[Obtention of a heterohybridoma for production of type IgM monoclonal antibodies against the D antigen of the Rh system].
The objective was to obtain a heterohybridoma capable of producing a monoclonal antibody with IgM type anti-D specificity (Rh system), that could be used as a reactive for hemoclasification. Mononuclear cells (MNC) were extracted from a blood sample of a highly sensitized woman, five days after giving birth to an Rh positive child. These were then transformed with the culture supernatant (CSN) of B05.8 cells, rich in Epstein Barr virus (EBV). Once transformed and in exponential growth, they were fused with K6H6/B5 line cells using PEG 4.000 as a fusing agent in a 1:1 proportion. After fusion, they were seeded in culture plates in order to evaluate the formation of hybrids and the secretion of specific antibodies in the CSN of each well. The efficiency of the fusion was 1.8 x 10(-6), making it possible to obtain an anti-D IgM producing clone, which we named BMS-9. This clone could be maintained in constant culture for three months, producing antibodies in a concentration of 4 microg/mL in de CSN. It was also possible to obtain antibodies with an Artificial Capilar System (ACS) reaching a concentration of 24 microg/mL. Potency was determined using Ror cells. In CSN at immediate centrifugation (IC): 1 x 32, score 52; 15' from incubation at room temperature (RT): 1 x 1,024 score 105. With that ACS product at IC: 1 x 32 score 54; 15' from incubation at RT: 1 x 8.192 score 136; and a 37 degrees C: 1 x 8,192 score 136. Reactivity was detected with red cells D(IIIa), D(IV), D(Va), D(VI) type IV, D(VII), DFR, DNU, STEM+, DAR and DAU. There was no reactivity with red cells D(IIIc), DI(Va), D(V) type II, D(VI) types I, II y III, Ro(HAR), DOL and weak D type II. During field study, no false negative or false positive reactions were detected. A stable heterohybridoma was obtained, producer of IgM type anti-D, with enough qualities to be used in blood typing. Given the excellent qualities of the antibody, we are evaluating dilution media and the addition of type IgG antibodies in order to manufacture a reactive for use in hemoclassification.
León-González
Graciela
G
Banco Municipal de Sangre del Distrito Capital, Esquina de Pirineos San José, Caracas, Venezuela. gonzaleo@cantv.net
Cruz
Carlos
C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D001788:Blood Grouping and Crossmatching; D002144:Callithrix; D002459:Cell Fusion; D002461:Cell Line, Transformed; D002999:Clone Cells / Q000276:immunology; D005260:Female; D004854:Herpesvirus 4, Human; D006801:Humans; D006825:Hybridomas / Q000378:metabolism*; D007075:Immunoglobulin M / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D007518:Isoantibodies / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D007963:Leukocytes, Mononuclear / Q000166:cytology; D049590:Postpartum Period; D011247:Pregnancy; D012203:Rh Isoimmunization / Q000097:blood; D012204:Rh-Hr Blood-Group System / Q000276:immunology*; D018029:Rho(D) Immune Globulin
D000911:Antibodies, Monoclonal; D007075:Immunoglobulin M; D007518:Isoantibodies; C061961:RHO(D) antibody; D012204:Rh-Hr Blood-Group System; D018029:Rho(D) Immune Globulin; C026410:Rho(D) antigen