Deelder A M, de Water R
J Histochem Cytochem. 1981 Nov;29(11):1273-80. doi: 10.1177/29.11.6798103.
For the application of beta-D-galactosidase-immunoglobulin conjugates in the enzyme-linked immunosorbent assay (ELISA), four techniques for the preparation of such conjugates were compared. Sheep immunoglobulin (Ig) (against soluble egg antigens of the trematode Schistosoma mansoni) was coupled to beta-D-galactosidase by means of 1) glutaraldehyde treatment, 2) the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), and 3,4) two different procedures using the coupling agent m-maleimidobenzoyl-N-hydroxysuccinimide ester(MBS). The prepared conjugates were then fractionated by gel filtration on Sepharose 6B and the resultant molecular weight fractions were tested in an ELISA for the detection of S. mansoni antigen. Optimal results were obtained with a conjugate that was synthesized according to one of the two techniques using MBS. With this conjugate, 10(-9) g antigen/ml could still be detected in an ELISA with a chromogenic substrate, which was at least ten times as sensitive as with the other conjugates. Application of a fluorogenic substrate resulted in a lower detection level of 10(-10) g antigen/ml.
为了将β-D-半乳糖苷酶-免疫球蛋白缀合物应用于酶联免疫吸附测定（ELISA），比较了四种制备此类缀合物的技术。通过以下方式将羊免疫球蛋白（Ig）（针对曼氏血吸虫的可溶性虫卵抗原）与β-D-半乳糖苷酶偶联：1）戊二醛处理；2）异双功能试剂N-琥珀酰亚胺基3-（2-吡啶基二硫代）丙酸酯（SPDP）；以及3、4）使用偶联剂间马来酰亚胺苯甲酰-N-羟基琥珀酰亚胺酯（MBS）的两种不同方法。然后通过在琼脂糖6B上进行凝胶过滤对制备的缀合物进行分级分离，并在ELISA中测试所得分子量级分以检测曼氏血吸虫抗原。使用根据两种使用MBS的技术之一合成的缀合物可获得最佳结果。使用这种缀合物，在使用显色底物的ELISA中仍可检测到10^(-9) g抗原/ml，其灵敏度至少是其他缀合物的十倍。使用荧光底物时，检测水平降低至10^(-10) g抗原/ml。
