The Levels of the Lectin Pathway Serine Protease MASP-1 and Its Complex Formation with C1 Inhibitor Are Linked to the Severity of Hereditary Angioedema.

C1 inhibitor (C1-INH) is known to form complexes with the lectin complement pathway serine proteases MASP-1 and MASP-2. Deficiency of C1-INH is associated with hereditary angioedema (HAE), an autosomal inherited disease characterized by swelling attacks caused by elevated levels of bradykinin. MASP-1 was shown to cleave high m.w. kininogen into bradykinin; therefore, we hypothesized that MASP-1 levels and the quantity of MASP-1/C1-INH complexes might be associated with different paraclinical and clinical outcomes of HAE. We measured MASP-1 serum concentrations and endogenous MASP-1/C1-INH complex levels in 128 HAE patients and 100 controls. Relatively high levels of pre-existing MASP-1/C1-INH complexes were observed in normal serum, and we found that both the serum levels of MASP-1 and the complex formation between MASP-1 and C1-INH were significantly reduced in HAE patients compared with matched controls (p < 0.0001). The level of MASP-1 and MASP-1/C1-INH complexes in HE patients correlated with the level of C1-INH (p = 0.0009 and p = 0.0047, respectively), the level of C4 (p = 0.0084 and p < 0.0001, respectively), and the number of attacks in the year of blood sampling (p = 0.0075 and p = 0.0058, respectively). In conclusion, we show that MASP-1/C1-INH complexes circulate in normal human blood. The levels of MASP-1 and MASP-1/C1-INH complexes are reduced in HAE patients compared with controls. Both MASP-1 and MASP-1/C1-INH complexes are related to the degree of complement C4 consumption, as well as the severity of disease. These results suggest that MASP-1 may exert a previously unrecognized role in the pathophysiology of HAE.

Plasma levels of OLFM4 in normals and patients with gastrointestinal cancer.

Olfactomedin 4 (OLFM4) is a secreted glycoprotein predominantly expressed in bone marrow and gastrointestinal tissues. Aberrant expression of OLFM4 has been shown in several cancers. However, the clinical significance hereof is currently controversial. OLFM4 has been proposed as a candidate biomarker of gastrointestinal cancers. To address this, we developed monoclonal antibodies against synthetic peptides representing various segments of OLFM4. We examined expression of OLFM4 in epithelial cells by immunohistochemistry and found that OLFM4 is highly expressed in proliferating benign epithelial cells and in some carcinoma cells. We developed an Enzyme Linked Immunosorbent Assay for OLFM4 and investigated whether plasma levels of OLFM4 reflect colorectal malignancies, but were unable to see any such association. Instead, we observed two populations of individuals with respect to OLFM4 levels in plasma, the majority with OLFM4 in plasma between 0 and 0.1 µg/ml, mean 0.028 µg/ml while 10% of both normals and patients with cancers had OLFM4 between 4 and 60 µg/ml, mean 15 µg/ml. The levels were constant over time. The background for this high plasma level is not known, but must be taken into account if OLFM4 is used as biomarker for GI cancers.

A novel mannose-binding lectin/ficolin-associated protein is highly expressed in heart and skeletal muscle tissues and inhibits complement activation.

The human lectin complement pathway involves circulating complexes consisting of mannose-binding lectin (MBL) or three ficolins (ficolin-1, -2, and -3) in association with three MBL/ficolin-associated serine proteases (MASP) (MASP-1, -2, and -3) and a nonenzymatic sMAP. MASP-1 and MASP-3 (MASP1 isoforms 1 and 2, respectively) are splice variants of the MASP1 gene, whereas MASP-2 and sMAP are splice variants of the MASP2 gene. We have identified a novel serum protein of 45 kDa that is associated with MBL and the ficolins. This protein is named MBL/ficolin-associated protein 1 (MAP-1 corresponding to MASP1 isoform 3). The transcript generating MAP-1 (MASP1_v3) contains exons 1-8 and a novel exon encoding an in-frame stop codon. The corresponding protein lacks the serine protease domains but contains most of the common heavy chain of MASP-1 and MASP-3. Additionally MAP-1 contains 17 unique C-terminal amino acids. By use of quantitative PCR and MAP-1-specific immunohistochemistry, we found that MAP-1 is highly expressed in myocardial and skeletal muscle tissues as well as in liver hepatocytes with a different expression profile than that observed for MASP-1 and MASP-3. MAP-1 co-precipitated from human serum with MBL, ficolin-2, and ficolin-3, and recombinant MAP-1 was able to inhibit complement C4 deposition via both the ficolin-3 and MBL pathway. In conclusion we have identified a novel 45-kDa serum protein derived from the MASP1 gene, which is highly expressed in striated muscle tissues. It is found in complex with MBL and ficolins and may function as a potent inhibitor of the complement system in vivo.

Development of ELISA-based methods to measure the anti-malarial drug chloroquine in plasma and in pharmaceutical formulations.

BACKGROUND: In Central and South America and Eastern and Southern Africa, Plasmodium vivax infections accounts for 71-81% and 5% of malaria cases, respectively. In these areas, chloroquine (CQ) remains the treatment of choice for P. vivax malaria. In addition, CQ has recently proven to be an effective HIV-1 therapeutic agent. There is a dire need to continue monitoring quality of CQ as there is a major influx of substandard and fake formulations into malaria-endemic countries. The use of fake/substandard drugs will result in sub-therapeutic levels endangering the patient and possibly select for parasite resistance. The aim of this study was to develop an inexpensive, simple antibody-based ELISA to measure CQ concentrations in tablets and in plasma. METHODS: A monoclonal antibody (MAb) that reacts with the N-side chain of the CQ molecule was prepared by use of a CQ analogue. A specific and reliable ELISA for detection of CQ was developed. The developed assay was validated by measuring CQ in tablets sold in Denmark, India and Sudan. Furthermore, kinetics of CQ concentrations in plasma of four volunteers, who ingested two tablets of Malarex® containing, 250 mg CQ base, were measured before drug intake, three hours later and thereafter at days 1, 3, 7, 14, 21 and 28. The same plasma samples were simultaneously measured by high performance liquid chromatography (HPLC). RESULTS: The ELISA proved an easy-to-handle and very sensitive tool for the detection of CQ with a lower limit of detection at 3.9 ng/ml. ELISA levels of CQ in plasma showed high agreement with the levels obtained by HPLC (r = 0.98). The specificity in the negative control group was 100%. CONCLUSION: The developed ELISA can be used for quality screening of CQ in pharmaceutical formulations and for drug monitoring in malaria and in other infectious diseases, such as HIV, where CQ proved to be an effective therapeutic agent. The methodology has been exploited to develop monoclonal antibodies for the drugs used in artemisinin-based combination therapy (ACT).

Sodium polyanethole sulfonate as an inhibitor of activation of complement function in blood culture systems.

Sodium polyanethole sulfonate (SPS; trade name, Liquoid) is a constituent in culture media used to grow bacteria from blood samples from patients suspected of bacteremia. SPS prevents the killing of bacteria by innate cellular and humoral factors. We analyzed the effect of SPS on the three complement activation pathways: the classical, alternative, and lectin pathways, respectively. Inhibition of complement activity by SPS is caused by a blocking of complement activation and is not a result of complement consumption. The classical pathway is inhibited at SPS concentrations greater than 0.1 mg/ml, and complete inhibition is seen at 0.4 mg/ml. An SPS concentration of 0.5 mg/ml completely inhibits the binding of C1q and subsequent incorporation of C3, C4, and C9. The same was observed for the alternative pathway with an inhibition at SPS concentrations from 0.1 mg/ml and a complete inhibition from 0.4 mg/ml. Here, properdin binding was completely absent, and no incorporation of C3 and C9 was observed. In contrast, the lectin complement pathway remains unaffected at these SPS concentrations, and inhibition is first observed from 0.7 mg/ml. A complete inhibition required concentrations greater than 1 mg/ml. SPS is used in growth media (e.g., BACTEC and BacT/Alert) at concentrations from 0.3 to 0.5 mg/ml. The well-known finding that certain bacteria are growth inhibited by blood factors could therefore be a consequence of the lectin pathway, which is not inhibited at these concentrations. In addition, our findings also open up the possibility of a new assay for the assessment of the functional capacity of the lectin complement pathway.

Evolutionary conservation of mannan-binding lectin (MBL) in bony fish: identification, characterization and expression analysis of three bona fide collectin homologues of MBL in the rainbow trout (Onchorhynchus mykiss).

The complement system of fish is generally as complex as in mammals, and in addition Teleost fish often possess several genes encoding different subtypes of a given complement component, such as C3-1, C3-3 and C3-4. Initiators of both the classical (C1) and alternative pathway (factor B) have been characterized in the rainbow trout but so far no molecules of the lectin pathway have been identified. Based on the generally accepted idea of complement evolution, which predicts that the alternative pathway predates the two other pathways, and that the lectin pathway developed before the classical, we set out to characterize members of the lectin pathway in fish. We identified and characterized three homologues of mannan-binding lectin (MBL) with a bona fide collectin structure. By means of RT-PCR and immunohistochemistry using monoclonal antibodies we found that they were synthesized in the spleen, the anterior intestine and the liver. In the liver, we saw co-expression with mannan-binding lectin associated serine protease (MASP). The MBL homologues 2 and 3 (MBL-H2,3) were also found in the vascular system of the rainbow trout. By means of gel size exclusion chromatography of serum we found that MBL-H2,3 oligomerized heterogeneously from monomers to tetramers of a trimeric collagenous subunit. Sequence comparison and phylogenetic studies showed that the homologues were more related with MBL than any other collectins, and that two previously characterized trout proteins, designated MBL1 and MBL2, should be reconsidered as MBL candidates.

Characterization of a polymorphism in the coding sequence of FCN3 resulting in a Ficolin-3 (Hakata antigen) deficiency state.

Ficolin-3 (Hakata antigen or H-ficolin) is a soluble pattern recognition molecule in the lectin complement pathway. We speculated whether common genetic variations in the FCN3 gene contribute to deficiency of Ficolin-3. The FCN3 gene was sequenced in 237 healthy Danish Caucasians. The relevance of polymorphisms was assessed with antibodies against Ficolin-3 in a novel ELISA system and by production of recombinant Ficolin-3 variants. Ficolin-3 serum profiles were analyzed by SDS-PAGE and western blotting. Ficolin-3 serum concentration varied 10-fold (median, 24microg/ml; range, 3-54microg/ml). Out of several polymorphisms one FCN3+1637delC causing a reading frame shift and a distortion of the C-terminal end of the molecule with an allele frequency of 0.011 was particularly interesting. In individuals heterozygous for the FCN3+1637delC deletion lowered Ficolin-3 concentration was observed (P=0.025). SDS-PAGE and western blotting of serum revealed a weak band corresponding to the truncated molecule in addition to the normal Ficolin-3 pattern. Characterization of recombinant Ficolin-3 derived from FCN3+1637delC showed that in the homozygous situation this allelic variant would lead to Ficolin-3 deficiency. In conclusion an FCN3+1637delC deletion variant disrupting the possibility for pattern recognition was detected. Characterization of recombinant variant Ficolin-3 shows that homozygosity for the FCN3+1637delC deletion may lead to Ficolin-3 deficiency and may thus be the basis for a novel complement deficiency state.

Dominant-negative SERPING1 variants cause intracellular retention of C1 inhibitor in hereditary angioedema.

Hereditary angioedema (HAE) is an autosomal dominant disease characterized by recurrent edema attacks associated with morbidity and mortality. HAE results from variations in the SERPING1 gene that encodes the C1 inhibitor (C1INH), a serine protease inhibitor (serpin). Reduced plasma levels of C1INH lead to enhanced activation of the contact system, triggering high levels of bradykinin and increased vascular permeability, but the cellular mechanisms leading to low C1INH levels (20%-30% of normal) in heterozygous HAE type I patients remain obscure. Here, we showed that C1INH encoded by a subset of HAE-causing SERPING1 alleles affected secretion of normal C1INH protein in a dominant-negative fashion by triggering formation of protein-protein interactions between normal and mutant C1INH, leading to the creation of larger intracellular C1INH aggregates that were trapped in the endoplasmic reticulum (ER). Notably, intracellular aggregation of C1INH and ER abnormality were observed in fibroblasts from a heterozygous carrier of a dominant-negative SERPING1 gene variant, but the condition was ameliorated by viral delivery of the SERPING1 gene. Collectively, our data link abnormal accumulation of serpins, a hallmark of serpinopathies, with dominant-negative disease mechanisms affecting C1INH plasma levels in HAE type I patients, and may pave the way for new treatments of HAE.

Association between combined properdin and mannose-binding lectin deficiency and infection with Neisseria meningitidis.

BACKGROUND: Individuals genetically deficient of properdin are more susceptible to meningococcal disease. Likewise low concentration or decreased biological activity of mannose-binding lectin (MBL) is associated with higher incidence of bacterial infections during childhood. In this study we report our findings in a Danish family with a remarkably high incidence of meningococcal meningitis-in total four cases, one of them fatal. METHODS: Properdin and MBL were quantified by ELISA and the properdin gene was screened for sequence variations using denaturing high-performance liquid chromatography (DHPLC) and subsequent sequencing of abnormal patterns. The MBL gene was genotyped for the three known variant alleles (B, C and D) as well as three promoter polymorphisms (-221Y/X, -550H/L and +4P/Q). RESULTS: Two out of six males with undetectable properdin activity had meningitis. They had also low MBL serum levels or carried an MBL variant allele, whereas high MBL concentrations were measured in three out of four properdin deficient males--without meningitis. A splice site mutation in exon 10 (c.1487-2A>G) was found in the properdin gene and co segregated with biochemically measured properdin deficiency. CONCLUSION: Our results indicate that a combined deficiency of both properdin and MBL increases the risk of infection with Neisseria meningitidis and stress the importance of epistatic genetic interactions in disease susceptibility.

Complement factor C4 activation in patients with hereditary angioedema.

OBJECTIVES: Low complement factor C4 is usually considered a valuable screening tool for patients with the potentially life-threatening hereditary angioedema with C1-inhibitor (C1-INH) deficiency (C1-INH-HAE). However, there are patients with C1-INH-HAE presenting with normal C4 levels. This means, that C1-INH-HAE may potentially be overlooked, if screening is performed only by measurement of C4. It has been suggested that measurement of C4 activation products is better suited to avoid false negative results. Our aim was to investigate whether total antigenic C4 or non-functional C4c is a better measure of the increased C4 activation in C1-INH-HAE patients. DESIGN AND METHODS: Two different monoclonal antibodies (mAb) to human C4 were produced: one had specificity for the ß-chain of C4 and would thus react with both functional and non-functional C4, and the other was developed against the factor I cleavage site on the α3-domain of C4 and was thus specific for activated, non-functional C4c. With these mAb we investigated plasma from 19 Danish C1-INH-HAE patients in three different enzyme-linked immunosorbent assays (ELISAs): a total antigenic C4 assay, a functional C4 assay and an assay measuring non-functional C4c. RESULTS: The amount of total antigenic C4 varied considerably between patients and 2 patients had total antigenic C4 levels in the normal area. Functional C4 was low in all C1-INH-HAE patients. A C4c/C4 ratio showed that around half the C4 measured in patients was non-functional and captured all C1-INH-HAE patients. CONCLUSIONS: This study shows that the C4c/C4 ratio seems to be a better diagnostic measure than total antigenic C4 alone. Our findings underline that screening with total antigenic C4 implies a risk of overlooking C1-INH-HAE patients.

Proteome-wide antigen discovery of novel protective vaccine candidates against Staphylococcus aureus infection.

Methicillin-resistant Staphylococcus aureus (MRSA) is a rapidly growing problem, especially in hospitals where MRSA cause increased morbidity and mortality and a significant rise in health expenditures. As many strains of MRSA are resistant to other antimicrobials in addition to methicillin, there is an urgent need to institute non-antimicrobial measures, such as vaccination, against the spread of MRSA. With the aim of finding new protective antigens for vaccine development, this study used a proteome-wide in silico antigen prediction platform to screen the proteome of S. aureus strain MRSA252. Thirty-five different S. aureus proteins were identified, recombinantly expressed, and tested for protection in a lethal sepsis mouse model using S. aureus strain MRSA252 as the challenge organism. We found that 13 of the 35 recombinant peptides yielded significant protection and that 12 of these antigens were highly conserved across 70 completely sequenced S. aureus strains. Thus, this in silico platform was capable of identifying novel candidates for inclusion in future vaccines against MRSA.

Deamidation and cross-linking of gliadin peptides by transglutaminases and the relation to celiac disease.

Activation of small intestinal gluten-reactive CD4+ T cells is a critical event in celiac disease. Such cells predominantly recognise gluten peptides in which specific glutamines are deamidated. Deamidation may be catalysed by intestinal tissue transglutaminase (TG2), a protein which is also the main autoantigen in celiac disease. Our aim was to study how the two main catalytic activities of transglutaminase--deamidation and transamidation (cross-linking) of an immunodominant gliadin epitope--are influenced by the presence of acceptor amines in the intestinal mucosa, and thereby contribute to further elucidation of the pathogenetic mechanisms in celiac disease. We prepared monoclonal antibodies, reacting specifically with the non-deamidated epitope QPFPQPQLPYPQPQ-amide and/or the deamidated epitope QPFPQPELPYPQPQ-amide. A solid phase immunoassay combined with gel filtration chromatography was used to analyse deamidation and cross-linking of these peptides to proteins. Our results show that QPFPQPQLPYPQPQ-amide was deamidated when incubated with purified TG2, with fresh mucosal sheets and with mucosal homogenates. Of other transglutaminases tested, only Streptoverticillium transglutaminase was able to generate the deamidated epitope. A fraction of the non-deamidated epitope was cross-linked to proteins, including TG2. The results suggest that intestinal TG2 is responsible for generation of the active deamidated epitope. As the epitope often occurs in a repeat structure, the result may be cross-linking of a deamidated, i.e., activated cell epitope. Alternatively, the deamidation may occur by reversal of the cross-linking reaction. The results provide a basis for the suggestion that binding of a peptide to a protein, in connection to its modification to a T cell epitope, might be a general explanation for the role of TG2 in celiac disease and a possible mechanism for the generation of autoantigens.

Mannose-binding lectin deficiency--revisited.

There is an emerging interest for mannose-binding lectin (MBL) due to its role in innate immunity. In this survey we present a mixture of old and new data describing the effect MBL polymorphisms may have on the level and function of the molecule. Three single nucleotide substitutions in exon 1 of the mbl2 gene cause a dominant decrease of functional MBL in the circulation. Additionally, promoter variants influence expression of MBL. It has been assumed that the structural variant alleles may disrupt the assembly of MBL trimers or accelerate the degradation of the protein, thereby causing the decrease in MBL serum concentrations. We have analysed 1183 different sera in a double sandwich antibody ELISA using the same antibody to capture and detect MBL and find the same results as have been presented previously showing that different MBL promoter alleles have profound effect of on the MBL serum concentration. The use of a new anti-MBL monoclonal antibody, however, has shown that the amount of MBL in the circulation is less dependent on the presence of structural variant alleles than previously anticipated. Molecular characterisation of MBL revealed that sera from donors homozygous for the normal MBL genotype predominantly contained high molecular weight MBL, while sera from individuals heterozygous for the variant alleles contained both high and low molecular weight MBL. The ratio between high and low molecular weight MBL was dependent on the MBL promoter type on the normal haplotype. Sera deriving from individuals homozygous for MBL variant alleles contained mainly low molecular weight MBL. Of the different oligomers of MBL only the high molecular weight forms bound mannan efficiently and activated complement. In contrast to a previous notion, we demonstrate that variant alleles give rise to relatively high levels of MBL in the circulation. However, the variant MBL has lower molecular weight and is dysfunctional compared to normal MBL. The physiological relevance of variant MBL remains to be established.

Antibodies with specificity for native and denatured forms of ovalbumin differ in reactivity between enzyme-linked immunosorbent assays.

In this study, polyclonal and monoclonal antibodies to native and denatured chicken ovalbumin (OVA) were produced to compare their dependency on continuous and three-dimensional epitopes. These antibodies were characterized with respect to reactivity to native and denatured OVA by enzyme-linked immunosorbent assay (ELISA) employing surface-bound OVA and streptavidin-capture ELISA to determine whether effects of different coating influence antibody specificity and with respect to epitope specificity by peptide ELISA, using overlapping peptides, covering the complete OVA sequence. Polyclonal antibodies to native OVA reacted strongly with native and denatured OVA in both assays, but did not react with the overlapping peptides. Polyclonal antibodies to denatured OVA reacted strongly with both OVA forms and with several of the overlapping peptides. Monoclonal antibodies to native OVA reacted preferentially with three-dimensional epitopes on native OVA and not with denatured OVA. Monoclonal antibodies to denatured OVA showed reactivity to both OVA forms. Two of these monoclonal antibodies, HYB 94-06 and 94-07, showed reactivity to overlapping peptides and their epitopes were identified as flexible structures constituting amino acids 130-135 and 136-141, respectively. Moreover, comparison of antibody reactivity to N OVA revealed that in the streptavidin-capture ELISA, antibody reactivity was notably reduced compared to ELISA employing surface-bound OVA. Collectively, immunization with native OVA preferentially generates highly specific antibodies reacting with three-dimensional epitopes, whereas immunization with denatured OVA generates antibodies occasionally reacting with continuous epitopes. Moreover, as differences in monoclonal antibody reactivity was found between the two assays, monoclonal antibodies always should be selected by an assay mimicking the desired use of the final antibodies as closely as possible.

Homology building as a means to define antigenic epitopes on dihydrofolate reductase (DHFR) from Plasmodium falciparum.

BACKGROUND: The aim of this study was to develop site-specific antibodies as a tool to capture Plasmodium falciparum-dihydrofolate reductase (Pf-DHFR) from blood samples from P. falciparum infected individuals in order to detect, in a sandwich ELISA, structural alterations due to point mutations in the gene coding for Pf-DHFR. Furthermore, we wanted to study the potential use of homology models in general and of Pf-DHFR in particular in predicting antigenic malarial surface epitopes. METHODS: A homology model of Pf-DHFR domain was employed to define an epitope for the development of site-specific antibodies against Pf-DHFR. The homology model suggested an exposed loop encompassing amino acid residues 64-100. A synthetic peptide of 37-mers whose sequence corresponded to the sequence of amino acid residues 64-100 of Pf-DHFR was synthesized and used to immunize mice for antibodies. Additionally, polyclonal antibodies recognizing a recombinant DHFR enzyme were produced in rabbits. RESULTS AND CONCLUSIONS: Serum from mice immunized with the 37-mer showed strong reactivity against both the immunizing peptide, recombinant DHFR and a preparation of crude antigen from P. falciparum infected red blood cells. Five monoclonal antibodies were obtained, one of which showed reactivity towards crude antigen prepared from P. falciparum infected red cells. Western blot analysis revealed that both the polyclonal and monoclonal antibodies recognized Pf-DHFR. Our study provides insight into the potential use of homology models in general and of Pf-DHFR in particular in predicting antigenic malarial surface epitopes.

Mannose-binding lectin (MBL) therapy in an MBL-deficient patient with severe cystic fibrosis lung disease.

Deficiency of mannose-binding lectin has been shown to be a risk factor for cystic fibrosis (CF) patients. We, therefore, decided to treat a patient with CF, mannose-binding lectin deficiency, severe bronchopulmonary Pseudomonas aeruginosa infection, and rapid deterioration of lung function with purified mannose-binding lectin in an attempt to ameliorate the course of the lung disease. The mannose-binding lectin used originated from pooled human donor plasma and was given as an intravenous infusion twice a week for a period of 3 months. The patients's clinical condition was stabilized during the treatment period, but was not improved. No adverse events were observed. However, the lung function assessed as percent forced expiratory volume in 1 sec (FEV1%) and percent forced vital capacirt (FVC%) correlated significantly with the mannose-binding serum lectin levels (rho=+0.68, P=0.008, and rho=+0.73, P=0.004). Additionally, an inverse correlation with the acute phase-reactant C-reactive protein and the proinflammatory cytokine IL-6 was observed (rho=-0.49, P=0.007 and rho=-0.41, P=0.04, respectively). The results emphasize the importance of mannose-binding lectin as a secondary disease modifier in CF. Moreover, purified mannose-binding lectin can safely be administered to chronically ill patients, and may be a potential treatment in CF and other diseases in which mannose-binding lectin deficiency plays a pathophysiological role.

A specific assay for quantification of human C4c by use of an anti-C4c monoclonal antibody.

The increasing evidence of the implication of the complement system in the pathogenesis of several diseases has emphasized the need for the development of specific and valid assays, optimized for quantitative detection of complement activation in vivo. In the present study, we have developed a mouse monoclonal antibody (mAb) that is able to detect fluid phase C4c without interference from other products generated from the complement component C4. The C4c specific mAb was tested in different enzyme-linked immunosorbent assay (ELISA) combinations with various types of in vitro activated sera and samples from factor I deficient patients. The specificity of the mAb was further evaluated by immunoprecipitation techniques and by analysis of eluted fragments of C4 after immunoaffinity chromatography. The anti-C4c mAb was confirmed to be C4c specific, as it showed no cross-reactivity with native (un-cleaved) C4, C4b, iC4b, or C4d. Also, no reaction was observed with C4 fragments in factor I deficient plasma or serum samples. We established and validated a sandwich ELISA based on this C4c specific antibody. The normal range of C4c in EDTA/futhan plasma collected from 100 Danish blood donors was measured, with a mean of 0.85mg/L and a range of 0.19-2.21mg/L. We believe that the C4c specific antibody and the ELISA might be important tools in the future assessment of in vivo activation in situations where the classical or the lectin complement pathways are involved in the pathogenesis.

Three monoclonal antibodies against the serpin protease nexin-1 prevent protease translocation.

Protease nexin-1 (PN-1) belongs to the serpin family and is an inhibitor of thrombin, plasmin, urokinase-type plasminogen activator, and matriptase. Recent studies have suggested PN-1 to play important roles in vascular-, neuro-, and tumour-biology. The serpin inhibitory mechanism consists of the serpin presenting its so-called reactive centre loop as a substrate to its target protease, resulting in a covalent complex with the inactivated enzyme. Previously, three mechanisms have been proposed for the inactivation of serpins by monoclonal antibodies: steric blockage of protease recognition, conversion to an inactive conformation or induction of serpin substrate behaviour. Until now, no inhibitory antibodies against PN-1 have been thoroughly characterised. Here we report the development of three monoclonal antibodies binding specifically and with high affinity to human PN-1. The antibodies all abolish the protease inhibitory activity of PN-1. In the presence of the antibodies, PN-1 does not form a complex with its target proteases, but is recovered in a reactive centre cleaved form. Using site-directed mutagenesis, we mapped the three overlapping epitopes to an area spanning the gap between the loop connecting α-helix F with ß-strand 3A and the loop connecting α-helix A with ß-strand 1B. We conclude that antibody binding causes a direct blockage of the final critical step of protease translocation, resulting in abortive inhibition and premature release of reactive centre cleaved PN-1. These new antibodies will provide a powerful tool to study the in vivo role of PN-1's protease inhibitory activity.

ELISA for determination of total coagulation factor XII concentration in human plasma.

Human blood coagulation factor XII (FXII) is the one chain 80 kDa zymogen form of the active serine protease α-FXIIa, which consists of a heavy and light chain linked by a disulfide bond, the light chain being responsible for the proteolytical activity. FXII is the first component of the contact dependent pathway of coagulation, but its physiological role is still subject to debate. In the present study we utilized two monoclonal antibodies against the heavy chain of FXII to establish a sandwich enzyme linked immunosorbent assay (ELISA) for quantification of total FXII concentration in human plasma samples. A unique characteristic of this assay is its equal recognition of FXII and inhibitor bound FXII. This is important, as inhibitor complexes of α-FXIIa are formed in vivo as well as during blood sampling and handling. Validation of the assay demonstrated a high sensitivity, with a limit of detection and quantification of 1.2 ng/mL and 2.6 ng/mL respectively. The coefficients of variation for the repeatability and within-laboratory standard deviations were 2.6% and 5.2% respectively. The reference interval determined from healthy volunteers (n=240) was 10.6-43 mg/L.

Serum concentration and interaction properties of MBL/ficolin associated protein-1.

Recently, a novel protein named MBL/ficolin associated protein-1 (MAP-1) derived from the MASP1 gene through differential splicing was identified. In the present study, we established biochemical characteristics, determined the serum level and assessed the interactions between the lectin complement pathway (LCP) recognition molecules and MAP-1. We expressed recombinant MAP-1 in CHO DG44 cells, developed a quantitative ELISA assay based on a MAP-1 specific monoclonal capture antibody and measured the serum levels in 100 Danish blood donors. In addition we assessed the association properties between MAP-1 and Ficolin-2, -3 and MBL in serum using ELISA and density gradient ultra centrifugation. When recombinant MAP-1 was subjected to N-glycosidase F treatment the molecular mass decreased from ∼45 kDa to ∼40 kDa equivalent with the calculated molecular mass from the deduced amino acid sequence without the signal peptide. We found that serum MAP-1 was very stable when subjected to repeated freeze and thaw cycles. The mean serum concentration of MAP-1 was found to be 240 ng/ml (range: 115-466 ng/ml). MAP-1 was predominantly found in complex with Ficolin-3 and to a lesser degree with Ficolin-2 and MBL and by use of density gradient ultra centrifugation we could show that the major part of serum MAP-1 circulates in complex with the LCP molecules. In conclusion, these results show that MAP-1 is a highly stable glycosylated human serum protein found in complex with Ficolin-3, Ficolin-2 and MBL.