Quality of horse F(ab')2 antitoxins and anti-rabies immunoglobulins: protein content and anticomplementary activity

Abstract

Background: Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab')₂ anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates.

Methods: Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p < 0.05.

Results: Horse F(ab')₂ antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates.

Conclusions: Anti-botulinum AB (bivalent), anti-diphtheric, antitetanic and anti-rabies horse F(ab')₂ immunoglobulins probably contain acceptable levels of aggregates, although other improvements on the preparations must be carried out. Protein profile analysis and in vitro anticomplementary activity of F(ab')₂ immunoglobulin preparations should be included as quality control steps, to ensure acceptable levels of aggregates, contaminants and whole IgG molecules on final products, reducing the chances of adverse reactions in patients.

Keywords: Anti-rabies; Antitoxins; Complement system; F(ab’)2 fragment; Heterologous immunoglobulin; Protein profile.

Fig. 1

Protein concentration of horse F(ab’)₂ antitoxins and anti-rabies immunoglobulins. The protein concentration of the samples was determined using the BCA method. Data represent the mean ± SD of two vials from the same batch for each serum type. *p < 0.05. Anti-Bot: Anti-botulinum AB; Anti-Diph: Anti-diphteric; Anti-Tet: Antitetanic; Anti-Rab: Anti-rabies

Fig. 2

Polyacrylamide gel electrophoresis and Western blots of horse F(ab’)₂ antitoxins and anti-rabies immunoglobulins. Serum samples were subjected to SDS-PAGE (a and c) and Western blot analysis (b and d) under non-reducing (a and b) and reducing (c and d) conditions. Molecular mass standards were included in all the runs and the relative molecular mass (Mr) are shown. Gels (a and c) were stained with silver and Western blot assays (b and d) were revealed with rabbit anti-horse IgG labelled with alkaline phosphatase. Anti-Bot: Anti-botulinum AB; Anti-Diph: Anti-diphteric; Anti-Tet: Antitetanic; Anti-Rab: Anti-rabies; H: heavy chain; L: light chain; pdH: pepsin-digested heavy chain

Fig. 3

Chromatographic profiles of horse F(ab’)₂ antitoxins and anti-rabies immunoglobulins. (a) Anti-botulinum AB, (b) anti-diphteric, (c) antitetanic and (d) anti-rabies sera were subjected to molecular exclusion chromatography on a Superose 12 HR 10/30 column at a 24 mL/h flow rate, and their protein content was monitored by recording the absorbance at 280 nm. The chromatograms were divided in four regions. The regions 1, 2 and 3 were considered for the calculation of the percentage of proteins in each region. The region 4 was considered to represent phenol used as preservative

Fig. 4

Detection of C5a/C5a desArg in NHS, after incubation with horse F(ab’)₂ antitoxins and anti-rabies immunoglobulins. Samples were incubated with NHS or saline (control) according to the volumes shown in Table 2. The concentration of C5a/C5adesArg was determined by ELISA. Data represent the mean ± SD of two independent experiments using two vials from the same batch for each serum type. Anti-Bot: anti-botulinum AB; Anti-Diph: Anti-diphteric; Anti-Tet: Antitetanic; Anti-Rab: Anti-rabies