Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1987-12-22
1991-10-22
Saunders, David
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 79, 435188, 436548, 436811, 530389, 530391, C07K 1528, G01N 33534, G01N 33535, G01N 33577
Patent
active
050595245
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to testing for the HLA-B27 antigen.
The disease ankylosing spondylitis (AS) is difficult to diagnose with certainty but it has been found that sufferers from AS have a probability of about 95% as being carriers of the gene coding for the HLA-B27 antigen.
The present invention provides a method of testing for the HLA-B27 antigen which comprises mixing with blood cells or blood cell lysates a first antibody capable of binding and blocking the HLA-B7 antigen and a second antibody capable of binding to the HLA-B27 antigen and wherein said second antibody is labelled so as to be detectable and thereafter detecting for the labelled said second antibody.
BACKGROUND OF THE INVENTION
Historically, only two methods for serological detection of HLA specificities have been popularly used since the introduction of routine tissue typing almost two decades ago. The first of these, a leukoagglutination technique, was replaced during the late 1960s by more sensitive and reliable cytotoxicity assays which were developed on a micro-scale by Kissmeyer-Nielson and by Terasaki, P. I. and McClelland, J. D.; Microdroplet assay for human serum cytotoxins, Nature, 206:998-1000 (1964). Microcytotoxicity remains standard today as it enables the simultaneous assessment of the reaction of a patient's cells with a very large number of scarce HLA typing sera which can be used in minute quantities.
Since the description of the close association of HLA-B27 with AS, a significant proportion of the labors of tissue-typing laboratories has been directed at assessment of HLA-B27 status, irrespective of the patient's full HLA phenotype. Requests for B27-typing by rheumatologists and other physicians have grown steadily despite frequent warnings that B27-status should rarely, if ever, be used as a diagnostic criterion for AS, as 8% of the (Caucasian) population is HLA-B27.sup.+ and only a small minority of these ever develop AS. At Royal Melbourne Hospital, for example, 45% of the 2500 requests for tissue typing during 1983 were solely for B27 status. Thus far, B27-typing has always been performed along routine tissue-typing lines, testing a panel of anti-B27 sera on the patient's lymphoctyes, and this has proven costly in terms of materials (especially antisera) and particularly because of recently escalating labor costs.
The aim of a full panel of standardized monoclonal HLA-typing sera is still a distant one, however, monoclonal antibodies may have much to offer in certain specific situations such as B27-typing. Accordingly, a number of procedures which have utilized an anti-HLA-B27 monoclonal antibody, have been developed, which should enable rapid, cheap and dependable assessment of B27 status. It is contended that these techniques will lead to a marked reduction in the costs of HLA-B27 typing, both in terms of staff labour and the costs of disposable reagents, allowing the direction of these resources to other services.
SUMMARY OF THE INVENTION
A method of testing for the HLA-B27 antigen which comprises mixing with blood cells or blood cell lysates a first antibody capable of binding and blocking the HLA-B7 antigen and a second antibody capable of binding to the HLA-B27 antigen wherein said second antibody is labelled so as to be detectable and thereafter detecting for the said labelled second antibody. The second antibody is specific to the HLA-B27 antigen. The HLA-B7 antigen binds in significant quantities to the second antibody. Therefore, it is necessary to block the HLA-B7 antigen with said first antibody.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: A titration of .sup.125 I-labelled anti-HLA-B27 monoclonal antibody on cell lysates of HLA-B27.sup.+ and B27.sup.- peripheral blood lymphocytes. Points on each curve represent the mean of triplicate assays .+-. standard error (cpm=counts per minute).
FIG. 2: A titration of .sup.125 I-labelled anti-HLA-B27 monoclonal antibody on leukocyte/platelet suspensions derived from HLA-B27.sup.+ and B27.sup.- subjects. Points on each curve represe
REFERENCES:
G. D. Johnson et al., in D. M. Weir (Ed.), Handbook of Experimental Immunology-3rd Ed., Blackwell Scientific Publications, Oxford, UK, 1978, pp. 15.1-15.7.
Hood et al., Immunology, 2nd Edition, Benjamin/Cummings Publishing Co., Inc., Menlo Park, Calif., 1984, pp. 66-68.
Sternberger, Immunocytochemistry, Prentice Hall, Inc., Englewood Cliffs, N.J., 1974, pp. 50-53.
Larsen, Transfusion (Phila.), 19, 219-221, 1979.
Trapani et al., Human Immunology, 1, 205-216, 1983.
Trapani et al., Immunol. Cell. Biol., 66, 215-219, 1988.
McKenzie Ian F. C.
Trapani Joe
Saunders David
The University of Melbourne
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