Revue Fran?aise de Transfusion et Immuno-hématologie最新文献

The MRC Blood group Unit offered to test the anti- glycophorin antibodies against cells with unsual MN phenotypes. The antibodies were first tested against cells of common MN phenotype to determine their apparent specificities and optimum dilution.

The antiglobulin test for the detection of red cell antibodies is included in almost all regimes for pretransfusion testing. The polyspecific reagents produced for this purpose usually contain anti-complement antibodies to enable the detection of certain complement-binding antibodies of clinical significance. In addition the diagnosis of Cold Auto-immune Haemolytic Anaemia (CHAD) is supported by a positive direct antiglobulin test (DAT) using a reagent containing anti-C3d. Anti-C3d is therefore a necessary component of a polyspecific reagent. Unfortunately, if the strength of anti-C3d from a polyclonal reagent is optimal, it tends to be associated with unwanted reactions with red cells that have been stored in serum or plasma [9]. Even when apparently pure antigen is used for immunization, animals may make large amounts of antibody against minor contaminants. The specificity and potency varies from one animal and even one breed to the next, making reagent production both time consuming and uncertain.

In recent years several monoclonal antibodies have been produced with a specificity for complement components or for certain fragments. These include an antibody to C3 which reacts with a C3d epitope which appears to be exposed on all complement-coated red cells encountered in blood transfusion serology, NBTS-BRIC 8 (Holt et al., 1985) [10] and an anti-C3c, WM1 [17]. Chaplin et al., 1980) [2, 3] has reported a comparative study of four monoclonal anti-C3d and a series of 32 antibodies to C3c and C3d have recently been described and detailed serological and immunochemical investigations reported [5]. Some antibodies react with fragments such as C3g (Lachmann et al., 1980) [11] which are not always present or exposed on complement-coated red cells.

In the present study the serological and immunochemical properties of antibodies 12 W 1, 12 W 2, 12 W 3, 12 W 4, 12 W 5 and 9 W 14 were investigated.

The aims of the work were three-fold :

• 1)

  to determine how many D epitopes were recognized by the 7 IgG anti-D monoclonals produced by our own group (4 of which were in the workshop collection) ;

• 2)

  to define the epitopes recognized by the other workshop anti-D antibodies ;

• 3)

  to determine the relationship between the D, c and E antigens.

Monoclonal antibodies have been shown to be very specific and useful reagents in serology, e.g. mouse monoclonal antibodies against blood group antigens A, B, M, N etc.

But using the mouse monoclonal technology there are only few reports concerning antibodies reacting with Rh-system antigens.

Because of the importance of the anti-Rh-antibodies the human monoclonal antibody technology has been improved and therefore most of the workshop antibodies are human monoclonal antibodies.

These antibodies were tested for their use in routine laboratory work.

The Rh antibodies were studied by manual serological techniques (see Daniels' report on Kell related antigens), using human red cells of common and rare Rh phenotypes in an attempt to identify their specificity and/or usefulness as reagents. The report is divided into 3 sections : section I anti-D antibodies, section II other Rh specifities, section III « anti-Rhantibodies.

Thirty-one antibodies submitted for testing were examined in order to determine with which glycophorin species they were reactive and what region of the target molecule(s) were involved in antigen recognition. Antibodies were assayed by direct and indirect aggmutination techniques, using cells of various MNSs phenotypes before and after exposure to various enzymes. In addition, antibodies were tested by immunoblotting and, in a small number of instances, by radioimmunoprecipitation, in order to determine or confirm the molecular species reactive with each antibody.

Our previous efforts with monoclonal antibodies against red blood cell antigens generally involved flow cytometric analysis and fluorescence activated cell sorting of erythrocytes using immunolabeling [1, 2, 3]. Therefore, our analyses performed for this workshop were by flow cytometry. Because flow cytometry is a technically exact method for measuring fluorescence from a large number of cells, specificity and sensitivity of labeling can be measured with high precision. However, the values determined with this technique may be much different from specificity and sensitivity determined using other measurements (e.g., hemagglutination or immunoblotting) or under different labeling conditions (e.g., different pH or ionic strength). Thus, we emphasize that evaluation of antibody characteristics depends on the measuring system.

Six monoclonal antibodies to complement components were evaluated, five detected components of C 3 and the other reacted with C 4.

The specificity and titre of antibodies to the various complement components is easily determined by the use of C 3 and C 4 coated red cells prepared by low ionic methods. These procedures have been reviewed by Voak and al. [5] and Engelfriet and al. [1] as a result of studies by a joint working party of the ISBT/ICSH on the standardization of anti-human globulin reagents.

The aim of this paper is to establish a simple procedure for the characterization of antibodies to C 3/C 4 complement components, as shown in Table I.

The Kell blood group presently includes 23 antigens [1], a null (K₀) phenotype, a number of phenotypes characterized by weak Kell antigen activity, and an independent antigen, designated Kx, which appears to play a role in Kell antigen expression.

Kell blood-group antigens are markers on the surface-exposed domain of a 93,000 daltons (93 kD) membrane glycoprotein [2]. Intrachain disulfide linkages [3], and probably the proper intra-membrane milieu, are important for the antigenic integrity of Kell 93 kD protein. Separated 93 kD Kell protein does not react by Western blot analysis with the Kell antibody used for its initial immuno-precipitation [1]. Incubation of intact red cells with solutions containing papain/DTT mixture [4] or 2-aminoethylisothiouronium bromide (AET) inactivates all antigens of the Kell complex except for Kx [5].

Five monoclonal antibodies have been examined for serological specificity within the Kell system and for their ability to recognize epitopes on a 93 kD red cell membrane protein by Western blot analysis.

The five Kell system-specific monoclonal antibodies (MABs) were subjected to a limited amount of testing because of the restricted number of variant Kell phenotype cells that were available to us. Nevertheless, the results obtained were as follows.