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D Stafford

Publications and source records attributed to D Stafford.

8 recordsLinked to original sources
PubMed1987

Carrier testing in hemophilia B with an immunoassay that distinguishes a prevalent factor IX dimorphism.

Immunoassays with a monoclonal antibody (A-1) detect a prevalent dimorphism in plasma coagulation factor IX. The antibody was shown to react with a dimorphic segment of the normal factor IX sequence as follows. First, A-1 bound to isolated activation peptide (residues 146 through 180) prepared from activated factor IX from a normal plasma pool. Second, binding of recombinant factor IXs with A-1 or factor IX from normal individuals was strong only when they had Threonine (Thr) at position 148; factor IXs with the Alanine (Ala) allele at that position were far less reactive. Third, immunoblot reactivity of Escherichia coli fusion proteins containing known segments of the factor IX sequence restricted the epitope to residues 147 through 153. In 120 hemophilia B pedigrees, the Ala immunoassay allele frequency was 0.19 and did not differ from the Ala frequency in normal males. In 22 of 49 families, immunoassay testing was informative for classification of obligate or possible carriers. In one large family, 4 obligate carriers were heterozygous for the dimorphism and 3 of their 7 daughters were classified as carriers. In other families, when the affected member had less than 1 nmol/L factor IX antigen, heterozygosity for Thr/Ala alleles excluded the carrier state even when DNA studies were not informative. Strong linkage disequilibrium of Thr/Ala alleles with the common TaqI DNA polymorphism was found. Nineteen of 75 normal and hemophilic factor IX genes had the 1.3-kilobase (kb) fragment and coded for the Ala allele; the rest had the 1.8-kb fragment and coded for Thr. In selected families, the A-1 immunoassay is an inexpensive and rapid method to confirm and supplement restriction fragment length polymorphism analyses of DNA for carrier testing.

Alleles↗
PubMed1986

Antigenic determinant in human coagulation factor IX: immunological screening and DNA sequence analysis of recombinant phage map a monoclonal antibody to residues 111 through 132 of the zymogen.

As an approach to the study of structure-function relationships in the normal and defective forms of human coagulation factor IX, we have begun to develop a series of monoclonal antibodies against specific sites on the protein. Zymogen and activated forms of normal factor IX were used initially as antigen for the preparation of monoclonal antibodies. Recombinant phage were prepared by cloning small (50- to 500-nucleotide) random DNA fragments from the coding region of a factor IX cDNA clone into the expression vector lambda gt11. Immunological screening of these recombinants with mixtures of monoclonal antibodies identified several immunoreactive phage. Further analysis showed that the monoclonal antibody designated IX-30 was generating the positive signals at a frequency of approximately 1/2,500 recombinants. Subcloning and sequence analysis of the inserted DNA in the immunoreactive phage revealed overlapping in-frame insertions, from which it could be inferred that the site in factor IX recognized by IX-30 is confined to residues 111 through 132 in the light chain. Similar mapping with other monoclonal antibodies should provide additional probes for the protein structure of human factor IX.

Amino Acid Sequence↗
PubMed1977

Platelet Autoimmunity.

Autoimmunity to platelets was looked for by complement fixation, antiglobulin consumption, transformation and migration-inhibition techniques. Only the migration-inhibition method gave consistently positive results. The test was positive in nine out of 14 patients with idiopathic thrombocytopenic purpura and all five patients with glandular fever.

Autoantibodies↗