Abelev G I, Karamova E R
Biull Eksp Biol Med. 1988 May;105(5):625-8.
Urinary proteins, concentrated and separated on cellulose-acetate membranes by counterflow isotachophoresis (ITP), were transferred by direct contact onto a strip of nitrocellulose membrane (NCM). ITP in the system of electrolytes: tris-HCl, pH 6.7 (the leading one) and tris-beta-alanine, pH 8.6 (the terminal one) gives rise to a strong electroendo-osmotic flow (EEF) in NCM, directed to the cathode. The rate of the counterflow in the zone, occupied by the leading electrolyte, exceeds the migration rate of any protein possessing anode mobility and present in the zone. Under these circumstances EEF serves as a "conveyer belt" transferring immunological reagents (antibodies, immunoconjugates, peroxidase) through the protein bands, "printed" on NCM. The immunoblots were developed in a standard way with 4-ethyl-1-naphthol as a substrate for antibody-bound peroxidase. The counterflow immunoblotting makes it possible to reveal and characterize light chains of immunoglobulins when they are present in the urine in the range of 20 ng/ml.
通过逆流等速电泳（ITP）在醋酸纤维素膜上浓缩和分离的尿蛋白，通过直接接触转移到硝酸纤维素膜（NCM）条带上。在电解质体系中进行ITP：pH 6.7的三羟甲基氨基甲烷 - 盐酸（先导电解质）和pH 8.6的三羟甲基氨基甲烷 - β-丙氨酸（终末电解质），在NCM中产生强大的电渗流（EEF），流向阴极。在先导电解质占据的区域中逆流的速率超过了具有阳极迁移率且存在于该区域中的任何蛋白质的迁移速率。在这种情况下，EEF充当“传送带”，将免疫试剂（抗体、免疫结合物、过氧化物酶）通过“印”在NCM上的蛋白条带进行转移。免疫印迹以标准方式用4-乙基-1-萘酚作为与抗体结合的过氧化物酶的底物进行显色。当免疫球蛋白轻链在尿液中的含量在20 ng/ml范围内时，逆流免疫印迹法能够揭示并表征它们。
