Wu C H, Hsieh M J, Huang J H, Luo S F
Department of Medical Research, Taichung Veterans General Hospital, Taiwan, Republic of China.
Ann Allergy Asthma Immunol. 1996 Feb;76(2):195-203. doi: 10.1016/S1081-1206(10)63422-9.
Previous studies have identified two allergic fractions (Cr-PI and Cr-PII) from crude American cockroach extract, from which the 72- and 78-kilodalton (kD) components of Cr-PI have been identified as major allergens.
The objective of this study was to identify important allergens in Cr-PII and produce monoclonal antibodies.
Sera of cockroach-sensitive atopic patients were examined for IgE binding to Cr-PII proteins and monoclonal antibodies were generated by fusion between spleen cells of BALB/c mice immunized with Cr-PII and FO cells.
Immunoblotting revealed that eight allergenic compounds in Cr-PII are able to bind specific IgE. Allergens of 18, 28, 32 and 45 kD bound 16.7% (2/12), 100% (12/12), 83.3% (11/12), and 100% (12/12) of the atopic sera tested, respectively. Cell fusion resulted in three stable subclones secreting monoclonal antibodies, and none of the monoclonal antibodies recognized any epitopes of German cockroach and Cr-PI of American cockroach. One monoclonal antibody reacted strongly with the 28- and 32-kD allergens of Cr-PII, and two monoclonal antibodies were able to bind the 18- and 12-kD components of Cr-PII with similar epitope specificities. All the epitopes defined by monoclonal antibodies are recognized by human IgE as demonstrated by a fluoroallergosorbent test (FAST) inhibition assay. Five commercial cockroach extracts were compared for Cr-PII levels by monoclonal antibody-base ELISA, and all extracts were found to contain detectable Cr-PII.
The 28-, 32-, and 45-kD components of Cr-PII may be considered as additional important allergens of American cockroach and monoclonal antibodies can be used to identify and define American cockroach allergens.
先前的研究已从美洲大蠊粗提物中鉴定出两种变应原成分（Cr-PI和Cr-PII），其中Cr-PI的72千道尔顿（kD）和78 kD成分已被确定为主要变应原。
本研究旨在鉴定Cr-PII中的重要变应原并制备单克隆抗体。
检测蟑螂敏感的特应性患者血清中IgE与Cr-PII蛋白的结合情况，并通过用Cr-PII免疫的BALB/c小鼠脾细胞与FO细胞融合来产生单克隆抗体。
免疫印迹显示Cr-PII中的8种变应原性化合物能够结合特异性IgE。18 kD、28 kD、32 kD和45 kD的变应原分别与16.7%（2/12）、100%（12/12）、83.3%（11/12）和100%（12/12）的受试特应性血清结合。细胞融合产生了3个分泌单克隆抗体的稳定亚克隆，且没有一种单克隆抗体识别德国小蠊和美洲大蠊Cr-PI的任何表位。一种单克隆抗体与Cr-PII的28 kD和32 kD变应原强烈反应，两种单克隆抗体能够以相似的表位特异性结合Cr-PII的18 kD和12 kD成分。荧光变应原吸附试验（FAST）抑制分析表明，单克隆抗体定义的所有表位均被人IgE识别。通过基于单克隆抗体的ELISA比较了5种市售蟑螂提取物中的Cr-PII水平，发现所有提取物均含有可检测到的Cr-PII。
Cr-PII的28 kD、32 kD和45 kD成分可能被视为美洲大蠊的其他重要变应原，单克隆抗体可用于鉴定和界定美洲大蠊变应原。
