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Chapter 15 : Evaluation of the Mannan-Binding Lectin Pathway of Complement Activation

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Evaluation of the Mannan-Binding Lectin Pathway of Complement Activation, Page 1 of 2

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Abstract:

Events in the mannan-binding lectin (MBL) pathway of complement activation involve the binding of MBL to patterns of carbohydrate structures presented on the surface of microorganisms and the following activation of the proenzymes of the complement system. The protocols for MBL antigen assay are described in this chapter and are useful for routine quantification of MBL and evaluation of the MBL pathway activity in clinical samples. The assays are based on the robust, highly sensitive and reproducible time-resolved immunofluorometric assay (TRIFMA). TRIFMAs are similar to enzyme-linked immunosorbent assays (ELISAs), with the only difference being the type of labeling of detecting molecules. Different approaches have been used to measure the MBL concentration in plasma or serum. The first is based on a modification of the conventional sandwich ELISA, in which microtiter wells are coated with anti-MBL antibody and then incubated with dilutions of plasma or serum and the amount of bound MBL is measured by using europium-labeled anti-MBL antibody (MBL antigen assay). The second assay quantifies MBL on the basis of its lectin-binding activity (lectin assay), in which microtiter wells are coated with mannan instead of anti-MBL antibody. The MBL antigen assay is more sensitive (down to 2 ng of MBL/ml of plasma) than the lectin assay (10 ng/ml).

Citation: Gadjeva M, Thiel S. 2006. Evaluation of the Mannan-Binding Lectin Pathway of Complement Activation, p 128-133. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch15

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Figures

Image of FIGURE 1
FIGURE 1

Quantification of MBL. (A) Quantification by the antigen assay. MBL is captured in the microtiter well by anti-MBL MAb and then detected by a biotinylated anti-MBL MAb. (B) Quantification by the lectin assay. MBL is captured in the microtiter well by mannan (high-affinity ligand for MBL) and is detected by biotinylated anti-MBL MAb. In both the antigen and the lectin assays, the biotinylated anti-MBL MAb step is followed by incubation with Eu-labeled streptavidin. (C)Histogram showing levels of MBL in plasma. MBL levels correlate with MBL polymorphisms. For illustrative purposes, the genotypes are given in a simplified form, as follows: A/A, wild-type gene, excluding XA/XA; A/O, heterozygotes for the mutations in exon 1 and in the promoter region, including also XA/XA; and O/O, homozygote mutants and compound heterozygote mutants. Individual MBL levels are represented as circles. Approximately 10% of the population appears to have MBL levels below 100 ng/ml, which can be recognized as deficiency.

Citation: Gadjeva M, Thiel S. 2006. Evaluation of the Mannan-Binding Lectin Pathway of Complement Activation, p 128-133. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch15
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Image of FIGURE 2
FIGURE 2

MBL pathway activity assay. (A) Schematic representation of the MBL-MASP pathway activity assay. MBL-MASP complexes bind to the mannan-coated microtiter wells. MASP-2 cleaves C4, splitting it into C4a and C4b. The C4b covalently deposits on mannan, and the bound C4b can be detected by use of biotinylated anti-C4 antibodies followed by streptavidin-Eu. (B) Relationship between C4 deposition activity and the MBL concentration in 100 normal plasma specimens. The genotypes of each individual are indicated. Linear regression, = 0.96.

Citation: Gadjeva M, Thiel S. 2006. Evaluation of the Mannan-Binding Lectin Pathway of Complement Activation, p 128-133. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch15
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Image of FIGURE 3
FIGURE 3

Quantification of MASP-2. (A) Schematic representation of MASP-2 assay. MASP-2 is captured on the anti-MASP-2 MAb-coated microtiter wells and detected by biotinylated anti-MASP-2 MAb. (B) Concentrations of MASP-2 in 100 healthy individuals. The individuals were genotyped for the presence of the MASP-2 D120G mutation and grouped according to the allele. A total of 86 individuals carried the wild-type allele, and the concentration of MASP-2 varied between 200 and 1,200 ng/ml. The 16 heterozygous individuals had less MASP-2 (between 50 and 400 ng/ml). While both the wild-type and the heterozygous groups consisted of healthy blood donors, the single individual homozygous for the G120 polymorphism was a patient ( ) who had no detectable levels of MASP-2.

Citation: Gadjeva M, Thiel S. 2006. Evaluation of the Mannan-Binding Lectin Pathway of Complement Activation, p 128-133. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch15
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