识别前列腺特异性抗原的单克隆抗体和多克隆抗体的特性：对夹心酶联免疫吸附测定设计的意义

Characterization of Monoclonal and Polyclonal Antibodies Recognizing Prostate Specific Antigen: Implication for Design of a Sandwich ELISA.

作者信息

Raoofi Mohseni Sahar, Golsaz-Shirazi Forough, Hosseini Mostafa, Khoshnoodi Jalal, Bahadori Tannaz, Judaki Mohammad Ali, Jeddi-Tehrani Mahmood, Shokri Fazel

机构信息

Department of Immunology, Faculty of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

Department of Epidemiology and Biostatistics, Faculty of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

出版信息

Avicenna J Med Biotechnol. 2019 Jan-Mar;11(1):72-79.

PMID:30800246

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6359702/

Abstract

BACKGROUND

Prostate cancer is the second most common cancer in men. Prostate-Specific Antigen (PSA) is a tumor-associated glycoprotein with enzymatic activity which is secreted by the prostate gland. Following entry to the blood, 70-90% of PSA forms complexes with protease inhibitors and its enzymatic activity is inhibited. The serum level of PSA is increased and the rate of free PSA (fPSA) to total PSA is decreased in prostate cancer patients. Therefore, measurement of PSA and fPSA in serum is very valuable for diagnosis and prognosis of prostate cancer.

METHODS

In the present study, five anti PSA monoclonal Antibodies (mAb) were characterized by Enzyme-Linked Immunosorbent Assay (ELISA) and immunoblotting. For designing a sandwich ELISA, epitope specificity of these antibodies was studied by a competition ELISA. Free PSA was purified by electroelution technique from seminal plasma and used to produce polyclonal anti-fPSA antibody in rabbit. Purified polyclonal antibody (pAb) and mAbs were conjugated with HRP enzyme and Biotin (Bio) to set up the sandwich ELISA.

RESULTS

Three of the mAbs were found to recognize PSA similarly. One of these mAbs (2G3) was paired with anti-fPSA pAb to detect fPSA in serum. Eventually, serum fPSA concentration of 356 subjects was measured and compared by our designed ELISA and a commercial ELISA kit. Our results demonstrated a significant correlation (r=0.68; p<0.001) between the two assays. Sensitivity and specificity of our designed ELISA was 72.4 and 82.8%, respectively.

CONCLUSION

These results imply suitability of our designed ELISA for detection of fPSA in patients with prostate cancer.

摘要

背景

前列腺癌是男性中第二常见的癌症。前列腺特异性抗原（PSA）是一种具有酶活性的肿瘤相关糖蛋白，由前列腺分泌。进入血液后，70 - 90%的PSA与蛋白酶抑制剂形成复合物，其酶活性受到抑制。前列腺癌患者血清中PSA水平升高，游离PSA（fPSA）与总PSA的比率降低。因此，检测血清中的PSA和fPSA对前列腺癌的诊断和预后非常有价值。

方法

在本研究中，通过酶联免疫吸附测定（ELISA）和免疫印迹对五种抗PSA单克隆抗体（mAb）进行了表征。为设计夹心ELISA，通过竞争ELISA研究了这些抗体的表位特异性。采用电洗脱技术从精浆中纯化游离PSA，并用于在兔体内制备多克隆抗fPSA抗体。将纯化的多克隆抗体（pAb）和单克隆抗体与辣根过氧化物酶（HRP）和生物素（Bio）偶联，以建立夹心ELISA。

结果

发现其中三种单克隆抗体对PSA的识别方式相似。其中一种单克隆抗体（2G3）与抗fPSA多克隆抗体配对用于检测血清中的fPSA。最终，通过我们设计的ELISA和商业ELISA试剂盒对356名受试者的血清fPSA浓度进行了测定和比较。我们的结果表明两种检测方法之间存在显著相关性（r = 0.68；p < 0.001）。我们设计的ELISA的灵敏度和特异性分别为72.4%和82.8%。