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Removal of IGM antibodies: DTT versus immunoreactive beads
Abstracts
137
C-7.1 #157
B-CELL RETRIEVAL FROM DYNABEADS(R) FOR DYE EXCLUSION AND FLUORESCENT METHODOLOGIES. LD Allen, DM Zeh, DO Crowe, DCI Laboratory, Erlanger Medical Center, Chattanooga, TN. Immunomagnetic beads have been used since the mid-198c)'s and have been useful for isolating cells bearing Class II antigens (generally lymphocyte "B cells"). Isolated cells are then used for HLA typing, antibody screening and crossmatching methodologies. However, the presence of beads in the B cell preparation is an occasional source of technical problems which may be related to inaccurate estimation of cell concentration and/or w e a k reactions which are difficult to score. Our lab utilizes a q u i c k and cost-efficient procedure to remove adherent B cells from Dynabeads(R). B cells are isolated using Dynabeads, according to the manufacturer's instructions. The cell/beads are resuspended in 0.75mi media and placed in a 37°C waterbath for 510 minutes. The cells/beads are periodically agitated with a Pasteur pipet during incubation. The suspension is then layered over 0.3 ml Ficoll-hypaque in a 1 ml Fisher tube and centrifuged at 2000g for 3-5 minutes. The B cell interface is washed 2-3 times with media and adjusted to the desired concentration. V i a b i l i t y of the cells after removal from the beads generally exceeds 95%. Methodologies are not limited to fluorescence techniques. The specificity of the antigen/antibody reaction and the strength of the titer are retained. Finally, the accuracy of manual and automatic scoring of antigen/antibody reactions is enhanced and more reproducible with the removal of the beads.
C-7.1 #158
REMOVAL OF IGM ANTIBODIES: DTT VERSUS IMMUNOREACTIVE BEADS. P Orchard, A Corba, A Asfour, K Brooks, A Thomas, R Haneke, and S. Valdya, Tissue Antigen Laboratory, UTMB, Galveston, TX We have previously reported that when T or B cells isolated with immunomagentic tIM) beads are used in lymphocytotoxic assay's (XM's) containing Dithiothreltol (DTT) treated sera, the frequency of false positive reactions increases (eg DTT treated IgM control remains cytotoxic). In this experiment we absorbed IgM from sera using goat anti human IgM coupled to agarose beads (Ab) or magnetic beads (Mb) and compared these to reduction of IgM by DTT in XM's using IM bead isolated lymphocytes. One milliliter of commercially prepared Ab or M b was able to bind 2.5 mg or >0.15 mg human IgM respectively. Each of 7 known IgM only sera were divided into 3 aliquots. The first aliquot was mixed in ab:sera (packed volume:volume) combinations of .25:1, 5:1, 1:1 nd 2:1. The second aliquot with Mb:sera combinations of 1:1, 10:1, 20:1 and 40:1. Each combination was further subdivided for incubations at 15 minutes/4 °, 15 mln./22°C, 30 min./4°C and 30 mln./22°C separately. The remaining allquot was treated with DTT at 0.005M final concentration. Our results showed that in 42 T and B cell xm's, Ab:sera at I:I 430 mln./4°C) provided 100% removal of IgM. At no concentration were Mb able to completely remove IgM and were ellminated from further study. To determine if Ab would nonspecifically adsorb IgG, we treated each of 6 known HLA specific sera (IgG only) with Ab 41:1, 30 min/4°C) or with DTT. In 16 out of 16 T and B cell XM's, treatment with Ab or DTT did not lower end point titers of the HLA specific sera at all. Our data suggest that absorbing sera with magnetic bead is not an effective means of removing IgM. However, agarose beads provides 100% removal of IgM when compared to DTT and leaves IgG reactivity intact as assessed by the lymphocytotoxlclty assay.
137
C-7.1 #157
B-CELL RETRIEVAL FROM DYNABEADS(R) FOR DYE EXCLUSION AND FLUORESCENT METHODOLOGIES. LD Allen, DM Zeh, DO Crowe, DCI Laboratory, Erlanger Medical Center, Chattanooga, TN. Immunomagnetic beads have been used since the mid-198c)'s and have been useful for isolating cells bearing Class II antigens (generally lymphocyte "B cells"). Isolated cells are then used for HLA typing, antibody screening and crossmatching methodologies. However, the presence of beads in the B cell preparation is an occasional source of technical problems which may be related to inaccurate estimation of cell concentration and/or w e a k reactions which are difficult to score. Our lab utilizes a q u i c k and cost-efficient procedure to remove adherent B cells from Dynabeads(R). B cells are isolated using Dynabeads, according to the manufacturer's instructions. The cell/beads are resuspended in 0.75mi media and placed in a 37°C waterbath for 510 minutes. The cells/beads are periodically agitated with a Pasteur pipet during incubation. The suspension is then layered over 0.3 ml Ficoll-hypaque in a 1 ml Fisher tube and centrifuged at 2000g for 3-5 minutes. The B cell interface is washed 2-3 times with media and adjusted to the desired concentration. V i a b i l i t y of the cells after removal from the beads generally exceeds 95%. Methodologies are not limited to fluorescence techniques. The specificity of the antigen/antibody reaction and the strength of the titer are retained. Finally, the accuracy of manual and automatic scoring of antigen/antibody reactions is enhanced and more reproducible with the removal of the beads.
C-7.1 #158
REMOVAL OF IGM ANTIBODIES: DTT VERSUS IMMUNOREACTIVE BEADS. P Orchard, A Corba, A Asfour, K Brooks, A Thomas, R Haneke, and S. Valdya, Tissue Antigen Laboratory, UTMB, Galveston, TX We have previously reported that when T or B cells isolated with immunomagentic tIM) beads are used in lymphocytotoxic assay's (XM's) containing Dithiothreltol (DTT) treated sera, the frequency of false positive reactions increases (eg DTT treated IgM control remains cytotoxic). In this experiment we absorbed IgM from sera using goat anti human IgM coupled to agarose beads (Ab) or magnetic beads (Mb) and compared these to reduction of IgM by DTT in XM's using IM bead isolated lymphocytes. One milliliter of commercially prepared Ab or M b was able to bind 2.5 mg or >0.15 mg human IgM respectively. Each of 7 known IgM only sera were divided into 3 aliquots. The first aliquot was mixed in ab:sera (packed volume:volume) combinations of .25:1, 5:1, 1:1 nd 2:1. The second aliquot with Mb:sera combinations of 1:1, 10:1, 20:1 and 40:1. Each combination was further subdivided for incubations at 15 minutes/4 °, 15 mln./22°C, 30 min./4°C and 30 mln./22°C separately. The remaining allquot was treated with DTT at 0.005M final concentration. Our results showed that in 42 T and B cell xm's, Ab:sera at I:I 430 mln./4°C) provided 100% removal of IgM. At no concentration were Mb able to completely remove IgM and were ellminated from further study. To determine if Ab would nonspecifically adsorb IgG, we treated each of 6 known HLA specific sera (IgG only) with Ab 41:1, 30 min/4°C) or with DTT. In 16 out of 16 T and B cell XM's, treatment with Ab or DTT did not lower end point titers of the HLA specific sera at all. Our data suggest that absorbing sera with magnetic bead is not an effective means of removing IgM. However, agarose beads provides 100% removal of IgM when compared to DTT and leaves IgG reactivity intact as assessed by the lymphocytotoxlclty assay.