Simultaneous Serodetection of 10 Highly Prevalent Mouse Infectious Pathogens in a Single Reaction by Multiplex Analysis

Antibodies to Plasmodium falciparum are classically measured using the enzyme-linked immunosorbent assay (ELISA). Although highly sensitive, this technique is labor-intensive when large numbers of samples must be screened against multiple antigens. The suspension array technology (SAT) might be an alterative to ELISA, as it allows measurement of antibodies against multiple antigens simultaneously with a small volume of sample. This study sought to adapt the new SAT multiplex system for measuring antibodies against nine malarial vaccine candidate antigens, including recombinant proteins from two variants of merozoite surface protein 1, two variants of apical merozoite antigen 1, erythrocyte binding antigen 175, merozoite surface protein 3, and peptides from the circumsporozoite protein, ring erythrocyte surface antigen, and liver-stage antigen 1. Various concentrations of the antigens were coupled to microspheres with different spectral addresses, and plasma samples from Cameroonian adults were screened by SAT in mono-and multiplex formats and by ELISA. Optimal amounts of protein required to perform the SAT assay were 10-to 100-fold less than that needed for ELISA. Excellent agreement was found between the single and multiplex formats (R > 0.96), even when two variants of the same antigen were used. The multiplex assay was rapid, reproducible, required less than 1 l of plasma, and had a good correlation with ELISA. Thus, SAT provides an important new tool for studying the immune response to malaria rapidly and efficiently in large populations, even when the amount of plasma available is limited, e.g., in studies of neonates or finger-prick blood.

mentioning

confidence: 62%

Section: Discussionmentioning

confidence: 99%

Multiplex Assay for Simultaneous Measurement of Antibodies to Multiple Plasmodium falciparum Antigens

Fouda

Leke

Long

et al. 2006

“…This multiplexing is the basis for the high-throughput capabilities of this diagnostic assay. Simultaneous serodetection of 10 mouse pathogens has recently been reported using this immunoassay format (16), and our laboratory currently uses this methodology to test for antibodies to 22 different mouse pathogens, including MNV-1. The MFI format also requires much less serum than an ELISA, since multiple agents can be screened using one aliquot of serum.…”

Section: Discussionmentioning

confidence: 99%

Development of a Microsphere-Based Serologic Multiplexed Fluorescent Immunoassay and a Reverse Transcriptase PCR Assay To Detect Murine Norovirus 1 Infection in Mice

Hsu

Wobus

Steffen

et al. 2005

131

124

Murine norovirus 1 (MNV-1) is a newly recognized pathogen of mice that causes lethal infection in mice deficient in components of the innate immune response but not in wild-type 129 mice. In this study, in vitro-propagated MNV-1 was used as antigen to develop a multiplexed fluorescent immunoassay (MFI) to detect antibodies to MNV-1 in infected mice. The MNV-1 MFI was 100% specific and 100% sensitive in detecting anti-MNV-1 antibody in sera from experimentally infected mice. Testing of a large number of mouse serum samples (n ‫؍‬ 12,639) submitted from contemporary laboratory mouse colonies in the United States and Canada revealed that 22.1% of these sera contained antibodies to MNV-1, indicating infection with MNV-1 is widespread in research mice. In addition, a reverse transcriptase PCR primer pair with a sensitivity of 25 virus copies was developed and used to demonstrate that MNV-1 RNA could be detected in the spleen, mesenteric lymph node, and jejunum from some experimentally infected mice 5 weeks postinoculation. These diagnostic assays provide the necessary tools to define the MNV-1 infection status of research mice and to aid in the establishment of laboratory mouse colonies free of MNV-1 infection.

“…Microbead sets (Luminex Corp, Austin, TX) were coupled through carbodiimide linkages with each of the M. tuberculosis recombinant antigens as previously described (14)(15)(16).…”

Section: Methodsmentioning

confidence: 99%

Plasma Antibody Profiles as Diagnostic Biomarkers for Tuberculosis

Khan

Ravindran

Krishnan

et al. 2011

Self Cite

Two billion people are infected with Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), worldwide. Ten million to 20 million of the infected individuals develop disease per year. TB is a treatable disease, provided that it is diagnosed in a timely manner. The current TB diagnostic methods are subjective, inefficient, or not cost-effective. Antibody-based blood tests can be used efficiently and cost-effectively for TB diagnosis. A major challenge is that different TB patients generate antibodies against different antigens. Therefore, a multiplex immunoassay approach is needed. We have developed a multiplex panel of 28 M. tuberculosis antigen-coated microbeads. Plasma samples were obtained from over 300 pulmonary TB patients and healthy controls in a country where TB is endemic, Pakistan. Multiplex data were analyzed using computational tools by multivariate statistics, classification algorithms, and cluster analysis. The results of antibody profile-based detection, using 16 selected antigens, closely correlated with those of the sputum-based diagnostic methods (smear microscopy and culture) practiced in countries where TB is endemic. Multiplex microbead immunoassay had a sensitivity and specificity of approximately 90% and 80%, respectively. These antibody profiles could potentially be useful for the diagnosis of nonpulmonary TB, which accounts for approximately 20% of cases of disease. Since an automated, high-throughput version of this multiplex microbead immunoassay could analyze thousands of samples per day, it may be useful for the diagnosis of TB in millions of patients worldwide.More than one-third of the world's population is infected with Mycobacterium tuberculosis (7, 26a). Annually, 10 million to 20 million of these individuals develop clinical symptoms, and about 2 million die of tuberculosis (TB) (4, 17a). The infected host typically mounts a vigorous immune response (25). Nevertheless, 10% of all infections result in active disease within 2 years. Another 10% of cases may experience disease after a latent phase spanning many years (8, 17a). Several Mycobacterium species (e.g., M. tuberculosis, M. bovis, and M. africanum) can infect and cause disease in humans (2, 24). In about 80% of active TB cases, direct involvement of the lung results in pulmonary disease (4a). However, M. tuberculosis can spread to other organs. In approximately 20% of cases, M. tuberculosis may cause nonpulmonary disease in various organ systems (urogenital system, nervous system, digestive system, skeletal system, etc.) with or without the lung involvement (7,18). TB is a treatable disease, provided that a timely and appropriate diagnosis is made (4a). Commonly used sputumbased methods for pulmonary TB diagnosis are subjective, insensitive, and/or inefficient. Furthermore, for the detection of pediatric pulmonary TB, a major limitation is that children often have difficulty producing usable quantities of sputum.Sputum smear acid-fast bacillus (AFB) microscopy is recommended by the World Health Organizatio...