Recombinant P53 Protein Research Articles

P53 immunoreactivity in human malignant melanoma and dysplastic naevi.

Expression of the tumour suppressor protein, p53, was determined in 77 cutaneous melanocytic lesions, and in five lymph node metastases from malignant melanoma, in an immunohistochemical study employing CM-1, an antiserum raised against recombinant human p53 protein. Because wild-type p53 protein is rapidly degraded in normal cells, p53 immunoreactivity suggests the presence of an abnormally stable p53 protein. This may occur through either post-translational mechanisms or gene mutation. A highly significant correlation was found between p53 immunoreactivity and malignancy in melanocytic lesions (P < 0.0001). Overall, p53 immunoreactivity was observed in 63% of tumour specimens examined, but not in benign melanocytic naevi, although occasional foci of weak nuclear p53 immunoreactivity were observed in a minority of dysplastic naevi and a solitary Spitz naevus. A significant correlation was also found between strong p53 immunoreactivity and malignant melanomas associated with a poor prognosis (P = 0.008). These data suggest an important role for p53 tumour suppressor protein in the biology of human cutaneous malignant melanoma.

Aberrant expression of p53 tumour-suppressor protein in non-melanoma skin cancer.

Expression of the cellular p53 tumour-suppressor protein was examined in 78 epidermal tumours, including basal and squamous cell carcinomas, keratoacanthomas, solar keratoses, Bowen's disease and viral warts. An immunohistochemical study was employed using the antibody CM-1, raised against recombinant human p53 protein. Positive staining for p53, not detectable in normal cells because wild-type p53 is rapidly degraded, reflects abnormal stabilization of p53 protein, and in many cases suggests p53 gene mutation. p53 immunoreactivity was not observed in normal skin or in viral warts. In contrast, positive staining for CM-1 was seen throughout the tumour in the majority of basal and squamous cell carcinomas and in Bowen's disease. Immunoreactivity to p53 was also observed in the majority of keratoacanthomas and solar keratoses, but was confirmed to areas of dysplastic basal epithelium. This study demonstrates that accumulation of p53 protein, suggestive in many cases of p53 gene mutation and hence loss of tumour-suppressor function, may occur as an important early step in the development of diverse epidermal cancers.

Immune Response to a Major Epitope of p24 during Infection with Human Immunodeficiency Virus Type 1 and Implications for Diagnosis and Prognosis

A sequential inhibition enzyme-linked immunoassay (SIEIA) using a peroxidase-conjugated monoclonal antibody reacting to the sequence AAEWDRVHP of p24HIV-1 (amino acids 209 to 217 of p55) was developed in order to detect and determine the titer of antibody to this epitope in various populations of human immunodeficiency virus type 1 (HIV-1)-positive patients. There was a good correlation between SIEIA and a commercially available competition assay that uses recombinant p24 protein and polyclonal antibody to HIV-1 antigen, demonstrating the importance of the described epitope. Analysis of sera from French patients showed a decline of antibody to the AAEWDRVHP sequence associated with the progression of AIDS. No decrease was observed with serum samples from African patients. An immune response to the epitope was detected by SIEIA early in the course of seroconversion. Although our SIEIA uses a single p24 epitope, these data are in accordance with previously published studies in which antibodies to the whole p24 were analyzed. Sera reacting to p24 only (indeterminate profiles by Western blot [immunoblot]) did not bind to AAEWDRVHP. This epitope, which is conserved between HIV-1 and HIV-2/simian immunodeficiency virus, appears to be a major antigenic domain of p24. The area containing the sequence AAEWDRVHP and the corresponding monoclonal antibody may serve as a convenient alternative to whole purified p24 and polyclonal antibody in diagnostic and prognostic assays.

Immune response to a major epitope of p24 during infection with human immunodeficiency virus type 1 and implications for diagnosis and prognosis

A sequential inhibition enzyme-linked immunoassay (SIEIA) using a peroxidase-conjugated monoclonal antibody reacting to the sequence AAEWDRVHP of p24HIV-1 (amino acids 209 to 217 of p55) was developed in order to detect and determine the titer of antibody to this epitope in various populations of human immunodeficiency virus type 1 (HIV-1)-positive patients. There was a good correlation between SIEIA and a commercially available competition assay that uses recombinant p24 protein and polyclonal antibody to HIV-1 antigen, demonstrating the importance of the described epitope. Analysis of sera from French patients showed a decline of antibody to the AAEWDRVHP sequence associated with the progression of AIDS. No decrease was observed with serum samples from African patients. An immune response to the epitope was detected by SIEIA early in the course of seroconversion. Although our SIEIA uses a single p24 epitope, these data are in accordance with previously published studies in which antibodies to the whole p24 were analyzed. Sera reacting to p24 only (indeterminate profiles by Western blot [immunoblot]) did not bind to AAEWDRVHP. This epitope, which is conserved between HIV-1 and HIV-2/simian immunodeficiency virus, appears to be a major antigenic domain of p24. The area containing the sequence AAEWDRVHP and the corresponding monoclonal antibody may serve as a convenient alternative to whole purified p24 and polyclonal antibody in diagnostic and prognostic assays.

The Cloning and Expression in Escherichia coli of Sequences Coding for p24, the Core Protein of Human Immunodeficiency Virus, and the Use of the Recombinant Protein in Characterizing a Panel of Monoclonal Antibodies against the Viral p24 Protein

The sequences encoding the p24 core protein of human immunodeficiency virus type 1 were identified in a cDNA library made from infected CEM cells. The nucleotide sequence of the DNA coding for p24 was shown to be very similar but not identical to the sequences of lymphadenopathy virus and human T-cell leukaemia virus type IIIb. These sequences were expressed in Escherichia coli at the amino terminus of beta-galactosidase and used to screen a panel of monoclonal antibodies raised against virus-expressed p24. Regions containing the epitopes of five of the monoclonal antibodies were located using a series of amino- and carboxy-terminal deletion mutants of the recombinant p24 protein.

Investigation of atypical western blot (immunoblot) reactivity involving core proteins of human immunodeficiency virus type

Serum specimens which originally exhibited a narrow (indeterminate) 24-kilodalton core protein (p24) or p24/p55 pattern of reactivity with human immunodeficiency virus (HIV) in the Western blot (immunoblot) test were studied to gather information on antibody specificity. A total of 12 specimens were initially reevaluated with an indirect immunofluorescence assay (IFA), three enzyme-linked immunosorbent assays (ELISAs), and Western blot analyses. Five of the specimens were IFA positive and contained anti-gp160/gp120 antibodies which were observed only when an HIV Western blot antigen rich in gp160 and gp120 was used. The remaining seven serum specimens were nonreactive by IFA and showed variable reactivity in HIV antibody ELISAs. The specimens did not cross-react with core antigens for human T-cell leukemia virus types 1 and 2 or contain detectable levels of HIV p24 antigen. The p24/p55 reactivity of six of the seven indeterminate specimens could be reduced or eliminated by preincubating the specimens with disrupted, HIV-infected H9 cells but not with uninfected H9 cells. The six specimens also exhibited discernible reactivity with recombinant HIV p24 antigen. When an additional 23 indeterminate specimens were assayed, all of the serum specimens were nonreactive by IFA while 65% (15 of 23) showed various degrees of reactivity with the recombinant p24 protein. There was no indication that any of the HIV core antibody reactivity was caused by HIV infection. Indeterminate results for five patients with specific p24 reactivity, who were retested after a period of weeks or months, remained indeterminate for HIV antibody with no significant change in ELISA or Western blot reactivity.