Antibody-based approach to high-volume genotyping for MIC-1 polymorphism.
Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the TGF-beta superfamily. There are at least two known alleles of MIC-1 that are due to a G-->C point substitution at position 6 of the mature protein, which alters a histidine to an aspartic acid (MIC-1 H and MIC-1 D). We have determined the phenotype of MIC-1 circulating in serum by exploiting the differences in the affinity of the two monoclonal antibodies to the H and D alleles of MIC-1. A PCR-RFLP-based method for genotyping MIC-1 is also described. We validate these two assays using DNA sequencing of 19 subjects as the standard. We then used the validated assay to determine the frequency of the two MIC-1 alleles in a population of 261 adult blood donors. Inter-assay and sequencing concordance was 100%. The frequency of the three common MIC-1 genotypes was homozygous (HH), 54%; heterozygous (HD), 39%; and homozygous (DD), 7%. This novel antibody-based assay confidently determines the genotype of MIC-1. It offers the advantages of an ELISA-ease of automation, high-volume throughput of samples, and ease of use in a routine, clinical laboratory.
St Vincent's Hospital, Sydney, Australia.
D A
DA
Brown
A R
AR
Bauskin
W D
WD
Fairlie
M D
MD
Smith
T
T
Liu
N
N
Xu
S N
SN
Breit
D000293:Adolescent; D000328:Adult; D000368:Aged; D000483:Alleles; D000818:Animals; D001483:Base Sequence; D016207:Cytokines / Q000097:blood* / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D005838:Genotype; D055436:Growth Differentiation Factor 15; D006801:Humans; D008297:Male; D051379:Mice; D008875:Middle Aged; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods
D016207:Cytokines; C526140:GDF15 protein, human; C526141:Gdf15 protein, mouse; D055436:Growth Differentiation Factor 15