Charlotte Sværke Jørgensen

Gc globulin (vitamin D‐binding protein) levels: an inhibition ELISA assay for determination of the total concentration of Gc globulin in plasma and serum

Gc globulin, also called vitamin D‐binding protein, is a plasma protein involved in the actin‐scavenger system. In this study, the total Gc globulin concentration in serum or plasma samples was determined using a new, fast, solid‐phase inhibition assay. Included in the study were 228 healthy volunteers (131 M, 97 F), 22 pregnant women, 90 cancer patients and 9 patients with chronic liver disease. Moreover, the degree of complexing with actin was determined in selected samples using crossed immunoelectrophoresis. The Gc globulin level in healthy controls was in the range 176–623 mg/L, showing no age dependency. The median level was found to be significantly higher in women than in men. Gc globulin concentrations were raised during pregnancy, showing a median value of 541 mg/L in the first trimester, and slightly raised to 574 mg/L in the second trimester. Cancer patients showed no changes in Gc globulin level, and there was no sign of increased amounts of complexing with actin. Chronic liver patients showed increased levels of Gc globulin following transplantation, but no signs of complexing with actin. This new solid‐phase inhibition assay is fast, it is a good complement to the existing quantification methods, and it is especially suitable for determination of the Gc globulin status in acute liver patients before and during treatment.

Interaction of C1q with the receptor calreticulin requires a conformational change in C1q.

The interaction between C1q and the chaperone calreticulin was studied under various conditions. When both proteins were present in equal amounts in solution, no interaction could be demonstrated. However, C1q immobilized on a hydrophobic surface, exposed to heat‐treatment or bound to immunoglobulins (Igs) showed a strong, rapid and specific binding of calreticulin. The interaction appeared to be a two‐step process, and the initial phase of interaction was sensitive to high concentrations of salt but not to a physiological salt concentration. The following strong binding was insensitive to salt and extremes of pH but sensitive to strongly denaturing agents (urea and guanidine). The sensitivity to salt during the initial phase of interaction was practically identical to that observed when calreticulin was bound to type V collagen. Binding between C1q and calreticulin could be inhibited by serum amyloid P component and by proteinase K‐digested ovalbumin, and the binding of calreticulin to proteinase K‐digested ovalbumin was shown to be inhibited by C1q. The data indicate that C1q binds stably to the peptide‐binding site of calreticulin and that the initial binding of calreticulin to C1q involves the collagen‐like domain of the C1q molecule. In conclusion, our results suggest calreticulin as a potential receptor for an altered conformation of C1q as occurs during binding to Igs. Thus, the chaperone and protein‐scavenging function of calreticulin may extend from the endoplasmic reticulum to the topologically equivalent cell surface, where it may contribute to the elimination of immune complexes and apoptotic cells.

Comparison of the sensitivity of the Legionella urinary antigen EIA kits from Binax and Biotest with urine from patients with infections caused by less common serogroups and subgroups of Legionella

The detection of urinary antigen is the most widely used method to diagnose Legionnaires’ disease (LD), so it is important that these assays have a high sensitivity for the disease. In this study, we compare two kits for their ability to detect urinary antigen in urine samples from patients infected with Legionella species and L. pneumophila sero- and subgroups not considered as the most common causes of LD. Urine samples (n = 33) from 30 culture-proven cases of L. pneumophila serogroup (sg) 1, subgroup non-Pontiac infection, and urine samples (n = 35) from 32 cases of non-L. pneumophila species or non-sg 1 infection were examined using the Binax EIA and Biotest EIA kits. For both groups, the overall diagnostic sensitivity of the Binax kit was significantly better than the sensitivity of the Biotest kits (P < 0.0001). For the non-Pontiac group, the sensitivity was 81.8 and 42.4%, respectively, and for the non-sg1 group, it was 51.4 and 28.6%, respectively. It was concluded that the Binax kit was more suitable for the general diagnosis of LD than the Biotest kit, but we still need urinary antigen detection methods with higher sensitivity for non-sg1 LD.

Non-specific binding in solid phase immunoassays for autoantibodies correlates with inflammation markers

Enzyme-linked immunosorbent assay (ELISA) is a validated and sensitive method for detection of human autoantibodies, but may have problems with specificity. Non-specific binding is a well-known problem often observed in tests for autoantibodies, when sera are incubated on plastic surfaces, e.g. an ELISA plate. To understand the mechanisms underlying non-specific immunoglobulin deposition, we here analyse the phenomenon in detail and we propose means of reducing false positive test results caused by non-specific binding. The level of non-specific binding, in sera with suspected autoreactivity, was analysed in non-coated and autoantigen-coated ELISA wells and 4-32% of sera showed a high level of non-specific binding depending on the assay conditions and serum properties. Non-specifically binding sera were found to contain increased concentrations of IgG and other inflammatory mediators. Moreover, non-specific binding could be induced in serum by increasing the concentration of IgG and incubating the serum at 40 °C. This suggests that non-specific binding immunoglobulins can be formed during inflammation with high immunoglobulin levels and elevated temperature. We show that the level of non-specific binding correlates with the IgG concentration and therefore propose that non-specific binding may be interpreted as an informative finding indicative of elevated IgG and inflammation.

Determination of new cutoff values for indirect immunofluorescence antibody test for Q fever diagnosis in Denmark

Q fever is a ubiquitous zoonosis caused by Coxiella burnetii. The disease is emerging in many parts of the world, likely because of increased awareness and availability of better diagnostics. The diagnosis is primarily based on serology. Because the prevalence of the disease varies worldwide, the establishment of local cutoff values is needed. A baseline for antibodies against C. burnetii in Denmark was defined by testing sera from healthy Danish volunteers using a commercially available immunofluorescence antibody test. Cross-reactivity was studied on sera obtained from patients experiencing clinically related diseases. The cutoff titers suggested by the manufacturer were found to result in very low specificity of the test. The specificity was, however, effectively increased by using cutoff titers based on the local baseline and equal to immunoglobulin M (IgM) phase I > or =128, IgM phase II > or =256, IgG phase I > or =512, and IgG phase II > or =1024.

Large-scale purification and characterization of non- glycosylated Gc globulin (vitamin D-binding protein) from plasma fraction IV

Gc globulin, also called vitamin D‐binding protein, is a plasma protein involved in the extracellular actin‐scavenger system. Low levels of Gc globulin have been found to correlate with multiple organ failure and nonsurvival of patients with fulminant hepatic failure and trauma. Therefore substitution therapy with Gc globulin might be beneficial for such patients, increasing their chance of survival. In the present study, we describe a large‐scale purification process for the production of a virus‐safe human plasma‐derived Gc globulin from Cohn fraction IV paste. The process includes three ion‐exchange‐chromatography steps, followed by a gel filtration, and two virus‐reduction steps are implemented. The Gc globulin product was characterized with respect to purity, functional activity, glycosylation and, finally, with respect to content of endotoxin. From the results, it can be concluded that human Gc globulin purified from Cohn fraction IV is non‐glycosylated. The purified Gc globulin is able to mask the presence of endotoxin by 20%.

Vaccination status and immune response to 13-valent pneumococcal conjugate vaccine in asplenic individuals

Overwhelming post-splenectomy infection (OPSI) is immediately life-threatening and vaccination against encapsulated bacteria, in particular pneumococci, decreases its incidence. First, we investigated the adherence to vaccination guidelines in a retrospective study of the hospital records of splenectomised patients. Second, patients were asked to complete a questionnaire and invited to participate in a study where 12-valent pneumococcal serotype-specific IgG concentrations were determined before and 4 to 6 weeks after vaccination with PCV13. Of 79 individuals who underwent splenectomy between 2000 and 2012: 81.0% received pneumococcal vaccine, 51.9% received vaccine against Haemophilus influenzae type B and 22.8% received meningococcal vaccine. 31 individuals were deceased. 33 individuals completed questionnaires and accepted participation in the second part of the study. The participants consisted of two groups: (1) prior PPV23 (n=24) and (2) prior PPV23+PCV13 (n=9). In group 1, pre-PCV13 GMCs≥0.35μg/mL were observed for serotypes 1, 4, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F, and GMCs<0.35μg/mL for serotypes 3 and 5, significant increases pre- to post-PCV13 were found for serotypes 1, 3, 4, 5, 7F, 18C, 19A, 23F (p≤0.001) and 19F (p=0.01) and all 12 serotypes-specific GMC were above 0.35μg/mL after vaccination. Group 2 did not receive vaccine in this study, but blood tests showed all 12 serotype-specific GMC>0.35μg/mL. Adherence to guidelines regarding primary pneumococcal vaccination was adequate but only a minority received the recommended meningococcal vaccination. High levels of pneumococcal serotype-specific antibodies were observed in the previous PPV23 vaccinated group, and more pronounced in the previous PCV13 group, and our data suggests that PCV13 is immunogenic for serotypes 1, 3, 4, 5, 7F, 18C, 19A, 19F and 23F, if used as a booster dose in asplenic patients with previous PPV23 vaccination.

Antigen-induced cytokine and chemokine release test for tuberculosis infection using adsorption of stimulated whole blood on filter paper and multiplex analysis.

Background: In vitro stimulation of whole blood or isolated blood cells with specific antigens is used for several purposes. Immediately following incubation with antigens, samples have to be centrifuged to stop the reactions by remaining cells and the supernatant refrigerated or analysed directly to preserve the analytes of interest, which makes samples difficult to prepare outside laboratories. We have tested whether spotting whole blood on filter paper after activation can be used in one of the tests for Mycobacterium tuberculosis infection (MTI), the QuantiFERON®-TB Gold In Tube test (QFT), where the spotting technique can make it suitable for use in locations without facilities like a centrifuge and a refrigerator. Materials and methods: Samples from 22 individuals undergoing screening for MTI and 10 healthy controls were incubated, centrifuged and IFN-γ measured by Enzyme-linked immunosorbent assay (ELISA), as described in the kit insert. In parallel, activated blood was spotted on filter paper (Schleicher & Schuell) and dried. The dried blood spot samples were analysed for 21 inflammatory markers with an in-house assay based on Luminex technology. Results: Our multiplex measurements of inflammatory markers in samples from suspected MTI patients confirmed the IFN-γ findings in the QFT. IL-2, GM-CSF, IL-5, and IL-1β were also found as useful markers for MTI. We were not able to distinguish between active tuberculosis and latent MTI. Conclusion: Applying blood on filter paper after incubation makes in vitro stimulation tests feasible in locations where heat and electricity is unavailable.

Seroincidence of Human Infections With Nontyphoid Salmonella Compared With Data From Public Health Surveillance and Food Animals in 13 European Countries

We developed a model that enabled a back-calculation of the annual salmonellosis seroincidence from measurements of Salmonella antibodies and applied this model to 9677 serum samples collected from populations in 13 European countries. We found a 10-fold difference in the seroincidence, which was lowest in Sweden (0.06 infections per person-year), Finland (0.07), and Denmark (0.08) and highest in Spain (0.61), followed by Poland (0.55). These numbers were not correlated with the reported national incidence of Salmonella infections in humans but were correlated with prevalence data of Salmonella in laying hens (P < .001), broilers (P < .001), and slaughter pigs (P = .03). Seroincidence also correlated with Swedish data on the country-specific risk of travel-associated Salmonella infections (P = .001). Estimates based on seroepidemiological methods are well suited to measure the force of transmission of Salmonella to human populations, in particular relevant for assessments where data include notifications from areas, states or countries with diverse characteristics of the Salmonella surveillance.

Immunosuppressive drugs impairs antibody response of the polysaccharide and conjugated pneumococcal vaccines in patients with Crohn's disease

BACKGROUND Patients with Crohns disease (CD) have a higher risk of infectious diseases including pneumococcal infections, and the risk increases with immunotherapy. The primary endpoint of this study was to investigate the specific antibody response to two pneumococcal vaccines in CD patients with and without immunosuppressive treatment four weeks post vaccination. METHODS In a randomized trial of the 23-valent pneumococcal polysaccharide vaccine (PPV23) and the 13-valent pneumococcal conjugated vaccine (PCV13), a group of CD patients treated with immunosuppressive drugs (IS) alone or in combination with TNF-α antagonists were compared to a group of CD patients not treated with any of these drugs (untreated). Specific pneumococcal antibody concentrations were measured against 12 serotypes common to the two vaccines before and 4 week after vaccination. RESULTS PCV13 induced a significantly higher antibody response for one serotype (23F) in IS treated patients and for two serotypes (9V and 23F) in untreated patients compared to CD patients vaccinated with PPV23. Untreated PPV23 recipients had higher responses for serotypes 9V and 18C compared to IS+TNF-α treated PPV23 recipients. Comparison between treatment groups showed that immunosuppressive treatment impaired the antibody response to both vaccines and that TNF-a treatment further conveyed additional impairment of the response. CONCLUSION PCV13 induces higher antibody response for some serotypes compared to PPV23. In addition, CD patients treated with immunosuppressive drugs alone or in combination with TNF-α antagonists had an impaired antibody response to both PPV23 and PCV13 compared to patients not receiving any of these treatments. The study has been registered in the European Clinical Trials Database (EudraCT, record no 2012-002867-86) and ClinicalTrials.gov (record no. NCT01947010).