An N-terminal glycosylation site and the peptide motif for complement C1q binding are present in CH2 of both isotypes. IgG1 isotypes are typical, with hinges containing the C-terminal Cys-Pro motif, but deletion and replacement of nucleotides (in the ancestral gene) of ruminant gamma 2 has shortened the IgG2 hinge, removing the Cys-Pro motif and the consensus high affinity Fc gamma RI receptor motif at the start of CH2. The majority of the differences between the isotypes occur in the hinge region and an evolutionary pattern for ruminant IgG hinges can now be identified. Both ovine gamma 1 and gamma 2 CH1 domains encoded two consecutive cysteine residues (Cys-127, -128, Kabat numbering), an arrangement which is deduced to form a pair of disulphide bridges, one to the L chain and one as an intra-chain bridge to the uppermost Cys of the hinge, as in rabbit and goat IgG. Ovine gamma 2 cDNA has 93% identity of nucleotides with ovine gamma 1. Of these, gamma 1 clones were positively selected and gamma 2 clones negatively selected with a gamma 1 hinge-specific probe. PCR products of the appropriate length were cloned and gamma positive clones selected with a CH1 conserved-region probe. Primers complementary to regions of CH1 conserved between ruminants were used for upstream priming, with downstream priming on the poly-A segment. If still negative, IgG sensitized cells are added.Ovine mesenteric lymph node mRNA was used for PCR amplification of DNA coding for immunoglobulin gamma 1 and gamma 2 heavy chain constant regions. The method consists of washing the cells well 3 - 4 times, adding polyspecific AHG serum, centrifuging,and reading macroscopically for agglutination, and microscopically (if negative). Unlike the IAT, no serum is added to the cells, and no incubation is required. The DAT detects only in vivo sensitization by IgG or C3. Quality Control of the IAT Direct Antiglobulin Test (DAT, DCT, DAGT) If negative, the test is read microscopically also, and if still negative, IgG sensitized cells are added. After the last wash has been thoroughly decanted, 2 drops of AHG serum are added and the test is centrifuged and read macroscopically for agglutination only (no serum remains to show hemolysis).Also, residual saline after the last wash will dilute the AHG serum and can cause a false negative. The washes are also decanted well, because residual saline will decrease the efficiency of protein removal. The washing stage is crucial, because if any unbound protein (IgG) remains, it could neutralize the AHG serum and cause a false negative. After reading the saline 37☌ part of the test, the red cells are washed well 3 - 4 times to remove all unbound protein.Note: the test could be read microscopically here, but it usually is not. This is the saline 37☌ part of the AHG test.
Following incubation, the test is read macroscopically only for agglutination and hemolysis.If the serum contains an antibody specific for an antigen on the red cells, the antibody will sensitize the cells (but not agglutinate them, if the antibody is IgG). 2 or 4 drops of serum (preferably 4) are incubated at 37☌ for 15-60 minutes with 1 drop of 3 - 5% red cells.This test is meant to detect in vitro sensitization, but will also detect in vivo sensitization if the red cells used already are sensitized with IgG. Today most labs use monospecific anti-IgG for IAT testing. Preparation and Source of AHG Serum Indirect Antiglobulin Test (IAT, IDC, IAGT) When doing DATs to detect in vivo sensitization with IgG or C3, blood banks must use polyspecific AHG serum containing anti-IgG, anti-C3b, and anti-C3d. The AHG serum that is routinely used by most labs for pretransfusion IATs is monospecific anti-IgG. Because some IgG and IgM antibodies also cause C3 to attach to red cells, polyspecific AHG serum also contains anti-C3, which can cause C3-coated red cells to agglutinate. Since most incomplete antibodies are IgG, polyspecific AHG serum contains anti-IgG. Principle of the AHG Testīecause antibodies are gamma globulins, an antibody to gamma globulin can form bridges between red cells sensitized with antibody and cause them to agglutinate ( Figure 3-1). It is the single most important test we have for detection of red cell antibodies. The antiglobulin (AHG) test was invented by Coombs, Race and Mourant in 1945 and is sometimes called the Coombs test after its main inventor.
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