Case M C, Burt A D, Hughes J, Palmer J M, Collier J D, Bassendine M F, Yeaman S J, Hughes M A, Major G N
Department of Medicine, The Medical School, University of Newcastle, Newcastle upon Tyne, UK.
J Immunol Methods. 1999 Feb 1;223(1):93-106. doi: 10.1016/s0022-1759(98)00207-5.
Our studies of DNA damage and repair in autoimmune disease, lymphomagenesis, and carcinogenesis, require identification of an immunoassay approach that is capable of ultrasensitive detection in a routine human tissue biopsy of several physicochemically diverse antigens, some of which will be present at very low level. Immuno-polymerase chain reaction (immuno-PCR) is a recently described method for ultrasensitive antigen detection that combines the amplification power of PCR with a method similar to a standard antibody capture, enzyme-linked immunosorbent assay (ELISA). As a test of the universality of immuno-PCR, and as an assessment of the suitability of this method for our studies, we used a single immuno-PCR protocol to assay purified forms of the following physicochemically diverse antigens: oligomeric pyruvate dehydrogenase complex (PDC; Mr 8.5 x 10(6)), the promutagenic DNA base adduct O(6)-methylguanosine (Mr 298) and its monomeric repair enzyme, O(6)-methylguanine-DNA methyltransferase (MGMT; Mr 22,000), and a peptide from the N-terminus of MGMT (Mr 2310). We found that all antigens could be ultrasensitively assayed using the single immuno-PCR protocol. Assay limits observed using antigen-specific (primary) antibodies at 1 microg/ml, were in the approximate range of 10(2)-10(9) molecules, with O(6)-methylguanosine being detected most sensitively. Sensitivity of the antigen assay appeared to positively correlate with primary antibody titres determined by ELISA. Furthermore, we observed a substantial increase in detection sensitivity for all antigens by the use of primary antibodies at the higher level of 10 microg/ml. The latter approach permitted antigen assay within the approximate range of 10(0)-10(7) molecules. The combination of higher titre primary antibodies and their use at higher input level, produced an increase of immuno-PCR assay sensitivity of up to four orders of magnitude greater than those previously reported through the use of this assay to measure other antigens. This represents up to a nine order of magnitude increase in immunoassay sensitivity compared to ELISA. Our findings provide compelling evidence that immuno-PCR is indeed a universal ultrasensitive antigen detection method. Using the indicated assay enhancements. immuno-PCR performed as detailed here can offer greatly increased sensitivity for antigen measurement compared to other methods. Thus, our findings suggest that parallel quantitation of several different antigens in very small samples of human tissue will be readily attainable using immuno-PCR.
我们对自身免疫性疾病、淋巴瘤发生和癌症发生过程中DNA损伤与修复的研究，需要一种免疫测定方法，该方法能够在常规人体组织活检中对几种物理化学性质各异的抗原进行超灵敏检测，其中一些抗原的含量会非常低。免疫聚合酶链反应（immuno-PCR）是一种最近描述的超灵敏抗原检测方法，它将PCR的扩增能力与一种类似于标准抗体捕获酶联免疫吸附测定（ELISA）的方法相结合。作为对免疫PCR通用性的测试，以及对该方法是否适合我们研究的评估，我们使用单一的免疫PCR方案来检测以下物理化学性质各异的纯化抗原：寡聚丙酮酸脱氢酶复合物（PDC；分子量8.5×10⁶）、前诱变DNA碱基加合物O⁶-甲基鸟苷（分子量298）及其单体修复酶O⁶-甲基鸟嘌呤-DNA甲基转移酶（MGMT；分子量22,000），以及MGMT N端的一个肽段（分子量2310）。我们发现，使用单一的免疫PCR方案可以对所有抗原进行超灵敏检测。使用浓度为1μg/ml的抗原特异性（一抗）抗体时观察到的检测限，大致在10² - 10⁹个分子范围内，其中O⁶-甲基鸟苷的检测最为灵敏。抗原检测的灵敏度似乎与ELISA测定的一抗效价呈正相关。此外，我们观察到使用浓度为10μg/ml的更高水平一抗时，所有抗原的检测灵敏度都有显著提高。后一种方法允许在大约10⁰ - 10⁷个分子范围内进行抗原检测。更高效价的一抗与更高输入水平的使用相结合，使得免疫PCR检测灵敏度比之前通过该测定法测量其他抗原所报道的灵敏度提高了多达四个数量级。与ELISA相比，这代表免疫测定灵敏度提高了多达九个数量级。我们的研究结果提供了令人信服的证据，表明免疫PCR确实是一种通用的超灵敏抗原检测方法。使用所示的测定增强方法，按照此处详细描述进行的免疫PCR与其他方法相比，能够大大提高抗原测量的灵敏度。因此，我们的研究结果表明，使用免疫PCR可以很容易地在非常小的人体组织样本中对几种不同抗原进行平行定量。
