Identification of Isoform p-53 Protein in CLL by Elisa 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. 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. For the men the results of protein concentration p-53 detection were following values, 20, 15, 18, 40, 10, 12, 14, 60, 30, 10, 13, 13, 5, 10, 15, 12, (µg / dl) and women's results of protein concentration p-53 were 140, 30, 13, 10, (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 being higher in men than in women, in ratio 2/1, in accordance with the data from the specialized literature. The overall frequency of positivity of the p53 protein, in the increased number of CLL cases studied, was 15% (3 out of 20 cases). The expression of the high-concentration p53 proteins in stage 2/3 of the disease was associated with a significantly weaker response to chemotherapy (p = 0.034). Statistical interpretations: the concentration of p-53 protein, in the 17 cases with p-53 protein expression, after excluding the 3 out-line cases present in the study, was calculated at the average value of 16.76µg / dL. In the CLL stage III/IV (n = 3 patients (Table 1), In the presented work with CLL, in the stage I/II, (n=17 patients) was registered in the average p-53 protein concentration in CLL, 16.76, (µg/dl), with mean values of hemogram: hematological parameters in peripheral blood, Leukocytes numberer = 35-50 x 10 3 / dL, Hb = 11.8g /dL; Platelet = 140 x 103/dL, Lymphocytes in peripheral blood = 65-80%. In some international studies, the immunological characteristics of patients with CLL having p-53 protein positive, were measured by immunohistochemistry, (IHC). In the second part of the study CLL on the stage I/II, (n=47 patients), the P-53 protein isoform concentration in reactive Lymphocyte B, the average p-53 protein concentration in CLL was on average 47 U/m with 16.7% samples studied, (7 out of 42 cases).

本研究使用的单克隆p53抗体PAb 240，在酶联免疫吸附试验（Enzyme-Linked Immunosorbent Assay, ELISA）体系中，可在变性条件下识别突变型与野生型p53蛋白。该抗体的物种反应性覆盖人类与恒河猴，符合实验预期。单克隆抗体PAb 240识别的表位，在p53野生型构象中处于结构隐藏状态，仅当p53蛋白发生变性或构象突变时才会暴露；p53基因的点突变会改变p53蛋白的末端结构，进而引发构象变化。
本ELISA试剂盒推荐用于血清、血浆或组织匀浆样本，不支持其他类型样本的检测。下述样本采集方案改编自相关参考文献。

本研究共纳入20例患者，其中男性14例，年龄区间为55~85岁；女性6例，年龄区间为39~85岁。所有患者在本次检测期间，均接受了针对慢性淋巴细胞白血病（Chronic Lymphocytic Leukemia, CLL）的细胞抑制剂治疗与特异性免疫治疗。
男性患者的p53蛋白浓度检测结果依次为：20、15、18、40、10、12、14、60、30、10、13、13、5、10、15、12 μg/dL；女性患者的p53蛋白浓度检测结果依次为：140、30、13、10 μg/dL。
3例p53水平显著升高的病理样本，其浓度对应突变型p53蛋白含量：2例男性样本分别为60 μg/dL与50 μg/dL，1例女性样本为140 μg/dL。慢性淋巴细胞白血病的男性发病率高于女性，比例约为2:1，与专业文献报道的数据一致。
本研究纳入的CLL病例中，p53蛋白阳性总检出率为15%（20例中3例）。p53蛋白高表达与疾病分期2/3期患者的化疗应答显著减弱相关（p=0.034）。

统计分析显示：本研究剔除3例异常值后，剩余17例存在p53蛋白表达的病例，其p53蛋白浓度平均值为16.76 μg/dL。其中，CLL分期III/IV期患者共3例（详见表1）；分期I/II期患者共17例，其p53蛋白平均浓度为16.76 μg/dL，相关外周血常规参数如下：白细胞计数为35~50×10³/dL，血红蛋白浓度为11.8 g/dL，血小板计数为140×10³/dL，外周血淋巴细胞占比为65%~80%。

既往部分国际研究中，针对p53蛋白阳性的CLL患者，已通过免疫组织化学（Immunohistochemistry, IHC）检测其免疫学特征。在本研究的第二部分，纳入分期I/II期的CLL患者共47例，对反应性B淋巴细胞中的p53蛋白亚型浓度进行检测；其中42例可评估样本的p53蛋白平均浓度为47 U/m，阳性检出率为16.7%（42例中7例）。