Lectin pathway deficiency assay
The invention refers to an in vitro method of functionally determining at physiological conditions deficiencies in the lectin pathway of the complement system, the method comprising the steps of (a) providing a sample of mammalian blood, serum, plasma or another body fluid; (b) preventing in the sample the activation of the classical pathway by contacting the sample with an inhibitor of a molecule of the C1 complex of the complement system; (e) preventing in the sample the activation of the alternative pathway; (d) activating the lectin pathway in the sample; and (e) determining in the sample any activation of the autologous C5b-9 complex. The invention also refers to a kit for functionally determining in a body fluid from a mammal deficiencies in the lectin pathway of the complement system, which kit comprises the separate items (a) an inert carrier and a substance activating the lectin pathway; (b) a diluent comprising an inhibitor of a molecule of the C1 complex; and an antibody against the autologous C5b-9 complex.
1. An in vitro method of functionally determining deficiencies in the Lectin Complement Pathway in a sample of blood, serum, or plasma obtained from a mammal, the method comprising the steps of
(a) adding a C1 complex inhibitor to the sample to inhibit the Classical Complement Pathway, the C1 complex inhibitor comprising:
an inhibitory C1q-binding proteins elected from the group consisting of C1 inhibitor, CRT, C1Qr, E. coli C1g binding protein, gC1qR, ghB3, decorin, chondroitin sulphate proteoglycan, and surfactant protein A,
a peptide inhibitor of C1q, C1r or C1s selected from the group consisting of TDGDKAFVDFLSDEIKEE (SEQ ID NO. 1), KDIRCKDD (SEQ ID NO. 2), AEAKAKA (SEQ ID NO. 3), VQVHNAKTKPR (SEQ ID NO. 4), WY, CEGPFGPRHDLTFCW (SEQ ID NO. 5), LEQGENVFLQATLL (SEQ ID NO. 6), CRWDGSWGEVRC (SEQ ID NO. 7), CMWVRMWGDVNC (SEQ ID NO. 8), CFWAGKFGLGTC (SEQ ID NO. 9), CKDRWVVEERCC (SEQ ID NO. 10), and CWNRFKKMDRC (SEQ ID NO. 11), or
an immunoglobulin that binds C1q, C1r or C1s;
(b) diluting the sample with a buffered aqueous media having a pH and ion strength corresponding to the physiological pH and ion strength of the blood, serum, or plasma from the mammal in order to inhibit activation of the Alternative Complement Pathway in the sample;
(c) adding a mannan-binding lectin (MBL) carbohydrate or ficolin binding carbohydrate to activate the Lectin Complement Pathway in the sample;
(d) adding a first antibody to the sample that specifically binds C5b-9 complex to detect formation of C5b-9 complex in the sample, and
(e) determining activation of the Lectin Complement Pathway in the sample at the physiological conditions by measuring the amount of C5b-9 complex detected in the sample.
2. The method according to claim 1 , wherein said immunoglobulin that binds to C1q, C1r, or C1s in step (a) is an immunoglobulin selected from the group consisting of polyclonal and monoclonal antibodies.
3. The method according to claim 1 , wherein said MBL carbohydrate in step (c) is an MBL carbohydrate of low molecular weight selected from the group consisting of mannose and fucose.
4. The method according to claim 1 , wherein said first antibody in step (d) is a polyclonal or a monoclonal antibody.
5. The method according to claim 1 , wherein the step in (d) comprises adding a second antibody that binds said first antibody, wherein said second antibody is a labeled antibody.
6. The method according to claim 1 , wherein said first antibody is a labeled antibody.
7. The method according to claim 1 , wherein said MBL carbohydrate in step (c) is a high molecular weight mannan.
8. The method according to claim 7 , wherein said mannan is glucomannan or galactomannan.