Using an Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of Streptococcus pneumoniae in Human Bodily Fluids

Jermaine D. Dorsey and Mamie T. CoatsDepartment of Biological Sciences, Alabama State University, Montgomery Alabama, USA, 36101

Abstract

Streptococcus pneumoniae is a major cause of bacteremia, meningitis, and pneumonia in both children and adults. Invasive pneumococcal disease (IPD) primarily affects young children, older adults (> 65 years of age), and individuals with comorbidities or impaired immune systems. The diagnosis of pneumococcal infections has been attempted using methods which detect specific antigens by the procedures of a quantitative enzyme-linked immunosorbent assay (ELISA). We will detect the antigen D39, WU2, and EF3030 for the diagnosis of Streptococcus pneumoniae infection.

Introduction

Streptococcus pneumoniae, pneumococcus, is a gram-positive pathogen found in the upper respiratory tract of healthy children and adults. Worldwide, S. pneumoniae remains one of the leading cause of community acquired bacterial infection for diseases like; pneumonia, otitis media, bacterial meningitis, and bacterium.

Pneumococcus polysaccharide capsule surrounds the pneumococcus and protects it against clearance by phagocytosis. More than 90 different capsule serotypes have been discovered thus far.

When pneumococcal infection is suspected time is very important in influencing patient outcomes. Classical identification takes several days to verify and molecular techniques are limited due to the variability of the capsular polysaccharide. The overall goal for this research is to develop a rapid, serotype independent assay to detect S. pneumoniae in body fluids in using an Enzyme-Linked Immuno-Sorbant Assay.  .

• Coated 96-well plate with PspC specific polyclonal capture antibody

• Blocked with Bovine Serum Albumin (BSA)

•Added 10X5 CFU/mL S. pneumoniae (strain D39, WU2)

•Added Detection antibody with incubation at 37°C

•Added labeled 2 antibody

•Added Substrate

•Read OD405

 Discussion and future plan

 • Overall, our experiment has shown that we

can detect PspC through ELISA Method.

• Use the same method in presence of bodily fluids to determine the sensitivity of this method of detection.

• Other virulence factors will be examined as potential capture antigens.

Acknowledgements

I would like to thank my mentor and MARC U STAR program, Biomedical Research and Training Program at Alabama State University, Montgomery, Al. 36104. This work was supported by the National Institutes of Health (NIH).

Methods and Results

Capture Antibody

Target Antigen

Detection Antibody

Labeled AntibodyBiotin

Substrate

Anticipated results

1 2 3 4 5 6 7 8 901234567

Free PspC

Log CFU/mL

Leve

l of P

spC

in E

nviro

nmen

t (m

edia

)

The level of PspC in the growth environment is directly proportional to the number of pneumococci present

The level of PspC in the growth patients’ samples is directly proportional to the level of infection and the site of infection

pathmicro.med.sc.edu

Uninfected sample

Low Moderate  High0

2

4

6

8

10

12

SputumBlood

Infection level

Amou

nt o

f Psp

C de

tect

ed