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XVII Procalcitonin (PCT)

Procalcitonin (PCT) is a 116 amino acid residuepeptide with molecular weight of about 13 kDa. Theamino acid sequence of PCT was firstly described byMoullec et al. in 1984 (1) (Fig. 37.). It belongs to agroup of related proteins including calcitonin gene-related peptides I and II, amylin, adrenomodulin andcalcitonin (CAPA peptide family). PCT, like otherpeptides of CAPA family, appears from the commonprecursor pre-procalcitonin consisting of 141 aminoacids by removal of 25 a.a.r. from N-terminus.

PCT is produced normally in C-cells of the thyroidglands. It undergoes successive cleavages to formthree molecules: N-terminal fragment (55 a.a.r.),

calcitonin (32 a.a.r.) and katacalcin (21 a.a.r.).

It has been shown that the level of PCT in serumincreases significantly during an infection of bacterialorigin (2). Today PCT is considered to be one of theearliest and most specific markers of sepsis. However,several studies revealed that elevated PCT level inhuman blood could be detected not only in case ofsepsis and infection, but also in cases of surgery,polytrauma, heat shock and cardiogenic shock (3).The importance of PCT measurements in combinationwith cTnT or cTnI during heart transplantation topredict an early graft failure has been proved recently(4).

Hybridomas producing anti-PCT MAbs weregenerated after immunization of Balb/c mice withthree PCT fragments - N-terminal fragment of PCT,calcitonin and katacalcin. For PCT immunodetectionantibodies specific to different fragments of the

molecule should be used. Calibration curves ofsandwich immunoassays for PCT detection in blood,utilizing MAbs with different epitope specificity, areshown on Fig. 38.

1. Anti-procalcitonin monoclonal antibodiesHost animal: mice BALB/cCell line used for fusion: Sp2/0Antigen: fragments of procalcitonin, conjugated with carrier proteinPurification method: protein A affinity chromatographyPresentation: MAb solution in PBS with 0.1 % sodium azideApplication: procalcitonin immunoassay

Fig. 37. Amino acid sequence and schematic diagram of human procalcitonin.

N - terminal region

1(3)– 5757(55) a.a.r.

60 - 9132 a.a.r.

96 - 11621 a.a.r.

calcitonin katacalcin

PROCALCITONIN – 116 a.a.r.

A P F R S A L E S S PA D PAT L S E D E A R L L L A A LV Q DY V Q M K A S E L E Q E Q E R E G S S L D S P R S KR-C G N L S T C M L G T Y T Q D F N K F H T F P Q TA I G V G A P G K K R D M S S D L E R D H R P H V S M P Q N AN

1000000

100000

10000

1000

Antigen concentration (in ml of plasma)

CP

S

3ug 1ug 0.3ug 0.1ug 30ng 10ng 3ng 1ng 0.3ng 0.1ng 30pg control

Fig. 38. Calibration curves of several PCT sandwich immunoassays.Capture MAbs: 1µg/wellAntigen: human recombinant PCTDetection Eu-labeled MAbs: 0.1 µg/well- 16B5 (central part) – 42 (N-terminal fragment)- 24B2 (central part) – 13B9 (central part)- 14C12 (C-terminal part) – 14A2 (central part)- 27A3 (N-terminal fragment) – 22A11 (C-terminal part)

49

The recommended pairs for sandwich immunoassay: The antibody pair 24B2 – 13B9 can be used for theimmunodetection of both PCT and free calcitonin.

MAbs may also be used for PCT detection inimmunoblotting. All MAbs recognize PCT in WesternBlotting after SDS-electrophoresis in reducingconditions (Fig 39).

Ordering information

Clone Cat. # Specificity (part of PCT) Subclass Application

6F10 4PC47 N-terminal fragment IgG1 EIA, Sandwich immunoassay (capture), WB27A3 4PC47 N-terminal fragment IgG2a EIA, Sandwich immunoassay (capture), WB38F11 4PC47 N-terminal fragment IgG1 EIA, Sandwich immunoassay (capture), WB44D9 4PC47 N-terminal fragment IgG2a EIA, Sandwich immunoassay (detection), WB42 4PC47 N-terminal fragment IgG2a EIA, Sandwich immunoassay (detection), WB13B9 4PC47 calcitonin (central part) IgG2a EIA, Sandwich immunoassay (detection), WB13F2 4PC47 calcitonin (central part) IgG1 EIA, Sandwich immunoassay (capture), WB13G11 4PC47 calcitonin (central part) IgG1 EIA, Sandwich immunoassay (capture), WB14A2 4PC47 calcitonin (central part) IgG1 EIA, Sandwich immunoassay (capture, detection), WB16B5 4PC47 calcitonin (central part) IgG2b EIA, Sandwich immunoassay (capture, detection), WB24B2 4PC47 calcitonin (central part) IgG1 EIA, Sandwich immunoassay (capture), WB14C12 4PC47 katacalcin (C-terminal part) IgG1 EIA, Sandwich immunoassay (capture), WB18B7 4PC47 katacalcin (C-terminal part) IgG1 EIA, Sandwich immunoassay (detection), WB22A11 4PC47 katacalcin (C-terminal part) IgG1 EIA, Sandwich immunoassay (detection), WB

Fig. 39. Immunodetection of human recombinant PCT in Westernblotting by monoclonal antibodies specific to different PCT fragmentsafter 15 % SDS-PAGE in reducing conditions.Antigen: 100 ng/track.

1 – MAb 6F102 – MAb 27A33 – MAb 38F114 – MAb 44D95 – MAb 426 – MAb 13B97 – MAb 13F28 – MAb 13G119 – MAb 14A210 – MAb 16B511 – MAb 24B212 – MAb 14C1213 – MAb 18B714 – MAb 22A11

References:1. Le Moullec J.M., et al. The complete sequence of human procalcitonin. // FEBS Lett., 167 (1), 93-97 (1984)2. Assicot M, et al. High serum procalcitonin concentrations in patients with sepsis and infection. // Lancet, 341, 515-18 (1993)3. Meisner M., Reinhart K. Is procalcitonin really a marker of sepsis? // Int. J. Intensive Care, 8 (1), 15-25. (2001)4. Potapov EV., et al. Elevated donor cardiac troponin T and procalcitonin indicate two independent mechanisms of early graft failure

after heart transplantation. // Int. J. Cardiol., 92 (2-3), 163-7 (2003)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

16B5 4214A2 4224B2 38F1113G11 4216B5 44D924B2 13B914C12 38F11