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Plate Coating for an indirect ELISA

Plate Coating for an indirect ELISA - ELISA Assay Forum

Plate Coating for an indirect ELISA - ELISA Forum. Discuss and post questions including optimization, troubleshooting, protocols and theory of ELISA assays using monoclonal, and polyclonal antibodies or peptides.


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Old 01-07-2013, 11:02 PM
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Default Plate Coating for an indirect ELISA



Is is possible to coat microplates with an non-purified vaccine? Has this ever been done by anyone? I know that people typically find antigens from the vaccines and coat the microplate with one antigen at a time. However, the composition of the vaccine I am working with is proprietary and I don not know the exact antigens it consists of.

Would it also be possible to separate the antigens from the adjuvant in the vaccine?
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Old 01-09-2013, 03:30 PM
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Default Re: Plate Coating for an indirect ELISA

Extraction modified protocols(1) antigens in vaccines formulated in aluminum hydroxide gel (AHG)
Vortex for 70 sec. at low speed for 2 cycles of every 30 sec., 1.0 ml vaccine in Al(OH)3. 0.3 ml of vaccine is immediately transferred to an suitable volume eppendorf microcentrifuge tube and 0.6 ml of extraction SDS citrate-phosphate buffer 1.15 M at pH 8.5 (0.60 M sodium citrate dihydrate and 0.55 M sodium phosphate dibasic and 30 mM sodium dodecyl sulfate [SDS]) is added. Mix tube by inversion 10 times and incubated for 2.5 hours at 60 C with a gently mixing every 20 minutes during the incubation. Centrifuge tube at 425 g for 2 minutes at room temperature. Measure concentration proteins in supernatant with BCA method and therefore dilute in coating buffer and to proceed at coated microplate step.
(1)Rinella JV, Workman RF, Hermodson MA, White JL, Hem SL. Elutability of proteins from aluminium-containing vaccine adjuvants by treatment with surfactants. J Colloid Interface Sci. 1998; 197:48–56.
Good luck and let me know
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Old 01-10-2013, 09:18 AM
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Default Re: Plate Coating for an indirect ELISA

Extraction modified protocols(1) antigens in vaccines formulated in oily adjuvants (OA)
Antigens for vaccine use can be put in solution with oily adjuvants such as squalene or oil components that a long storage they allow and same time assures that vaccine remains for many months in inoculation site. To extract the antigen from the solution vaccinate in oily adjuvants to verify quality and typology of it, benzyl alcohol is used for breaks the emulsion and the antigens is released and recovered in the resultant aqueous layer. For procedure in preheated (37 C) or pre-cooled (4 C) test-tubes, 9 ml of formulated vaccine in mixture wateril (30:70 or 50:50) with coinciding temperature is distributed and add 1 ml of benzyl alcohol, per 10% final concentration. Vortex for 20 mins at maximum speed using a bench-top vortex and then centrifuge at 16100 g for 10 mins or at 10900 rpm for 20 mins to form three layers. Protein is recovered in the aqueous (middle) layer and they concentration measured with BCA method and therefore dilute in coating buffer and to proceed at coated microplate step.
(1)Aron P. Miles, Allan Saul : Extraction and Characterization of Vaccine Antigens from Water-in-Oil Adjuvant Formulations. Methods in Molecular Biology – Vol. 308 – Therapeutic Proteins, 2005, pp. 293-300
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Old 01-14-2013, 09:09 PM
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Default Re: Plate Coating for an indirect ELISA

Thanks so much for your reply! As soon as I determine which formulations are used in the vaccines I am working with I will try one of these protocols out.

Do you know if there is any way to tell which of these mediums the vaccine is formulated with? In the MSDSs for two of the vaccines I am working with, Aluminum Hydroxide is listed as an ingredient. Is this sufficient information to assume that the antigens are formulated in the aluminum hydroxide gel?

I am also thinking about performing a test for lipids on the vaccines.

Thanks!
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Old 01-15-2013, 10:01 AM
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Default Re: Plate Coating for an indirect ELISA

Dear Moscow13
If in MSDSs vaccine foresees aluminum hydroxide as ingredient is certain that this salt as adjuvant is used, therefore the protocol to be employed is that relate to it.
Good luck and let me know
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