Figure 3.1. Chromatogram of L-ficolin elution from the CysNAc column. The relative L-ficolin concentration (closed circles, left Y-axis, arbitrary units) and the ionic strength (open circles, right Y-axis, mM NaCl) are shown. As can be observed the relative L-ficolin concentration in the eluate is increased when the ionic strength is decreased. For a detailed description of the experimental setup see section

Fraction ml Fraction ml Fraction ml Fraction ml Fraction ml Fraction mlFraction ml Fraction ml Fraction ml A B Figure 3.2. MonoQ purification step of L-ficolin. Panel A shows the chromatogram from the elution of bound material from the monoQ column. Panel B shows the SDS-PAGE analysis of the protein content of the first 9 gradient fractions eluted. As can be observed in the chromatogram very little protein is eluted from the column but when analyzed on SDS-PAGE, L-ficolin (35 kDa band) is found in the fractions between 69 and 73 ml. In the initial two fractions (69-71) no contaminants are visible while in the later fractions (71-73) contaminants corresponding to the observed shoulder of the peak centered at 71 ml becomes visible. For a detailed description of the experimental setup see section

L-ficolin non-reduced (1 μg) L-ficolin reduced (1 μg) Figure 3.3. Purified L-ficolin analyzed reduced and non-reduced on SDS-PAGE. Non-reduced, three predominant bands can be observed one at 35 kDa and two of more than 212 kDa. The top bands are L-ficolin multimers composed of several L- ficolin polypeptide chains while the 35 kDa band is the L-ficolin monomer. In between the top bands and 158 kDa marker other bands can be observed which most likely are low-abundance oligomeric forms of L-ficolin. Reduced, the L-ficolin monomeric band increases in intensity compared to the non-reduced lane confirming the top bands as being L-ficolin oligomers. Four other bands are detected in the reduced lane but these are low abundance contaminants and only visible due to prolonged silver staining. For a detailed description of the experimental setup see section

Figure 3.4. Cross-reactivity of anti-L-ficolin antibodies. To assess the cross- reactivity of the anti-L-ficolin antibodies towards H-ficolin, wells coated with L- ficolin or His-tagged rH-ficolin were developed with biotinylated anti-L-ficolin antibodies. As can be observed the L-ficolin antibodies have about 10% cross- reactivity towards H-ficolin at 1 μg/ml while at 40 ng/ml the signal from the his- tagged rH-ficolin is at background level. This indicates that only a small proportion of the L-ficolin antibodies are capable of recognizing H-ficolin and that they are unlikely to affect any future measurements. For a detailed description of the experimental setup see section

Figure 3.5. Determination of the coating antibody concentration for the L-ficolin assay. A microtiter plate coated with 10, 5, 2.5, 1 or 0 μg/ml of anti-L-ficolin antibodies was incubated with a 3-fold dilution series of serum and developed with 10 μg/ml biotinylated anti-L-ficolin antibodies to determine which concentration of coating antibody resulted in the broadest dynamic range. As can be observed all four different concentrations resulted in the same 27-fold dynamic range and therefore the 1 μg/ml concentration was selected. The negative controls non-immune IgG and no antibody (0 μg/ml) do not show any dose-dependent signal. For a detailed description of the experimental setup see section

Figure 3.6. The serum concentration of L-ficolin in healthy volunteers (HV) (n=112) compared to a schizophrenic population (SP) (n=103). Medians, interquartiles (boxes) and 95% confidence intervals around the medians (- - -), as well as means and 95% confidence intervals around the means ([) are indicated. Using the Mann- Whitney test the difference between the two groups was found to be significant (p=0.0024). This figure was made by Dr. K Mayilyan.

Figure 3.7. The L-ficolin serum concentration of 78 healthy Danish volunteers. The median value was 6.7 μg/ml, the interquartiles were 5.1 and 10 μg/ml and the 95% confidence intervals 3 and 24.6 μg/ml. For a detailed description of the experimental setup see section

Figure 3.8. Analysis of L-ficolin by analytical ultracentrifugation. The fit of the L- ficolin to a single species model when using three amounts of L-ficolin (27.5 (A), 13.8 (B) and 6.9 (C)  g of protein.) The lower diagram shows the fit of the acquired data to a single species model (Red line) while the upper diagram displays the distribution of data points according to this model. If the model is correct the data points in the top diagram should be distributed randomly. The data suggest that the majority of the protein preparation is a single species but also that other species are present due to the lack of randomly distributed data points. For a detailed description of the experimental setup see section ABC

AB Figure 3.9. Sucrose density gradient ultracentrifugation of serum. The position of L- ficolin in the 1 M salt and EDTA gradient (A) and in the low salt Ca 2+ gradient (B). The arrows labeled A, B and C indicate the position of the standards thyroglobulin (19.2 S), bovine catalase (11.3 S) and BSA (4.4 S), respectively. Based on the signals from the L-ficolin assay the L-ficolin peak center was estimated to 3.2 ml for the high ionic strength and EDTA gradient. At physiological conditions two peaks could be detected at 4.7 and 4.1 ml. Using the position of the standard proteins the sedimentation coefficient was estimated for the three peaks to 17.2 (3.2 ml), 20.6 (4.1 ml) and 18.7 (4.7 ml), respectively. This shows that L-ficolin devoid of MASPs sediments as a smaller protein than L-ficolin in complex with MASPs. Note Panel A and B are from different sets of gradients, and the position of the standard proteins are not identical. For a detailed description of the experimental setup see section

Figure Elution volume of L-ficolin after gel filtration chromatography. The peak fractions from the sucrose gradient centrifugation (3.2 ml, figure 3.9A and 4.1 ml, figure 3.9B) were processed on a Superose 6 column and the amount of L-ficolin in each of the fractions was estimated in the N-acetyl BSA (B) based L-ficolin assay. The open circles indicate the L-ficolin trace from the peak fraction from the 1 M NaCl, EDTA gradient while the closed circles show the same for the 140 mM NaCl, Ca 2+ gradient. Based on the chromatogram the elution position of L-ficolin was estimated to 10.8 and 10.3 ml for the 1 M NaCl and the 140 mM NaCl gradients, respectively. The arrows indicate the elution volume of the standard proteins that were used to calculate the Stokes radius for L-ficolin: A) thyroglobulin (85 Å, 669 kDa), B) apoferritin (61 Å, 460 kDa), C) BSA (40 Å, 66 kDa) and D) carbonic anhydrase (27 Å, 29 kDa). For a detailed description of the experimental setup see section

A B Figure L-ficolin binding to the different carbohydrate structures on the glycan array chip. In panel A the overall binding profile of L-ficolin is shown. The error bars indicate the standard error of measurement. In panel B the four glycan structures to which L-ficolin binds best are displayed together with glycan structures illustrating aspects of structural requirements for L-ficolin binding. The glycan number matches with the X-axis in figure 3.11A. The average and the standard error of measurement (STE) are based on 4 individual experiments. The Sp8 and Sp0 are linkers through which the glycans are attached to the chip. For a detailed description of the experimental setup see section Glycan No.Glycan NameAverageSTE 499-O-AcNeu5NAcα2-6Galβ1-4GlcNAcβ-Sp GlcNAcα1-6Galβ1-4GlcNAcβ-Sp GlcNAcα1-3Galβ1-4GlcNAcβ-Sp Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1- 3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ-Sp GlcNAcβ1-3(GlcNAcβ1-4)(GlcNAcβ1-6)GlcNAc-Sp β-GlcNAc–Sp Galβ1-4GlcNAcβ–Sp GlcNAcβ1-6Galβ1-4GlcNAcβ-Sp Neu5Acα2-6Galβ1-4GlcNAcβ–Sp

Figure Purified L-ficolin binding to LPS from E. coli, S. typhimurium, N. meningitidis and H. influenzae. Microtiter wells coated with the respective LPS were developed with a 3-fold dilution series of purified L-ficolin starting at 1 μg/ml. All four LPS display dose-dependent binding of L-ficolin but since wells without LPS and only the blocking agent BSA display equally high binding potential the signals are most likely not due to specific L-ficolin binding. For a detailed description of the experimental setup see section