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. 2007 Jun 30;323(2):147-59.
doi: 10.1016/j.jim.2007.04.004. Epub 2007 May 11.

Fluorochrome-linked immunoassay for functional analysis of the mannose binding lectin complement pathway to the level of C3 cleavage

Mary C Walsh  1 , Lisa A Shaffer, Benjamin J Guikema, Simon C Body, Stanton K Shernan, Amanda A Fox, Charles D Collard, Michael Fung, Ronald P Taylor, Gregory L Stahl
Affiliations

Fluorochrome-linked immunoassay for functional analysis of the mannose binding lectin complement pathway to the level of C3 cleavage

Mary C Walsh et al. J Immunol Methods. .

Abstract

The humoral response to invading pathogens is mediated by a repertoire of innate immune molecules and receptors able to recognize pathogen-associated molecular patterns. Mannose binding lectin (MBL) and ficolins are initiation molecules of the lectin complement pathway (LCP) that bridge innate and adaptive immunity. Activation of the MBL-dependent lectin pathway, to the level of C3 cleavage, requires functional MASP-2, C2, C4 and C3, all of which have been identified with genetic polymorphisms that can affect protein concentration and function. Current assays for MBL and MASP-2 lack the ability to assess activation of all components to the level of C3 cleavage in a single assay platform. We developed a novel, low volume, fluorochrome linked immunoassay (FLISA) that quantitatively assesses the functional status of MBL, MASP-2 and C3 convertase in a single well. The assay can be used with plasma or serum. Multiple freeze/thaw cycles of serum do not significantly alter the assay, making it ideal for high throughput of large sample databases with minimal volume use. The FLISA can be used potentially to identify specific human disease correlations between these components and clinical outcomes in already established databases.

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Figures

Figure 1
Figure 1. The Lectin Complement Pathway
This figure demonstrates the key components in LCP activation that can be analyzed using the MBL-dependent LCP FLISA. Oval outlines delineate the endpoints directly analyzed by the assay.
Figure 2
Figure 2. Quantitation of MBL in the standard (PLS)
A pooled human serum standard (PLS) was standardized by using MBL-deficient serum reconstituted with a human MBL standard (Staten Serum Institut). Correlation between detected units of integrated intensity (II) at 800nm and ligand binding was performed using a linear regression model to establish MBL-dependent binding in ng/ml (panel a). Specificity of functional MBL binding was demonstrated by blocking with mAb 3F8 (panel b). Using the PLS to establish functional MBL levels (panel c), triplicate values of each donor (DN 1 = donor 1, DN 2 = donor 2, DN 3 = donor 3, DN 4 = donor 4) serum sample diluted in binding buffer are expressed as mean +/− SE.
Figure 3
Figure 3. Quantitation of MBL-dependent C4b deposition
The amount of MBL-dependent C4b that was bound to the mannan coated plates following incubation of the PLS was revealed as a log-linear relationship between the integrated intensity of activated PLS deposited C4b and known amounts of C4b (panel a). Specificity for MBL-dependent C4b deposition was verified by inhibition with mAb 3F8 (panel b). Using the PLS to establish MBL-dependent C4b levels (panel c), triplicate values of each donor (DN 1 = donor 1, DN 2 = donor 2, DN 3 = donor 3, DN 4 = donor 4) serum sample diluted in binding buffer are expressed as mean +/− SE of the PLS for C4b (horizontal bars) and MBL (vertical bars).
Figure 4
Figure 4. Quantitation of MBL-dependent C3b deposition
The amount of MBL-dependent C3b that was bound to the mannan coated plates following incubation of the PLS was revealed as a log-linear relationship between the integrated intensity of activated PLS deposited C3b and known amounts of C3b (panel a). Specificity for MBL-dependent C3b deposition was verified by inhibition with mAb 3F8 (panel b). Using the PLS to establish MBL-dependent C3b levels (panel c), triplicate values of each donor (DN 1 = donor 1, DN 2 = donor 2, DN 3 = donor 3, DN 4 = donor 4) serum sample diluted in binding buffer are expressed as mean +/− SE of the PLS for C3b (horizontal bars) and MBL (vertical bars).
Figure 5
Figure 5. Functional MBL, and MBL-dependent LCP activation, correlated to MBL genotype
Serum samples from donors with the indicated MBL haplotype were evaluated for functional MBL (panel a), C4b deposition (panel b) and C3b deposition (panel c). Differences between medians of haplotype groups for MBL, MBL-dependent C4b, and MBL-dependent C3b are statistically significant; Kruskal-Wallis ANOVA on ranks (p<0.001). There is a direct correlation to functional MBL concentrations with C4b deposition (panel d) and C3b deposition (panel e). The MBL-dependent LCP phenotype can be summarized by evaluating MBL binding, MBL-dependent C4b and C3b deposition together (panel f).
Figure 6
Figure 6. MBL-dependent AP amplification
MBL-dependent C4b deposition evaluated in 3–25% serum is inhibited by 3F8 and not anti-factor D (panel a). Alternatively, MBL-dependent AP ‘tick over’ amplification occurs approaching physiologic serum concentration (13 and 25%), as anti-human MBL mAb 3F8 together with anti-human factor D mAb can inhibit C3b deposition (panel b), but neither of these mAb can completely inhibit independently. Analysis of MBL-dependent AP amplification at 25% serum shows that although MBL-dependent C4b deposition remains compromised in DN1 (MBL-low/deficient) compared to DN 4 (MBL-high) (panel c), deficiency in MBL-dependent LCP activation can be rescued at the level of C3 convertases (panel d) when DN 1 is compared with DN 4 at 25% serum concentration. Figures 3c and 4c represent the same donors at 3% serum and demonstrated MBL-dependent C4b and C3b deposition, respectively.
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