma samples containing lower citrate concentrations were more prone to activation of hemostasis or fibrinolysis. CONCLUSION: A reduction in the final citrate concentration of plasma collected by automated plasmapheresis results in higher yields of factors V, VIII, and IX without activation of hemostasis. More comprehensive studies should confirm previous work dealing with the establishment of the lowest citrate concentration acceptable in plasma used as therapeutic fresh-frozen plasma or as starting material for the manufacture of plasma derivatives.

Determination of free apolipoprotein(a) in serum by immunoassay and its significance for risk assessment in patients with coronary artery disease.

This paper describes a new enzyme-linked ligand sorbent assay (ELLSA) to quantify free apolipoprotein(a) (apo(a)). The new test immobilizes free apo(a) utilizing a specific peptide that carries the amino acid sequence of a non-covalent apo(a) binding site on apoB3375-3405 (ligand-peptide). The ligand-peptide coupled to Sepharose was used in affinity chromatography to separate free apo(a) from whole serum. Isolated free apo(a) consisted of full length apo(a) and smaller apo(a). Additionally, free apo(a) levels determined by ELLSA as well as by electroimmunodiffusion correlated moderately well. Significantly increased serum concentrations of free apo(a) were found in coronary artery disease. The mean value of free apo(a) was three times higher in patients than in controls while the lipoprotein(a) (Lpla)) concentration was doubled. Utilizing receiver operating characteristic diagrams, it was shown that the free apo(a)-ELLSA had a better diagnostic test performance in atherosclerotic risk assessment than the Lp(a)-test: specificity free apo(a)-ELLSA 0.77, Lp(a)-test 0.81 [with (a:a)-enzyme immunoassay (EIA)] to 0.83 [with (a:B)-EIA]; sensitivity free apo(a)-ELLSA 0.57, Lp(a)-test 0.36 to 0.40. In conclusion, the new free apo(a)-ELLSA allows for the specific quantification of free apo(a). This provides an interesting indicator for atherosclerotic risk assessment.

Tick-borne encephalitis diagnosis in patients with inflammatory changes in the cerebrospinal fluid in a region with very low prevalence.

Tick-borne encephalitis (TBE)-IgG antibodies are used for the serologic detection of antigen contact caused by TBE infection or immunization. In the present study, enzyme-linked immune sorbent assay (ELISA) results from a group of patients with inflammatory changes in the cerebrospinal fluid (CSF) were re-examined using Western blot technology. The result of the TBE-IgG-ELISA was positive in 47 of the 904 sera samples tested. Retesting the sera with a Western blot confirmed this result in only 31.8% of the positive cases. In 134 of the 904 sera, the ELISA result was borderline. In 5.5% of these sera, the Western blot reacted specifically. The remaining 723 sera samples tested negative with the ELISA. Of these sera, 15 were selected randomly and retested with the Western blot; none of them tested positive. The high number of false positive ELISA results can be explained by the highly selected group of patients and the low prevalence of TBE in the region studied. In patients with meningitis or encephalitis with positive ELISA results and uncharacteristic clinical symptoms, the treating physician should consider the possibility of nonspecific reactions involving inflammatory mediators or cross-reactivity with other flaviviruses. The ELISA-mediated diagnosis of TBE should therefore be verified by means of the patient's history and clinical symptoms, as well as further serologic tests including the Western blot, the hemagglutination test and the neutralization test.

Diagnosis of tick-borne encephalitis: evaluation of sera with borderline titers with the TBE-ELISA.

Tick-borne encephalitis (TBE) is a member of the Flaviviridae family. Strong cross-reactions can occur between members of this family, so that it may be difficult to diagnose specific flavivirus infections, especially when tests with frequent cross-reactions e.g. ELISA tests are used. We tested 238 sera with borderline titers for TBE using the indirect immunofluorescence or neutralization test for other flaviviruses (yellow fever, dengue, West Nile) to detect cross-reactions due to other flavivirus infections or flavivirus vaccination. Only one serum reacted against all the flaviviruses tested, indicating cross-reactivity due to infection with any of the flaviviruses. Two other sera exhibited low antibody titers against yellow fever, which could be confirmed by the neutralization test, indicating recent yellow fever vaccination. None of the other sera reacted at all against any of the flaviviruses tested in the tests used, which indicates false positive reactions with the TBE-ELISA. Sera with borderline titers in the TBE-ELISA in particular should be retested using other test systems (preferably neutralization) and for other flaviviruses (yellow fever, dengue, West Nile) to detect cross-reactions and to confirm positive results.

[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.

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.

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.

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.

Human monoclonal IgM antibodies from foetal B-cell hybridomas directed against a surface antigen on human tumour cells.

In order to assess the existence of B lymphocytes capable of producing anti-tumour antibodies in non-tumour-bearing individuals, human lymphocytes derived from foetuses and adults were fused with the heteromyeloma cell line CB-F7. By indirect immunofluorescence, 29 out of 4,472 IgM-producing hybridomas (from 8 foetuses and 8 adults) were shown to produce antibodies which bind to colon carcinoma lines Colo205 and SW620, Raji lymphoma cells and small cell carcinoma of the lung. In vitro growth of tumour cells recognized by these antibodies was inhibited. The antibodies also mediated complement-dependent cytotoxicity. All antibodies tested recognized a cell surface molecule of 55 kDa. Southern blot hybridization analysis of hybridoma DNA with a human JH probe showed that the hybridomas were derived from clonally unrelated B cells. These results demonstrate that human foetal and adult B cells from non-tumour-bearing individuals are able to produce IgM antibodies recognizing defined cell surface molecules expressed on some tumour cells.

[Autoimmune phenomena during Trypanosoma equiperdum inoculation of the laboratory mouse]. / Autoimmunphänomene bei der Trypanosoma equiperdum-Inokulation der Labormaus.

In the course of the Trypanosoma equiperdum-infection of mice an increase of IgM antibodies against the autoantigens dsDNA, keratin and collagen as well as against a protozoan foreign antigen consisting of Sarcocystis gigantea-extract could be observed with a maximum level between 4th and 8th day p. i. The IgG-antibodies did not significantly change during the investigation time. A splenomegaly appeared after the infection. The weight of spleen was four times higher than normal. It was suggested that splenomegaly as well as induction of antibodies against several autoantigens and a foreign antigen were due to a polyclonal activation of lymphocytes.

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.

Human hybridomas derived from CD5+ B lymphocytes of patients with chronic lymphocytic leukemia (B-CLL) produce multi-specific natural IgM (kappa) antibodies.

Great numbers of CD5+ B lymphocytes were detected in the peripheral blood of patients with B-CLL. To study the antibody repertoire of this immune cell subpopulation on a monoclonal level, we fused the lymphocytes derived from five different donors to a highly efficient HAT-sensitive heteromyeloma line (CB-F7). A fusion frequency of up to 10(-5) allowed us to analyse hundreds of initial hybridoma lines per fusion. In all culture supernatants in three out of five fusions IgM lambda antibodies were detected, in two experiments only IgM kappa was measured, suggesting monoclonality of the primary hybridoma cell lines. The later fusions resulted in hybridomas producing multi-specific antibodies against both an autoantigen and an infectious agent: (i) dsDNA/influenza virus haemagglutinin; (ii) dsDNA/class V outer membrane protein type C from Neisseria meningitidis. However, no antibodies of the described specificity were detected in blood sera of patients, indicating a 'switch-on' of the immunoglobulin secretion capacity of malignant B cells during fusion to a myeloma partner. We discuss the results as further evidence for the natural multi-reactive antibody repertoire of CD5+ B cells.

Development of a solid phase enzyme immunoassay for the detection of anti-Ro autoantibodies.

An enzyme immunoassay was developed to detect anti-Ro(SS-A) autoantibodies. Both Ro-antigen components (52 and 60 kD) were purified from a pig spleen extract, using fast protein liquid chromatography (FPLC). Anti-La, anti-RNP, anti-DNA and anti-Sm antibodies do not react to the purified antigen. There was a strong correlation between anti-Ro activity in EIA and the titers in counter immunoelectrophoresis (rs = 0.893). Anti-Ro antibodies were found in 54 (69.2%) of 78 SLE sera by the developed EIA.

A solid-phase enzyme immunoassay for the detection of tetanus toxin using human and murine monoclonal antibodies.

Three human and three murine monoclonal antibodies were tested for their reactivity to tetanus toxin and toxoid and used to establish an enzyme immunoassay specific for tetanus toxin. The dissociation constants of the monoclonal antibodies were between 3.91 x 10(-9) and 8.48 x 10(-12). Two human monoclonal antibodies recognized conformation determinants on the toxin, whereas the others reacted to the heavy chain. Only a combination of antibodies of the two species allowed the development of an enzyme immunoassay for the detection of tetanus toxin with a lower detection limit of 1.2 micrograms/l.

[Detection of autoantibodies to extractable nuclear antigens in autoimmune diseases of rheumatic origin using immunoblotting--comparison with counter-electrophoresis]. / Nachweis von Autoantikörpern gegen extrahierbare nukleäre Antigene (ENA) bei Autoimmunerkrankungen des rheumatischen Formenkreises mittels Immunoblotting--Vergleich mit der Gegenstromelektrophorese.

Sera from 82 patients with rheumatic autoimmune disease were tested for anti-ENA antibodies by immunoblotting and counterimmunoelectrophoresis (CIE), using HeLa cell extract and rabbit thymus extract, respectively, as antigens. Anti-ENA antibodies were more frequently detected and could be better differentiated by immunoblotting rather than by CIE. There was especially an increase in anti-Sm antibodies (23, in contrast to four positive results), which was only detectable in serum samples for the 59 SLE patients. The sera of the six patients with MCTD all reacted with the 68 U1-RNP antigen. The sera of the five patients with Sjögren's syndrome only recognized the 50k La antigen, while other anti-ENA antibodies were not observed. In SLE anti-La antibodies were often associated with other anti-ENA antibodies. Seven out of eight SLE patients showing a combined detection of antibodies against Sm, U1-RNP and La by immunoblotting demonstrated severe organ involvements, especially lupus nephritis. Therefore, the characterization of anti-ENA antibodies by immunoblotting may contribute to improve the differentiation of connective tissue diseases.