Tissue Specificity of Porcine Zona Pellucida Antigen(s) Tested by Radioimmunoassay

Palm, Viktor; Sacco, Anthony G.; Syner, Frank N.; Subramanian, Marappa G.

doi:10.1095/biolreprod21.3.709

Cited by 41 publications

(17 citation statements)

References 10 publications

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“…SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in slab gels using the buffer system of Laemmli (1970 Radioimmunoassay s. Radiolabelling of ZP3 and deglycosylated-ZP3 with Na125I by the chloramine-T procedure (Greenwood, Hunter & Glover, 1963), was carried out as described elsewhere (Palm, Sacco, Syner & Subramanian, 1979 Production of antisera. Five different rabbit antisera produced against various preparations of pig zonae pellucidae proteins were used.…”

Section: Methodsmentioning

confidence: 99%

Carbohydrate influences the immunogenic and antigenic characteristics of the ZP3 macromolecule (Mr 55 000) of the pig zona pellucida

Sacco¹,

Yurewicz²,

Subramanian³

1986

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Summary. The glycoprotein molecule ZP3 of the pig zona pellucida (Mr 55 000) was deglycosylated by trifluoromethane\p=n-\sulphonic acid. While gas chromatography established that deglycosylation resulted in >91% carbohydrate removal, deglycosylated ZP3 (DG-ZP3) still retained significant immunogenic potential as shown by its ability to elicit antibody production in the rabbit. Several lines of evidence, however, demonstrated that deglycosylation produced significant modifications in the ZP3 macromolecule which profoundly altered its antigenicity: (1) 125I-radiolabelling procedures using chloramine-T consistently resulted in 125I-labelled ZP3 with a higher specific activity than 125I-labelled DG-ZP3; (2) in competitive binding radioimmunoassays (RIAs) using an 125I-labelled-ZP3 vs anti-ZP3 system, unlabelled DG-ZP3 successfully displaced ZP3 in a dose-related manner; (3) in direct binding RIAs comparing the reaction of labelled ZP3 and DG-ZP3 versus 5 different zona antisera, higher titres were consistently achieved with 125I-labelled ZP3; (4) as compared to ZP3, 2-dimensional immunoelectrophoresis using DG-ZP3 as antigen yielded significantly modified precipitin arc patterns; (5) with electrophoretic blotting procedures, antisera to ZP3 or DG-ZP3 cross-reacted with the heterologous antigen; (6) treatment of pig zonae with anti-DG-ZP3 serum produced a dense precipitation layer on the zona surface. When evaluated collectively, these results provide grounds for an important role for protein and carbohydrate in establishing the immunological characteristics of the Mr 55 000 macromolecule of the pig zona pellucida.

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Section: Methodsmentioning

confidence: 99%

Carbohydrate influences the immunogenic and antigenic characteristics of the ZP3 macromolecule (Mr 55 000) of the pig zona pellucida

Sacco¹,

Yurewicz²,

Subramanian³

1986

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“…Specificity of the porcine zona antigen(s) has been confirmed by a radioimmunoassay system (Palm, Sacco, Syner & Subramanian, 1979 (Mori et al, 1978 (Sacco, 1977b (Baranska, Koldovsky & Koprowski, 1970) or with mouse vitelli , although much larger number of eggs were used than in the present study. However, the detection of such antibodies is not feasible because of the limited supply of human ova.…”

Section: Methodsmentioning

confidence: 60%

Heteroimmunization with isolated human ova

Takai¹,

Mori²,

Noda³

et al. 1981

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“…Consequently, antiserum produced against SIZP is zona specific (Gwatkin & Williams, 1978;Gwatkin, Anderson & Williams, 1980;Dunbar & Raynor, 1980). The zona specificity of the remaining two antisera used in this investigation, anti-IZP and anti-PPZA, has previously been demonstrated (Palm et al, 1979;Subramanian et al, 1981). Therefore, precipitin arc patterns resolved in this investigation by use of these three antisera are probably representative of only zona-specific antigen-antibody reactions.…”

Section: Line Immunoelectrophoresismentioning

confidence: 72%

“…Two additional preparations of ZP3 were studied and differed primarily from the above PPZA by the inclusion of SDS in the purification protocol and by the homogeneity of the product (Palm et al, 1979;Subramanian et al, 1981) and by formation of the precipitation layer on the outer surface of the zona after antibody treatment of zonae-encased oocytes (Sacco, 1981 Garrels (1979). Radioiodination of PPZA and electrophoresis conditions as applicable to the zona system have previously been detailed .…”

Section: Antigen Preparationmentioning

confidence: 99%