[Obtaining antibodies and development of an immunoassay for the detection of Escherichia coli proteins in preparations of human recombinant gamma interferon].
The present paper refers to the obtainment of polyspecific antisera directed against Escherichia coli host strain used to produce recombinant human gamma interferon (rec. hum. gamma IFN). The antisera were obtained by the cascade immunization method. The animals (n = 3) were initially immunized with an E. coli protein preparation (EcPp) of the host strain obtained from a blank run which assures the absence of the rec. hum. gamma IFN protein. Afterwards, consecutive immunizations were carried out with the less immunogenic proteins. To obtain those proteins, EcPp is passed through a column containing antibodies purified from previous inoculations coupled to a gel matrix. In this way, the proteins that have not been recognized by the immune system in that moment (e.g. do not have their corresponding antibodies coupled to the column) are separated an used to reimmunize the animals. The analyses of the antisera by Western blotting show a progressive recognition of the host strain proteins by the antisera with the progression of the cascade method. The recognition is evident through all the molecular weight range. Those antisera were used as quality control of the recombinant protein, by quantification of the host strain protein contaminants using a multiantigenic ELISA. The detection limit of that system was 3.125 ng/mL and the quantification limit 6.25 ng/mL.
Díaz
N
N
Departamento de Inmunoquímica, Centro de Ingeniería Genética y Biotecnología, La Habana, Cuba. niubel.diaz@.cigb.edu.cu
Santana
H
H
Cruz
M
M
Brito
R
R
Maso
Y
Y
Pérez
E
E
Furrazola
G
G
D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology* / Q000302:isolation & purification; D000942:Antigens, Bacterial / Q000276:immunology / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification; D015153:Blotting, Western; D004340:Drug Contamination; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000276:immunology*; D005260:Female; D006801:Humans; D007106:Immune Sera; D007114:Immunization / Q000379:methods; D007163:Immunosorbent Techniques; D007371:Interferon-gamma / Q000302:isolation & purification*; D011817:Rabbits; D011994:Recombinant Proteins
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D007106:Immune Sera; D011994:Recombinant Proteins; D007371:Interferon-gamma