Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1997-05-13
2002-03-05
Scheiner, Laurie (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S007100, C422S067000
Reexamination Certificate
active
06352826
ABSTRACT:
TECHNICAL FIELD
The present invention relates to an improved method and kit for detecting antibodies in whole blood of individuals who test seronegative by conventional assay techniques. More particularly, the present invention relates to an assay for detecting possible retrovirus infection, such as infection by the HIV virus, which utilizes a mitogen in whole blood to stimulate antibody production by peripheral blood mononuclear cells. The present invention also relates to an improved assay kit which does not require the separation of peripheral blood mononuclear cells from whole blood prior to culture with pokeweed mitogen.
BACKGROUND OF THE INVENTION
As used herein, mitogen means any substance capable of activating B-cells and/or T-cells. The term “whole blood” means blood collected with heparin, EDTA, or any other substance that prevents coagulation and clotting. The term whole blood as used herein also includes blood collected from and animal or human with heparin, ethylenediaminetetraacetate, or any other substance that prevents coagulation and clotting. “Whole blood” can also mean blood wherein the red blood cells have been lysed while maintaining the viability of the remaining white blood cells.
Serological detection of antibodies against a variety of infectious disease agents is considered evidence of exposure to and/or active infection by the agent. Serological detection of antibodies could also be useful for early detection of cancer and for predicting the success of organ or tissue transplants. Enzyme-linked immunosorbent assay (ELISA) commercial kits are commonly used as screening tests for serological detection of antibodies. The western blot technique has been the method most widely used to confirm ELISA-reactive serum samples, although other methods such as immunofluorescence, may also be applicable. Polymerase chain reaction (PCR) technique may also be used to confirm results of a preliminary assay.
As part of standard ELUSA procedure, test serum is incubated with specific antigens that are immobilized on beads or wells. Non-specific antibody in the serum is removed by washing, but the antibodies with affinity for the antigens present in the system remain bound. When the appropriate developing reagents are added, spectrophotometrically detectable color is produced, the optical density of which is proportional to the amount of antibodies bound. The standard optical density is established by the manufacturer of the ELISA kit and affects both the sensitivity and specificity of the assay. Generally, a sample that is positive is retested twice and deemed “positive” only if at least one of the subsequent two tests is also reactive. Due to its subjective nature, the prediction value of a positive ELISA varies depending on the degree of ELISA reactivity and the probability of infection. Additionally, results may be affected by the presence of a variety of other conditions, including autoimmune disease.
Western blot technique is widely used to confirm ELISA-reactive serum samples. In the Western blot test, the desired antigens are electrophoretically separated into discrete bands that are then transferred onto nitrocellulose paper. Particular antigens will exhibit identifiable and characteristic banding patterns. The nitrocellulose test strips are then incubated with donor serum specimens. Antibodies present in the sample will bind with specific antigenic bands and thus facilitate separation and identification of the antibodies present. Prepared nitrocellulose test strips are commercially available for a variety of tests. The Western blot technique is considered more specific than the ELISA technique, yet it is usually less sensitive.
Current serological techniques, however, do not identify individuals who are infected but lack detectable levels of reactive antibodies. Examples of conditions in which detectable levels of reactive antibodies are lacking include autoimmune diseases, where antibody may be present only a portion of the time and suppressed the remainder of the time or where antibodies are bound to the antigen forming immune complexes and thus may be nondetectable in serum; some forms of cancer, where antibody production against the tumor may be suppressed by some specific process in the development of the cancer, organ and tissue transplants, where the recipient is not producing antibodies against the potential donor but would suffer rapid graft rejection because of recall stimulation of the immune system due to a cross-reaction of the donor's antigens with antigens the recipient was previously exposed to; cytomegalovirus, which causes a reduction in antibody production; and, a host of other infections in which antibody production is subsequently suppressed. A variety of viruses can interfere with immunological functions as well. The inhibition that is induced may be specifically related to immune reactions to the virus or may be non-specific and affect many components of the general immune system of the host.
For example, recently a new class of human retroviruses which infect a subset of lymphocytes has been shown to cause profound immunological suppression and to cause an individual who has been infected with the virus to develop susceptibility to many pathogenic organism. Human Immunodeficiency Virus (HIV) infects T-lymphocytes belonging to the helper cell subset. The infection and subsequent loss of T-helper cells is thought to lead to immunosuppression and the resulting acquired inmmunodeficiency syndrome (AIDS).
AIDS was first reported by the Center for Disease Control (CDC) in 1981. Individuals were defined as having AIDS if the following conditions were present: (1) a reliably diagnosed disease such as
P.Carinii
pneumonia, other opportunistic infection, or Kaposi's sarcoma in a person less than 60 years of age that suggested an underlying cellular immune defect, and (2) occurrence of the disease in the absence of a cellular immune deficiency that could be ascribed to another factor (Samter, M., ed. “Immunological Diseases”, 4th ed, p. 445 (1988)). Two related disorders were also noted which manifested a variety of signs and symptoms suggestive of AIDS but did not meet the criteria established by the CDC. These syndromes are described by the terms AIDS-related complex (ARC) and chronic lymphadenopathy. ARC is characterized by fatigue, fever, night sweats, diarrhea, unintentional weight loss, oral candidiasis, generalized lymphadenopathy, leukopenia, and anemia, accompanied by immunological abnormalities similar to AIDS. Chronic lymphoadenapathy syndrome describes a condition of chronic lymphadenopathy of at least 6 months duration and affecting two or more extrainguinial sites in the absence of an illness or drug use known to cause lymphadenopathy. “Immunological Diseases”, supra., at p.445-446.
AIDS and its related syndromes are attributed to a lymphocytotrophic retrovirus designated: human immunodeficiency virus (HIV). HIV can be readily recovered from individuals with early stages of AIDS but cannot always be recovered intact from individuals in the late stages of AIDS. It is postulated that this is because the subset of T cells thought to harbor the virus has been depleted.
Serological screening techniques are being utilized worldwide for the detection of human immunodeficiency virus type 1 (HIV-1). The presence of antibody against human immunodeficiency virus type 1 (HIV-1) is considered a strong indicator of HIV-1 infection. An ELISA assay is currently being utilized on serum samples in most hospitals and screening rooms to make this determination. A similar assay is being used to detect the presence of simian immunodeficiency virus.(SIV), a virus similar to HIV found in nonhuman primates. If the serum sample is positive, an aliquot of the sample is screened by a Western blot assay kit for confirmation. The presence of antibody against two to three of the major protein bands of the virus is considered a positive confirmation and identification that the serum sample donor is infected.
Experimental results indicate that the currentl
Eitan Pearl Latzer & Cohen-Zedek
Parkin Jeffrey S.
Scheiner Laurie
Yoreh Biotechnologies Ltd.
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