Polyclonal Antibodies in Microplates to Predict the Maximum Adsorption Activities of Enzyme/Mutants from Cell Lysates

Yiran Feng, Xiaolan Yang, Deqiang Wang, Xiaolei Hu, Huimin Chong, Juan Liao, Chang guo Zhan, Fei Liao

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


With microplate-immobilized polyclonal antibodies against a starting enzyme or its active mutant bearing consistent accessible epitopes, the maximum activity of an adsorbed enzyme/mutant (Vs) was predicted for comparison to recognize weakly-positive mutants. Rabbit antisera against Escherichia coli alkaline phosphatase (ECAP) were fractionated with 33% ammonium sulfate to yield crude polyclonal antibodies for conventional immobilization in 96-well microplates. The response curve of the activities of ECAP/mutant adsorbed by the immobilized polyclonal antibodies to protein quantities from a cell lysate was fit to an approximation model to predict Vs. With 0.4 μg crude polyclonal antibody for immobilization, Vs was consistent for ECAP in cell lysates bearing fourfold differences in its apparent specific activities when its abundance was greater than 0.9%. The ratio of Vs of the mutant R168K to that of ECAP was 1.5 ± 0.1 (n = 2), consistent with that of their specific activities after affinity purification. Unfortunately, the prediction of Vs with polyclonal antibodies that saturated microplate wells was ineffective to Pseudomonas aeruginosa arylsulfatase bearing less than 2% specific activity of ECAP. Therefore, with microplate-immobilized polyclonal antibodies to adsorb enzyme/mutants from cell lysates, high-throughput prediction of Vs was practical to recognize weakly-positive mutants of starting enzymes bearing fairly-high activities.

Original languageEnglish
Pages (from-to)212-219
Number of pages8
JournalProtein Journal
Issue number3
StatePublished - Jun 1 2017

Bibliographical note

Publisher Copyright:
© 2017, Springer Science+Business Media New York.


  • Maximum adsorption activity
  • Microplate
  • Polyclonal antibodies
  • Positive mutant
  • Prediction

ASJC Scopus subject areas

  • Analytical Chemistry
  • Bioengineering
  • Biochemistry
  • Organic Chemistry


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