PMID- 7876565
OWN - NLM
STAT- MEDLINE
DCOM- 19950404
LR  - 20190723
IS  - 0022-1759 (Print)
IS  - 0022-1759 (Linking)
VI  - 179
IP  - 2
DP  - 1995 Feb 27
TI  - Purification of immunoglobulin E (IgE) antibodies from sera with high IgE titers.
PG  - 153-64
AB  - A three stage method for the ultrapurification of polyclonal IgE from human serum 
      is reported using anion exchange chromatography followed by monoclonal antibody 
      based positive and negative affinity chromatography. Following dialysis of 25-100 
      ml of serum (2.3-14 micrograms IgE/ml, n = 4) against 0.05 M Tris pH 8, each 
      specimen was subjected to diethylaminoethyl (DEAE)-cellulose chromatography 
      (serum/matrix = 1/4). IgE was eluted with 0.05 M Tris, 0.05 M NaCl pH 8, yielding 
      an IgE recovery of 61-93%, with removal of approximately 90% of other serum 
      proteins and an IgE purity ([IgE]/[Igs]) of 0.1-1.1%. After adjusting to 0.1 M 
      NaCl and concentrating approximately 30-fold, the eluted IgE was further purified 
      by affinity chromatography using a panel of IUIS/WHO-documented mouse monoclonal 
      anti-human immunoglobulin antibodies (alpha hIg-MAbs). First, the IgE-enriched 
      DEAE-cellulose chromatography fraction was incubated in a batch mode with two 
      alpha hIgE-Fc MAbs (HP6029, HP6061) coupled to CNBr-Sepharose, CL-4B. IgE was 
      eluted with 0.05 M glycine pH 2.8 and immediately neutralized. The IgE recovery 
      was 32-52% and IgE purity was 72-97%. Silver-stained SDS-PAGE and noncompetitive 
      solid-phase two-site immunoenzymetric assays for total human IgA, IgE, IgG and 
      IgM indicated that IgA, IgG and IgM were the only contaminants. Next, the IgE was 
      concentrated 10-30-fold in the presence of 0.1% HSA. One IgE specimen was 
      ultrapurified in a batch mode by negative selection chromatography using three 
      pairs of alpha hIg-MAbs (alpha hIgA: HP6111 + HP6123; alpha hIgG: HP6017 + 
      HP6046; alpha hIgM: HP6081 + HP6083) coupled to CNBr-Sepharose, CL-4B. IgE purity 
      increased from 91% to > 99.9% with approximately 70% recovery of IgE for this 
      step. The ultrapurified IgE antibody was shown to be functionally reactive for 
      allergen and Fc epsilon RI receptors on human basophils. We conclude that alpha 
      hIg-MAbs are powerful tools to facilitate the affinity purification of 
      functionally active human IgE from serum; however, when the analyte is present in 
      low concentration, a carrier protein needs to be added to minimize non-specific 
      loss of the material during this process.
FAU - Kleine-Tebbe, J
AU  - Kleine-Tebbe J
AD  - Department of Medicine, Johns Hopkins University School of Medicine, Johns 
      Hopkins Asthma and Allergy Center, Baltimore, MD 21224-6801.
FAU - Hamilton, R G
AU  - Hamilton RG
FAU - Roebber, M
AU  - Roebber M
FAU - Lichtenstein, L M
AU  - Lichtenstein LM
FAU - MacDonald, S M
AU  - MacDonald SM
LA  - eng
GR  - R01AI01290/AI/NIAID NIH HHS/United States
GR  - SR29AI32651/AI/NIAID NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - Netherlands
TA  - J Immunol Methods
JT  - Journal of immunological methods
JID - 1305440
RN  - 0 (Antibodies, Anti-Idiotypic)
RN  - 0 (Antibodies, Monoclonal)
RN  - 37341-29-0 (Immunoglobulin E)
SB  - IM
MH  - Antibodies, Anti-Idiotypic
MH  - Antibodies, Monoclonal
MH  - Chromatography, Affinity
MH  - Chromatography, Ion Exchange
MH  - Humans
MH  - Immunoglobulin E/*isolation & purification
EDAT- 1995/02/27 00:00
MHDA- 1995/02/27 00:01
CRDT- 1995/02/27 00:00
PHST- 1995/02/27 00:00 [pubmed]
PHST- 1995/02/27 00:01 [medline]
PHST- 1995/02/27 00:00 [entrez]
AID - 0022175994002796 [pii]
AID - 10.1016/0022-1759(94)00279-6 [doi]
PST - ppublish
SO  - J Immunol Methods. 1995 Feb 27;179(2):153-64. doi: 10.1016/0022-1759(94)00279-6.