Yukawa N, Kohno T, Ishikawa E, Takahama K
Department of Legal Medicine, Medical College of Miyazaki, Japan.
Forensic Sci Int. 1987 Dec;35(4):253-65. doi: 10.1016/0379-0738(87)90097-1.
A highly specific and sensitive sandwich enzyme immunoassay for human hemoglobin A (Hb A) is described. A rabbit anti-human Hb A IgG-coated polystyrene ball was incubated with human Hb A and subsequently with affinity-purified anti-human Hb A Fab'-horseradish peroxidase conjugates, which had been prepared before and after absorption with Japanese monkey Hb-Sepharose 4B and dog Hb-Sepharose 4B. Bound peroxidase activity was measured by fluorimetry using 3-(4-hydroxyphenyl)propionic acid as a substrate. The detection limits of human Hb A using the conjugate before and after the absorption were 0.65 pg/tube (3 X 10(10)-fold dilution of whole blood) and 2.0 pg/tube (1 X 10(10)-fold dilution of whole blood), respectively. Human Hb A could be discriminated from Hb of animals such as Japanese monkey, dog, cat, pig, horse, sheep, chicken and cow by measuring bound peroxidase activity in the presence and absence of the conjugates prepared before and after the absorption. Human Hb A in bloodstains on cotton gauze could be discriminated from Hb of animals described above even after seven washings. Human Hb A in 220,000-fold diluted bloodstains on cotton gauze could also be discriminated from Hb of animals described above.
本文描述了一种用于人血红蛋白A（Hb A）的高特异性和高灵敏度夹心酶免疫测定法。将包被有兔抗人Hb A IgG的聚苯乙烯球与人Hb A一起孵育，随后与亲和纯化的抗人Hb A Fab'-辣根过氧化物酶缀合物孵育，该缀合物是在与日本猕猴Hb-琼脂糖4B和犬Hb-琼脂糖4B吸收前后制备的。使用3-(4-羟基苯基)丙酸作为底物，通过荧光法测量结合的过氧化物酶活性。吸收前后缀合物对人Hb A的检测限分别为0.65 pg/管（全血稀释3×10(10)倍）和2.0 pg/管（全血稀释1×10(10)倍）。通过测量吸收前后制备的缀合物存在和不存在时结合的过氧化物酶活性，可以将人Hb A与日本猕猴、犬、猫、猪、马、羊、鸡和牛等动物的血红蛋白区分开来。即使经过七次洗涤，棉纱布上血迹中的人Hb A也能与上述动物的血红蛋白区分开来。棉纱布上220,000倍稀释血迹中的人Hb A也能与上述动物的血红蛋白区分开来。
