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The principle of the MAIEA technique depends on the binding of two antibodies made in different species to different determinants on the same membrane component to form of a tri-molecular complex [4]. Briefly, a murine monoclonal antibody (MAb) and human antibody are incubated simultaneously with red cells. Excess antibody is removed, the sensitized cells are solubilised with Triton, so the tri-molecular complex is released into solution. The complex is detected by an ELISA type assay. The tri-molecular complex is captured by an anti-mouse globulin precoated onto a microtitre plate. The human antibody is then detected by a peroxidase-conjugated anti-human IgG. A positive reaction gives a high absorbance value and a negative reaction gives a low absorbance value. A negative result is obtained when the antibodies used bind to different membrane components, so no tri-molecular complex is formed. A negative result is also obtained when the monoclonal antibody and human antibody compete for the same epitope. Results can be represented as ratios of absorbances for antigen positive to antigen negative cells or as bar charts. In these studies a murine anti-CR1 (E11) and human anti-Kna and other Knops system antibodies were used against antigen positive and antigen negative cells. Absorbances for antigen positive cells with anti-Kna, anti-McCa anti-Sla and anti-Yka were high and results for the antigen-negative cells were low [8]. Comparison of chymotrypsin treated Kn(a+) cells with Kn(a-) cells showed that chymotrypsin did indeed destroy Kna antigen; chymotrypsin treated cells, therefore, were suitable cells to use as antigen negative cells when cells of rare phenotype were not available [8]. These reactions gave significantly positive ratios (Table I). In contrast, low absorbances were recorded for Cs(a+) and Cs(a-) cells with anti-Csa, the 1:1 ratio indicating a negative result (Table I). Serologically the Helgeson phenotype cells have a Knops null phenotype, all 4 antigens are negative but the antigens could be detected by flow cytometry and in immune precipitation [6,7]. Moulds and colleagues provided an explanation for this when they found that such cells did not completely lack CR1 but had a low copy number of CR1 molecules per cell [9]. Had it not been known already, the presence of Knops system antigens on Helgeson phenotype cells could have been deduced from the MAIEA results. The absorbance values for Helgeson phenotype cells were significantly higher than for antigen negative cells for Kna, McCa and Yka [8]. MAIEA has confirmed that Kna, McCa, Sla and Yka but not Csa are associated with the CR1 molecule in the red cell membrane and can detect weak expression of CR1 antigens on Helgeson phenotype cells [8]. MAIEA is useful for investigating problem antibodies suspected to be Knops system antibodies and can also be used to Knops phenotype cells with poor expression of Knops system antigens.
DOI link for The principle of the MAIEA technique depends on the binding of two antibodies made in different species to different determinants on the same membrane component to form of a tri-molecular complex [4]. Briefly, a murine monoclonal antibody (MAb) and human antibody are incubated simultaneously with red cells. Excess antibody is removed, the sensitized cells are solubilised with Triton, so the tri-molecular complex is released into solution. The complex is detected by an ELISA type assay. The tri-molecular complex is captured by an anti-mouse globulin precoated onto a microtitre plate. The human antibody is then detected by a peroxidase-conjugated anti-human IgG. A positive reaction gives a high absorbance value and a negative reaction gives a low absorbance value. A negative result is obtained when the antibodies used bind to different membrane components, so no tri-molecular complex is formed. A negative result is also obtained when the monoclonal antibody and human antibody compete for the same epitope. Results can be represented as ratios of absorbances for antigen positive to antigen negative cells or as bar charts. In these studies a murine anti-CR1 (E11) and human anti-Kna and other Knops system antibodies were used against antigen positive and antigen negative cells. Absorbances for antigen positive cells with anti-Kna, anti-McCa anti-Sla and anti-Yka were high and results for the antigen-negative cells were low [8]. Comparison of chymotrypsin treated Kn(a+) cells with Kn(a-) cells showed that chymotrypsin did indeed destroy Kna antigen; chymotrypsin treated cells, therefore, were suitable cells to use as antigen negative cells when cells of rare phenotype were not available [8]. These reactions gave significantly positive ratios (Table I). In contrast, low absorbances were recorded for Cs(a+) and Cs(a-) cells with anti-Csa, the 1:1 ratio indicating a negative result (Table I). Serologically the Helgeson phenotype cells have a Knops null phenotype, all 4 antigens are negative but the antigens could be detected by flow cytometry and in immune precipitation [6,7]. Moulds and colleagues provided an explanation for this when they found that such cells did not completely lack CR1 but had a low copy number of CR1 molecules per cell [9]. Had it not been known already, the presence of Knops system antigens on Helgeson phenotype cells could have been deduced from the MAIEA results. The absorbance values for Helgeson phenotype cells were significantly higher than for antigen negative cells for Kna, McCa and Yka [8]. MAIEA has confirmed that Kna, McCa, Sla and Yka but not Csa are associated with the CR1 molecule in the red cell membrane and can detect weak expression of CR1 antigens on Helgeson phenotype cells [8]. MAIEA is useful for investigating problem antibodies suspected to be Knops system antibodies and can also be used to Knops phenotype cells with poor expression of Knops system antigens.
The principle of the MAIEA technique depends on the binding of two antibodies made in different species to different determinants on the same membrane component to form of a tri-molecular complex [4]. Briefly, a murine monoclonal antibody (MAb) and human antibody are incubated simultaneously with red cells. Excess antibody is removed, the sensitized cells are solubilised with Triton, so the tri-molecular complex is released into solution. The complex is detected by an ELISA type assay. The tri-molecular complex is captured by an anti-mouse globulin precoated onto a microtitre plate. The human antibody is then detected by a peroxidase-conjugated anti-human IgG. A positive reaction gives a high absorbance value and a negative reaction gives a low absorbance value. A negative result is obtained when the antibodies used bind to different membrane components, so no tri-molecular complex is formed. A negative result is also obtained when the monoclonal antibody and human antibody compete for the same epitope. Results can be represented as ratios of absorbances for antigen positive to antigen negative cells or as bar charts. In these studies a murine anti-CR1 (E11) and human anti-Kna and other Knops system antibodies were used against antigen positive and antigen negative cells. Absorbances for antigen positive cells with anti-Kna, anti-McCa anti-Sla and anti-Yka were high and results for the antigen-negative cells were low [8]. Comparison of chymotrypsin treated Kn(a+) cells with Kn(a-) cells showed that chymotrypsin did indeed destroy Kna antigen; chymotrypsin treated cells, therefore, were suitable cells to use as antigen negative cells when cells of rare phenotype were not available [8]. These reactions gave significantly positive ratios (Table I). In contrast, low absorbances were recorded for Cs(a+) and Cs(a-) cells with anti-Csa, the 1:1 ratio indicating a negative result (Table I). Serologically the Helgeson phenotype cells have a Knops null phenotype, all 4 antigens are negative but the antigens could be detected by flow cytometry and in immune precipitation [6,7]. Moulds and colleagues provided an explanation for this when they found that such cells did not completely lack CR1 but had a low copy number of CR1 molecules per cell [9]. Had it not been known already, the presence of Knops system antigens on Helgeson phenotype cells could have been deduced from the MAIEA results. The absorbance values for Helgeson phenotype cells were significantly higher than for antigen negative cells for Kna, McCa and Yka [8]. MAIEA has confirmed that Kna, McCa, Sla and Yka but not Csa are associated with the CR1 molecule in the red cell membrane and can detect weak expression of CR1 antigens on Helgeson phenotype cells [8]. MAIEA is useful for investigating problem antibodies suspected to be Knops system antibodies and can also be used to Knops phenotype cells with poor expression of Knops system antigens.
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