Enzyme-Linked Immunosorbent Assay For The Detection Of Alloantibodies To Factor IX In Hemophilia B

1981 ◽  
Author(s):  
K H Örstavik ◽  
I Örstavik
Keyword(s):  
Immunosorbent Assay ◽  
Solid Phase ◽  
Factor Ix ◽  
Hemophilia B ◽  
Negative Reaction ◽  
Enzyme Linked Immunosorbent Assay ◽  
Nitrophenyl Phosphate ◽  
Human Factor Ix ◽  
Coagulation Assay

A solid phase enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantitative determination of acquired inhibitors to factor IX. Wells of polystyren Micro-ELISA plates were coated with the IgG fraction of a sheep antiserum to human factor IX. After incubation with pooled normal plasma as a factor IX source, the wells were incubated with test plasma. The binding of alloantibodies to the factor IX-sheep-anti-factor IX complexes was then detected by incubation with alkaline phophatase conjugated antiserum to human IgG. As substrate was used p-nitrophenyl phosphate.Plasma samples from five patients with severe hemophilia B and acquired inhibitors to factor IX were examined. All samples gave a positive reaction in the ELISA. The titers as determined in the ELISA were in good agreement with the titers as determined in a coagulation assay (0.1-800 U/ml). Plasma from 13 patients with hemophilia B and no detectable inhibitor in a coagulation assay all gave a negative reaction in the ELISA. A negative reaction was also found in plasma from four patients with hemophilia A and acquired inhibitors to factor VIII, and in plasma from 15 healthy persons.It is concluded that the ELISA is a simple and sensitive technique for the determination of acquired inhibitors to factor IX in hemophilia B.

scholarly journals Use of a BamHI polymorphism in the factor IX gene for the determination of hemophilia B carrier status

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1508-1511 ◽  
Author(s):  
CW Hay ◽  
KA Robertson ◽  
SL Yong ◽  
AR Thompson ◽  
GH Growe ◽  
...  
Keyword(s):  
Human Factor ◽  
Factor Ix ◽  
Hemophilia B ◽  
Carrier Status ◽  
X Chromosomes ◽  
Human Factor Ix ◽  
History Of

Abstract A BamHI polymorphism has been identified in the human factor IX gene. This polymorphism, which occurs in approximately 6% of X chromosomes, has been used to determine the carrier status of a female in a family with a history of hemophilia B. This family was uninformative for the previously reported TaqI and Xmnl polymorphisms in the factor IX gene.

Reduced bleeding events with subcutaneous administration of recombinant human factor IX in immune-tolerant hemophilia B dogs

Blood ◽  
2003 ◽  
Vol 102 (13) ◽  
pp. 4393-4398 ◽  
Author(s):  
Karen E. Russell ◽  
Eva H. N. Olsen ◽  
Robin A. Raymer ◽  
Elizabeth P. Merricks ◽  
Dwight A. Bellinger ◽  
...  
Keyword(s):  
Human Factor ◽  
Subcutaneous Administration ◽  
Chronic Administration ◽  
Factor Ix ◽  
Hemophilia B ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bleeding Events ◽  
Human Factor Ix ◽  
Immune Tolerant ◽  
Recombinant Human Factor Ix

AbstractIntravenous administration of recombinant human factor IX (rhFIX) acutely corrects the coagulopathy in hemophilia B dogs. To date, 20 of 20 dogs developed inhibitory antibodies to the xenoprotein, making it impossible to determine if new human FIX products, formulations, or methods of chronic administration can reduce bleeding frequency. Our goal was to determine whether hemophilia B dogs rendered tolerant to rhFIX would have reduced bleeding episodes while on sustained prophylactic rhFIX administered subcutaneously. Reproducible methods were developed for inducing tolerance to rhFIX in this strain of hemophilia B dogs, resulting in a significant reduction in the development of inhibitors relative to historical controls (5 of 12 versus 20 or 20, P < .001). The 7 of 12 tolerized hemophilia B dogs exhibited shortened whole blood clotting times (WBCTs), sustained detectable FIX antigen, undetectable Bethesda inhibitors, transient or no detectable antihuman FIX antibody titers by enzyme-linked immunosorbent assay (ELISA), and normal clearance of infused rhFIX. Tolerized hemophilia B dogs had 69% reduction in bleeding frequency in year 1 compared with nontolerized hemophilia B dogs (P = .0007). If proven safe in human clinical trials, subcutaneous rhFIX may provide an alternate approach to prophylactic therapy in selected patients with hemophilia B. (Blood. 2003;102:4393-4398)

Use of a Heterologous Solid-Phase Antigen in an Indirect Competitive Antibody-Capture Enzyme-Linked Immunosorbent Assay for T-2 Mycotoxin

1997 ◽  
Vol 60 (3) ◽  
pp. 321-327 ◽  
Author(s):  
DANUTA KIEREK-JASZCZUK ◽  
RONALD R. MARQUARDT ◽  
DAVID ABRAMSON
Keyword(s):  
Immunosorbent Assay ◽  
Solid Phase ◽  
Egg Yolk ◽  
Enzyme Linked Immunosorbent Assay ◽  
Optical Signals ◽  
Low Concentrations ◽  
Nitrophenyl Phosphate ◽  
Reporter Enzyme ◽  
Short Period ◽  
Solid Phase Assay

Anti- T-2 toxin (T-2) antibodies raised in chicken hosts were isolated as total IgY (chicken immunoglobulins of IgY isotype) from serum and the egg yolk. They were used for quantitation of T-2 toxin in an indirect competitive enzyme-linked immunosorbent assay (ELISA) which employed molecularly distinct toxin-carrier conjugates as solid-phase assay antigens. The sensitivities of the assays utilizing any of the purified IgY or unfractionated serum were similar but varied with respect to the type of conjugate used and could be improved by lowering the concentration of assay antigens or antibodies. Low concentrations of both immunoreactants resulted in assays of superb sensitivity, although the standard curves generated with some assay antigens decreased markedly in slopes and impractically long incubation times were required to develop sufficiently high read-out signals. However, high optical signals were obtained in a very short period of time when an amplified substrate system instead of para-nitrophenyl phosphate (pNPP) was used for the detection of a reporter enzyme, alkaline phosphatase. The use of an amplified substrate and of a solid-phase antigen that was prepared with Iso T-2 toxin attached directly to a carrier protein yielded an ELISA that had an improved ability to detect T-2.

scholarly journals Enzyme-linked immunosorbent assay determination of specific rubella antibody levels in micrograms of immunoglobulin G per milliliter of serum in clinical samples

1978 ◽  
Vol 8 (4) ◽  
pp. 419-423
Author(s):  
P O Leinikki ◽  
I Shekarchi ◽  
P Dorsett ◽  
J L Sever
Keyword(s):  
Immunosorbent Assay ◽  
Immunoglobulin G ◽  
Solid Phase ◽  
Laboratory Diagnosis ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibody ◽  
Standard Curve ◽  
Antibody Levels

A "microgram assay" is described in which solid-phase enzyme-linked immunosorbent assay is used for the determination of specific rubella immunoglobulin G (IgG) antibody levels in micrograms per milliliter of serum. The quantitation was based on a standard curve obtained by using a reference serum, for which the specific IgG content was assayed by immunochemical purification. IgG was first purified and specific rubella antibodies were separated by an immunoadsorbent prepared by linking rubella virus antigens to Sepharose 4B. By using IgG-specific conjugate, the levels of specific rubella IgG antibodies could then be determined from clinical samples. Seronegative samples showed antibody levels less than 1 microgram/ml, whereas levels up to several hundred micrograms per milliliter were detected in some postinfection sera. The correlation between microgram antibody levels and hemagglutination inhibition titers was linear. The method offers a simple and sensitive antibody assay which could be used both for the laboratory diagnosis of acute rubella and for the evaluation of immunity.

Isolation and Characterization of Canine Factor IX

1996 ◽  
Vol 75 (03) ◽  
pp. 450-455 ◽  
Author(s):  
Yuichi Sugahara ◽  
James Catalfamo ◽  
Marjory Brooks ◽  
Eri Hitomi ◽  
S Paul Bajaj ◽  
...  
Keyword(s):  
Amino Acid ◽  
Human Factor ◽  
Factor Ix ◽  
Hemophilia B ◽  
Enzyme Linked Immunosorbent Assay ◽  
Amino Terminal ◽  
Isolation And Characterization ◽  
Human Factor Ix ◽  
Plasma Factor ◽  
Canine Plasma

SummaryCanine plasma factor IX was purified to homogeneity by a combination of barium citrate precipitation and three-step column chromatographies of DEAE sepharose, heparin agarose and a monoclonal antifactor IX antibody-linked agarose. Canine factor IX has an apparent molecular size of 61 kDa, which is slightly smaller than that of human factor IX, as determined by denatured polyacrylamide gel electrophoresis. Its amino acid composition, amino-terminal and carboxyterminal amino acid sequences agreed well with those predicted from the reported cDNA. Unlike purified human factor IX, canine factor IX preparation often showed a discrete smaller molecular species (∼50 kDa) which was generated by a specific proteolytic cleavage between Arg310 and Val311. When purified canine factor IX was utilized as a standard for enzyme linked immunosorbent assay, the concentration of canine factor IX in the pooled normal dog plasma was determined to be 5.3 Μg/ml with 11.2% carbohydrate content (or 4.7 Μg/ml for its polypeptide chain moiety). Concentration of plasma factor IX antigen was measured in six severely affected, unrelated hemophilia B dogs. Four had factor IX antigen of less than 1% of the normal, and two had undetectable levels. The latter two had gross molecular abnormalities in their factor IX genes. Three obligate carrier females had variable but proportionately reduced factor IX antigen and factor IX coagulant activity levels. These results provide a quantitative method for measuring canine factor IX antigen which is a prerequisite for studying hemostasis and development of gene transfer approaches in the canine model of hemophilia B.

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