Simionescu L, Radu I, Costache L, Dimitriu V, Zamfir-Grigorescu D, Aman E, Dumitriu L, Zamfirescu I, Ursu H I
Endocrinologie. 1984 Apr-Jun;22(2):125-34.
double antibody radioimmunoassay (RIA) system for human myoglobin (hMb) was developed using our own reagents. The antigen (hMb) was isolated from human muscle, purified and stored frozen until needed for immunization, radiolabeling or reference preparation. The anti-hMb serum raised in rabbits was used at 1:2.10(4) dilution (initial). The Chloramine-T method was used for the hMb labeling obtaining at 10-15 muCi/micrograms (370-550 KBq/micrograms) specific activity. Working standards were prepared having concentrations in the range of 2.0 to 500 ng/ml. The reagents were incubated at +4 degrees C for 48 plus 24 hrs. The specificity and accuracy of our hMb-RIA system were validated using for parallel assays an already validated immunochemical system, the hemagglutination inhibition (HI) technique and the parallelism test using serum dilutions from patients with acute myocardial infarction (AMI). The serum hMb concentration in normal subjects (no = 23) was 54.14 +/- 15.08 ng/ml (X +/- SD), being higher in short-term hypothyroidism (no = 13), 87.95 +/- 20.90 ng/ml (p less than 0.0005) or in treated hyperthyroidism (no = 5), 80.03 +/- 21.81 ng/ml. In AMI (no = 6) the serum hMb concentration varied in the range of 123 to 1510 ng/ml. The sensitivity of our hMb-RIA system is 2 ng/ml and the intraassay average error (coefficient of variability % in %B) is 2.26%. Trials to shorten the incubation time showed that adequate binding of labelled Mb may be obtained with 2 plus 4 hr intervals at room temperature. It is necessary to establish, in our conditions, the variation limits for serum hMb in normal subjects according to sex and age as a comparison basis for the study of its physiological and pathological variations.
使用我们自己的试剂开发了用于人肌红蛋白（hMb）的双抗体放射免疫分析（RIA）系统。抗原（hMb）从人肌肉中分离出来，纯化后冷冻保存，直到用于免疫、放射性标记或制备标准品。兔抗hMb血清以1:2.10(4)稀释度（初始）使用。采用氯胺-T法对hMb进行标记，比活度为10-15 μCi/μg（370-550 KBq/μg）。制备工作标准品，浓度范围为2.0至500 ng/ml。试剂在+4℃下孵育48加24小时。我们的hMb-RIA系统的特异性和准确性通过与已验证的免疫化学系统、血凝抑制（HI）技术进行平行测定以及使用急性心肌梗死（AMI）患者血清稀释液进行平行试验来验证。正常受试者（n = 23）的血清hMb浓度为54.14±15.08 ng/ml（X±SD），短期甲状腺功能减退患者（n = 13）中较高，为87.95±20.90 ng/ml（p < 0.0005），或在治疗的甲状腺功能亢进患者（n = 5）中为80.03±21.81 ng/ml。在AMI患者（n = 6）中，血清hMb浓度在123至1510 ng/ml范围内变化。我们的hMb-RIA系统的灵敏度为2 ng/ml，测定内平均误差（%B中的变异系数%）为2.26%。缩短孵育时间的试验表明，在室温下以2加4小时的间隔可以实现标记Mb的充分结合。在我们的条件下，有必要根据性别和年龄确定正常受试者血清hMb的变化范围，作为研究其生理和病理变化的比较基础。
