Development and validation of a robust multiplex serological assay to quantify antibodies specific to pertussis antigens

Abstract

Despite wide spread vaccination, the public health burden of pertussis remains substantial. Current acellular pertussis vaccines comprise upto five Bordetella pertussis (Bp) antigens. Performing an ELISA to quantify antibody for each antigen is laborious and challenging to apply to pediatric samples where serum volume may be limited. We developed a microsphere based multiplex antibody capture assay (MMACA) to quantify antibodies to five pertussis antigens; pertussis toxin, pertactin, filamentous hemagglutinin and fimbrial antigens 2/3, and adenylate cyclase toxin in a single reaction (5-plex) with a calibrated reference standard, QC reagents and SAS^® based data analysis program. The goodness of fit (R²) of the standard curves for five analytes was ≥0.99, LLOQ 0.04–0.15 IU or AU/mL, accuracy 1.9%–23.8% (%E), dilutional linearity slopes 0.93–1.02 and regression coefficients r² = 0.91–0.99. MMACA had acceptable precision within a median CV of 16.0%-22.8%. Critical reagents, antigen conjugated microsphere and reporter antibody exhibited acceptable (<12.3%) lot-lot variation. MMACA can be completed in <3 h, requires low serum volume (5μL/multiplex assay) and has fast data turnaround time (<1 min). MMACA has been successfully developed and validated as a sensitive, specific, robust and rugged method suitable for simultaneous quantification of anti-Bp antibodies in serum, plasma and DBS.

Introduction

Pertussis is an acute respiratory disease caused by Bordetella pertussis. This disease is also referred as ‘whooping cough’ due to its characteristic cough. Pertussis was a major cause of childhood morbidity and mortality in children during the first half of the 20th century until the introduction of whole cell pertussis vaccines, followed by replacement with acellular pertussis vaccines in many countries beginning in 1981 [1]. Even though there has been an over 90% decrease in the incidence of severe disease, pertussis incidence in the U.S. has increased steadily since the 1980s. Furthermore, epidemic cycles are reported every 2–3 or 5 years [2]. Despite enormous progress made in understanding the epidemiology, control and prevention of pertussis, the disease continues to be poorly controlled among infants [3]. In 2014, World Health Organization (WHO) 2014, estimated global pertussis cases in children <5 years to be 24.1 million and 160,700 deaths annually despite 86% diphtheria-tetanus-pertussis (DTP) vaccine coverage [4]. In the United States, during 2013–2015, between 20,762 and 32,791 cases were reported which makes pertussis the most prominent emerging vaccine-preventable disease [5].

This resurgence of pertussis in the US warrants better understanding on the host-pathogen relationship. There have been several possible hypotheses suggested for the continued incidence or resurgence of pertussis, including antigenic changes in the organism, better or intensified diagnosis, and/or waning vaccine immunity from acellular pertussis vaccines [6,7]. Current acellular pertussis vaccines (aP) may comprise up to four Bp antigens; pertussis toxin (Pt), pertactin (Prn), filamentous hemagglutinin (Fha) and fimbrial antigen 2/3 (Fim2/3). Several additional vaccine candidate antigens might be considered to be combined with these antigens to increase the vaccine effectiveness. Even though most of these antigens induce a robust humoral immune response leading to the elaboration of IgG, IgM, and IgA, the combination and concentration of antibodies to confer immunity is poorly understood [8]. Lack of a confirmed immunological correlate of protection and standardized method of antibody response assessment have hampered the progress in understanding the aP vaccine response [9,10]. Although there is no definitive threshold of antibody response established for protection, it has been suggested that high levels of antibodies to pertussis toxin, pertactin, and fimbrial agglutinogens protect singly and synergize. In other words, having antibodies to any one of these antigens gives some protection and better protection is given by antibodies to 2 or all 3 of the antigens [11].

At present, antibody responses to pertussis vaccines are evaluated using sensitive and specific ELISAs for each antigen [[12], [13], [14], [15]]. ELISA has several technical caveats especially when required to quantify the immune response to multivalent vaccines or natural infection with the pathogen presenting multiple virulence factors. In these situations, performing an ELISA for each antigen is time consuming and poses limitations when assessing immune response in children where the volume of serum samples may be limited. In vitro serological assays with the capacity to detect and quantify several analytes in a single reaction have broad acceptance and application [[16], [17], [18], [19], [20], [21], [22], [23], [24]]. With the increase in the number of multivalent vaccines and concurrent vaccinations, it is crucial to have serological techniques capable of quantifying the antigen specific immune response in as few reactions as plausible [14,[22], [23], [24]]. A major factor that challenges the use of serological antibody quantification assays is the inter-laboratory reproducibility. The assay formats, reportable values and interpretation often vary significantly between laboratories, leading to difficulties in data comparison between studies [[25], [26], [27]].

The objective of the present study was to address this variability by creating a standardized technology platform for the quantification of IgG antibodies to Bp antigens in a single reaction. The anti-Bp antigen specific IgG microsphere based multiplex antibody capture assay (MMACA) was developed and validated to a level of standardization that facilitates its use in a variety of laboratories, making this technology and critical reagents available thereby providing a framework for qualitative and quantitative comparison of pertussis vaccine responses and diagnostic tests. This article reports the development, performance characteristics and validation of MMACA for human serum in a robust and rugged format. Assay development experiments were described in section 2.1 and the summary of assay performance characteristics presented in Table 3. Section 2.2 describes assay validation experiments with the data summarized in Table 4. The validated MMACA was assessed for the serological screening of plasma and dried blood spot (DBS) as described in section 2.3 Plasma vs serum, 2.4 Dried blood spot vs serum.