B protein of factor XIII: differentiation between free B and complexed B.
Plasma factor XIII is a complex of A and B proteins noncovalently linked in a tetramer, A2B2, Enzyme-linked immunosorbent assays (ELISA) were developed to measure the separate factor XIII proteins and the complex. All of the A protein in plasma is in the zymogen complex. The B assay measures the total amount of B protein in plasma (both free B and complexed B). This was confirmed by nondenaturing gel electrophoresis and immunoblotting, which showed two bands for B in plasma with this antibody. Two assays were developed to measure A2B2 complex specifically. One assay used a monoclonal antibody to B to bind antigen and measured B protein in the zymogen complex only and hence the concentration of the complex. The specificity of this antibody was also shown by immunoblotting. In the second assay, the capture antibody was to B and the tag antibody was to A. These two assays gave identical results for the concentration of A2B2 (0.07 mumol/L, 21.6 micrograms/mL in normal plasma). Thus, for the first time, differentiation and quantitation of free B and complexed B in plasma was possible. The assays were used to measure factor XIII proteins in plasma from normal controls, homozygous-deficient factor XIII patients, and their heterozygous relatives. The normal concentration of A in plasma is 0.13 to 0.16 mumol/L (approximately 11 micrograms/mL), all of which is in A2B2. The total B concentration is 0.26 to 0.28 mumol/L (approximately 21 micrograms/mL), half of which is complexed. The free B concentration is 0.13 mumol/L (approximately 10 micrograms/mL). Homozygous-deficient patients have essentially no A protein, but their free B concentration is 0.11 mumol/L. Heterozygotes have decreased A2B2, but their free B is 0.11 mumol/L. These results indicate that the concentration of free B is remarkably constant and does not depend on the concentration of A2 or A2B2.
Yorifuji
H
H
Department of Pathology, Beth Israel Hospital, Boston, MA 02215.
Anderson
K
K
Lynch
G W
GW
Van de Water
L
L
McDonagh
J
J
D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D004306:Dose-Response Relationship, Immunologic; D004792:Enzyme Precursors / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D005176:Factor XIII / Q000032:analysis* / Q000276:immunology; D006801:Humans; D015394:Molecular Structure; D008970:Molecular Weight
D000911:Antibodies, Monoclonal; D004792:Enzyme Precursors; D005176:Factor XIII