ELISA

1. Development of an antigen-capture
enzyme-linked immunosorbent assay for
Clostridium perfringens beta2-toxin in
porcine feces and the neonatal piglet
intestine
Guevarra; Olivar; Santos; Tan; Virata

2. Clostridium perfringens
•third most common cause of food
poisoning though it can sometimes
be ingested and cause no harm
• Generally affect the health of your
livestock. Killing them and preventing
their usage as commodity.

3. Clostridium perfringens
•5 toxinotypes (A-E), A being most
important in neonatal pigs
•In toxinotype C, beta2-toxin (CPB2)
is involved in the virulence
• 2 forms: CONSENSUS and ATYPICAL
• Expressed in ALL toxinotypes
• Believed to be MARKER for
presence of toxinotype A

4. Methodology
Purification of
CPB2 protein
Preparation of
Monoclonal
Antibodies
Development
and optimization
of ELISA
polyclonal capture
antibody (rabbit
anti-rCPB2)E. coli D5α

5. Development and Optimization of
ELISA
• Assessment of the optimal concentrations allowing antigen-antibody
interaction
• Optimization of incubation conditions
• Selection of blocking buffers
• Selection of coating buffers

6. Extraction Protocols
Intestines Stool Sample
Centrifugation at 4 C at
17,500 x g for 30-60 min
until clear supernatant is
collected
Centrifugation at 17,500 x
g until clear supernatant is
collected

7. Assessment of the optimal
concentrations allowing antigen-
antibody interaction
•captured CPB2 detected
was between 2.0 and 3.5
ng/ml
•pAb diluted 1:2,500 for 2
hr at 37°C followed by
overnight at 4°C was
optimal for coating the
wells

8. Development and Optimization of
ELISA
ELISA Components Used in Experiment
Antibody rabbit anti-rCPB2
Antigen Beta2-toxin
Enzyme-linked Antibody horseradish peroxidase (HRP)-labeled monoclonal detection antibody
(mouse anti-rCPB2 mAb-HRP)
Colorless substrate 2,2’-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS)
Wash buffer phosphate buffered saline [PBS], 0.05% Tween 20, 0.5% fish skin gelatin
Blocking buffers PBS, 0.05% Tween 20, 2% BSA
Coating buffers PBS
Positive color GREEN intensity measured at 405 nm
OPTIMUM condition: incubation of pAb diluted 1:2,500 for 2 hr at 37°C followed by overnight at 4°C was optimal
for coating the wells and that a 1:5,000 dilution of mAb–HRP incubated for 1 hr at room temperature was
optimal for detecting captured antigen.

9. Fight against proteases
Agent Action
amidinophenyl-methanesulfonyl fluoride INHIBITOR, binds with serine
protease inhibitor mix INHIBITOR
Processing at 4 C Slows down activity of protease
pH effects Changes ionic interaction of proteases
*Before any procedures done, dead tissue samples were “SPIKED” (incubated at -70 C)
to allow CLUMPING of different proteins
**Freezing yielded best results since it was able to detect the toxin at low
concentrations and it provided the least “noise”.

10. Conclusion
•With appropriate sample preparation, antigen-
capture ELISA can detect beta2-toxin in the
intestinal content and feces of neonatal piglets.