Naslov
A novel method for precise quantification of IgG in hyperimmune horse plasma

Autori
Halassy, Beata ; Kurtović, Tihana ; Lang Balija, Maja ; Tunjić, Monika ; Brgles, Marija

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
2017 Annual Meeting of the Croatian Immunological Society with EFIS on Tour / - , 2017, 50-50

Skup
2017 Annual Meeting of the Croatian Immunological Society with EFIS on Tour

Mjesto i datum
Zagreb, Hrvatska, 20.10.2017. - 21.10.2017

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

Ključne riječi
IgG quantification ; Hyperimmune horse plasma

Sažetak
The hyperimmune horse plasma (HHP), prepared by an active immunisation of horses with an antigen of interest, is a starting material for antitoxin production (tetanus antitoxin, rabies antitoxin, antivenom). Antitoxins are pure horse immunoglobulins or their fragments, with the ability to neutralise respective antigen (pathogen, toxin, venom). Precise determination of the IgG quantity in the starting plasma is a prerequisite for accurate estimation of process efficiency by in vitro methods. The currently used methods are SDS-PAGE with the densitometric analysis of electropherograms or the gel filtration chromatography with peak area analysis. However, these methods are not accurate so methods that measure antibody activity have to be used for precise production process yields monitoring. In the case of antivenoms, these methods are in vivo methods in mice. In order to completely avoid the usage of animals in yield calculation during antivenom downstream processing, we aimed to develop in vitro method for precise and accurate measurement of IgG content in HHP. The principle of our approach was to measure IgG population specific for venom in the HHP, that would serve as a predictor of the whole IgG population. An ELISA was developed in which affinity-purified IgGs from venom-specific HHP served as a standard. The IgG fraction of a plasma is a heterogenous population of immunoglobulins that differ in class, subclass, or specificity. In addition, each plasma sample has a different distribution of these different immunoglobulin molecules. Any change in class or subclass distribution in a sample can affect the ELISA measurement, in which detection is made by a polyclonal anti-horse IgG reagent. So we introduced an individual internal reference for each particular plasma pool measurement. It is prepared by a fast, cheap and 100% efficient protocol (based on the caprylic acid precipitation of non-Ig proteins) for isolation of whole IgG population from the particular plasma pool. The isolation procedure does not change either IgG specificity or class distribution, as we proved by in vivo potency/IgG ratio assesment that showed equal values for plasma and pure IgG sample. Such an individual internal IgG reference sample for each plasma was more than 90% pure as determined by gel filtration HPLC and it was possible to precisely measure its IgG concentration (by total protein determination). Such well-characterised IgG sample from each plasma pool served as an internal reference in ELISA, for correction of ELISA results of the respective plasma pool. The newly developed method was validated and proven to fullfill regulatory requirements of pharmaceutical industry for precision, accuracy, linearity and specificity.

Izvorni jezik
Engleski

Znanstvena područja
Kemija

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