Role of the Isoform p-53 Protein in Failed Cases of Chronic Lymphocytic Leukemia

Introduction

It was discovered in the last few years that the production of some percentage of mutant p-53 proteins, with the increased stability in type B lymphocytes, leads to the carcinogenesis process. This discovery led to the identification and quantification of the p-53 protein by different methods such as immuno-histochemistry (IHC), polymerase chain reaction (PCR), single-stranded peptide microarray, (SSPMa), next-generation sequencing (NGS), and the sandwich enzyme-linked immunosorbent assay (sandwich ELISA).

Method

Using the ELISA technique, the frequency of p-53 protein expression in 20 representative patients diagnosed with CLL-B was analyzed, in order to investigate the relationship of the p-53 protein at the different stages of the disease and the impact on patient survival.

ELISA Kit Component: Coated 96-well, Strip Plate 1, Standard (Lyophilized) 2 vials Assay, Diluent (5x) 1 vial x 15 ml, Biotinylated Detection Antibody 2 vials, HRP-Streptavidin Conjugate (800x) 1 vial x 200 µl, Wash Buffer (20x) 1 vial x 25 ml, TMB Substrate 1 vial x 12 ml, Stop Solution H₂SO₄ 1 vial x 8 ml, Plate Sealers 4.

Molecular biology technology used in the method

The monoclonal p-53 antibody, PAb 240, used in the ELISA method recognizes both mutant and wild-type p-53 under denaturing conditions. Species reactivity is for human or rhesus monkey in conformity with the prospect. The monoclonal antibody PAb 240 recognizes an epitope that is structurally hidden in the wild-type conformation of p-53 and becomes exposed by denaturing the p-53 protein or the mutant conformations of p-53, where point mutations in the P-53 gene alter the terminal structure of the p-53 protein.

This ELISA kit is recommended for use in serum, plasma or tissue homogenates. Using other types of sample is not supported. The sample collection protocols below were adapted from the references.

1.Full physical examination of patients diagnosed with CLL

All patients were admitted to hospital with symptoms and clinical features of CLL, such as cough, night sweating, and retrosternal pain. Clinical examination and ultrasounds revealed adenopathy and/or splenomegaly, with spleen enlargement of 3 cm above the normal diameter.

2.Immunophenotyping using monoclonal antibodies (Flow Cytometry)

CLL-B diagnosis was confirmed by immunophenotyping: monoclonal antibodies in CD5⁺, CD19⁺, CD20⁺, CD23⁺, CD28⁺ receptors, and with B lymphocytes expressing IgM or IgG heavy chains with kappa or lambda light chains.

3.Laboratory Exams:

For each of the cases, a 5 Diff Hematology Analyzer was used to perform a haemogram, and blood smear cytology exams on peripheral blood and medullary bone marrow were carried out by May-Grunwald Giemsa staining. The leukemia cells found in the peripheral blood smear had characteristic microscopic morphology, with the small nuclei having mature lymphocytes with full or partially aggregated chromatin and lacking nucleoli.

Results

Of the 20 patients studied, 14 men have aged 55-85 years and 6 women aged 39-85 years. Patients were treated at the time of these investigations with cytostatic and immunotherapy specific for CLL.

Male results: Protein concentration p-53 / µg / dl: 20, 15, 18, 40, 10, 12, 14, 60, 30, 10, 13, 13, 5, 10, 15, 12.

Women's results; Protein concentration p-53 / µg / dl: 140, 30, 13, 10, [Graphic 1].

Statistical interpretations: The probability index (NORMDIST) p, was calculated in the value of p = 0.034. The reference interval was established between the values 10-40µg / dl, (m = 24.5 µg / dl.

The pathological values in the 3 cases of highly elevated p-53, reflecting the concentration of p-53 protein mutant, were calculated in 2 men in the amount of 60 µg / dl, respectively in 50 µg / dl, and in the female case, it was calculated in the amount of 140 µg / dl, the frequency of chronic lymphocytic leukemia with mutant p-53 protein being higher in men than in women, in ratio 2/1, in accordance with the data from the specialized literature.

Conclusions

In the context of a heterogeneous malignant disease, such as CLL-B, a simple and inexpensive ELISA method, such as employed in this study, proves useful for identifying patients to be considered as candidates for personalized therapeutic strategies, based on the mutation of the TP- 53 gene and the presence of p-53 isoform protein.

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