1. Validation of Opsonophagocytic killing assay
for “Streptococcus Pneumoniae’’
Internal Guide: Dr ASIT RANJAN GHOSH
Professor, SBST
External guide: Dr ASHA MALLYA
DY DIRECTOR SERUM INSTITUTE OF INDIA LTD.
PUNE 411028
Email.Id:asha.mallya@seruminstitute.com
Kishor kulkarni-14MBT0022
Email Id:
kishorkulkarni92@gmail.com
(9422914177)

2. INTRODUCTION
Streptococcus Pneumoniae:
 It is a species of genus streptococcus which belongs to the family of Streptococcaceae.
 It is a gram –positive diplococcal bacterium.
 First isolated by Pasteur in 1881.
 Currently 92 Pneumococcal serotypes are recognized.
 It is a fastidious bacterium, growing best in 5% carbon dioxide. at 37°C and has a doubling
Time of 20-30 min.
Clinical features :
 The major clinical syndromes of pneumococcal disease are pneumonia which is to be a major
cause of morbidity and mortality among both the elderly and the very young.
 Global Estimates suggest that approximately 10 million childhood deaths per year result from
pneumonia,
 Adults over 65 years old and people with chronic health problems bear the greater burden of
pneumonia
Fig1 Chains of Streptococcus pneumoniae bacteria.

3. INTRODUCTION
 The symptoms of infectious pneumonia are which
include the high fever (up to 105 degrees),
Drowsiness, Rapid breathing, Chest pain, Cough with
mucus.
Vaccination:
 The antibiotic resistant for Pneumococcal bacteria is also one of the main reason.
 The development of an effective vaccine for the prevention of pneumococcal infection
plays a crucial role in reducing the disease burden worldwide.
Pneumococcal vaccines are classified into two major categories those are :
 Pneumococcal Polysaccharide Vaccine (PPV)
 Pneumococcal Conjugate Polysaccharide Vaccine (PCV).
 During this infection pus and fluid fill the in one
or both lungs, and this interferes with oxygen
absorption, making breathing difficult.
Fig2 Pneumonia Infection

4. 4
Incubation in RT 20 min
Streptococcus pneumoniae
Serum
C1q
+
Baby rabbit sera
HL-60 cells
Plated
Incubation at 37oC for 45 min
INTRODUCTION
Opsonophagocytic Assay (OPA):
The present work relates to the field of Opsonophagocytic Activity (OPA) assays for Gram
Positive bacteria, in particular S. pneumoniae
The principle of the Opsonophagocytic assay is based on the detection of the functional
antibodies against Streptococcus pneumoniae after complement mediated opsonization through
the classical pathway by antibodies in serum samples.
The OPA used in the analysis of pneumococcal vaccines is designed to mimic the in vivo
opsonophagocytosis which involves bacteria, antibodies, complement, and phagocytic cells.

5. INTRODUCTION
Significance of OPA :
 Functional antibodies are measured by Opsonophagocytic Assay (OPA) rather than by ELISA
Which is helpful to increase the efficacy of the vaccine that's why OPA is considered as de
facto gold standard in this field.
 This assay provides an exact in vivo condition in in-vitro which involves bacteria, antibodies,
complement, and phagocytic cells, that's why this method is widely accepted for measuring
the protective capacity of pneumococcal antibodies.
 Study shows that there is a substantial of evidence that the rapid clearance of pneumococcal
from the blood is due to opsonophagocytosis mediated by type-specific antibody and
complement.
 This assay has been used extensively over the last 12 years and has effectively become the
standard assay .

6. 2.AIM AND OBJECTIVE
AIM : The aim of this research project is the “Validation of
Opsonophagocytic killing Assay for Streptococcus Pneumoniae.”
Objectives-The main objectives of the present study include:
Validating the performance of the assay in accordance with International Conference
for Harmonization guidelines, including its -
 Precision -
 The degree of agreement of OPA results when serum specimens are tested multiple times.
 Linearity -
 The ability of the OPA to yield titers that are directly proportional to the concentration of antibody.
 Accuracy -
 The closeness of agreement of a value obtained from the assay to the reference or known value.
 Robustness-
 The susceptibility of these results to deliberate changes in assay conditions.
 Specificity-
 The degree of PnPs-specific opsonophagocytic activity or nonspecific killing.

7. 3.MATERIALS AND METHODS
The critical components of OPA:
 Serum - 007sp (Human Anti-Pneumococcal Reference serum).
 Bacteria - The source of the target S. pneumoniae strains used in the
OPA were serotype ‘A’.
 Complement -Multiple lots of baby rabbit serum .
 Culturable phagocytes (HL-60)- Differentiated HL60 promyelocytic leukemia
(HL60) cells were used as effector cells, they were maintained, passaged, and
differentiated into granulocytes (with 100 mM dimethyl formamide [DMF]).
 Opsono buffer-Hanks Balanced Salt Solution with of 2% gelatin.

8. 3.MATERIALS AND METHODS
PROCEDURE FOR OPSONOPHAGOCYTIC ASSAY (OPA):
 Step: 1 Preparation and Addition of opsono buffer.
 Step: 2 Addition of test serum sample.
 Step: 3 Two fold dilution of test serum sample.
 Step: 4 Addition of bacteria.
 Step: 5 Incubation (20 min).
 Step: 6 Preparations of HL-60 cells.
 Step: 7 HL-60 cell counting under Haemocytometer (10X)
Total no of cell * D.F * Correction factor
Total no of Square

9. 3.MATERIALS AND METHODS
 Step: 8 Resuspension of HL- 60cells.
 Step: 9 Addition of complement.
 Step: 11 Addition of HL-60 cells.
 Step: 12 Incubation of microtitre plate . (45 min at 37 degree with constant shacking)
 Step: 13 Termination of reaction.
 Step: 14 Spotting On THYE Agar Plates.
(Placed in CO2 incubator at 370 C for 16-24hr)
 Step: 15 Titre assignment.
Fig 3 Bacterial Colonies grown after overnight incubation

10. 3.MATERIALS AND METHODS
Final
Serum
Dilution
Sample
1
Sample
2
Sample
3
Sample
4
QC serum Controls
1 2 3 4 5 6 7 8 9 10 11 12
1:8
A 20 µl serum(for dilution) , 20 µl bacteria, 10 µl complement, 40 µl HL-60
cells
C’
Ctrl
C’
Ctrl
1:16 B
All wells finally contain
10 µl of serum diluted in OPB
20 µl of bacterial suspension
10 µl complement source
40 µl of HL-60 cells
1:32 C
1:64 D
1:128 E
1:256 F
1:512 G
1:1024 H
Fig 4 Plate Format For OPA:
Fig 4 Plate Format For OPA

11. Results and Discussion
1 PRECISION
GRAPH 1.1 PRECISION
64
128
256

12. 1. PRECISION
Result:
The serum sample (007 sp) are tested multiple times, the results obtained showed
median titre 64 (n=20) and other titre we got is also + two fold of median titer i.e
128 (n=11 )and 256 (n=2) so 100% of the data was in accordance of qualification
criteria.
.
Calculation:
The results for precision are plotted with the No of repetitions on the X-axis and the
OPA titre obtained on the Y-axis the median titre is calculated according to the
statistical method. This median titre is highlighted with Red line.

13. 2.Accuracy
Table 2.1 Table 2.2
NS-Negative sera
Table 2.3 Table 2.4

14. 2.Accuracy
Result:
The final complied table shows the results for accuracy where in the
obtained or observed titers for the serotype ‘A’ were compared with the
median titer and a percentage agreement was calculated
The results obtained showed 100% agreement (agreement is + two
fold titer =64) accuracy for serotype ‘A’ .
Calculation:
Table 2.5

15. 3 LINEARITY
Table 3.1
Table 3.2
Graph 3.1
Graph 3.2

16. 3 LINEARITY
Table 3.3
Serotype A Linearity Run-4
3
5
6.0
5
y = -0.9415x + 6.0585
R2
= 0.9487
0.0
2.0
4.0
6.0
8.0
10.0
0 0.5 1 1.5 2 2.5 3 3.5
Serum Dilution
Titre
Table 3.4
Graph 3.3
Graph 3.4

17. 3 LINEARITY
Calculation:
The results for Linearity are plotted by first converting the values for the serum
dilution (007 sp) and the titers obtained in to LOG 2 and then are plotted on the X-
axis and the Y-axis respectively. The regression coefficient and the resulting linear
equation are obtained through Microsoft EXEL.
Result:
The correlation coefficient of the serotype ‘A’-specific OPAs ranged from 0.948 to
1.000 and All slopes were within the acceptable range ,Thus the data support
good linearity.

18. 4 ROBUSTNESS
Table 4.1
E-Effector, T-Target
Table 4.2
Graph 4.1
Graph 4.2

19. 4 ROBUSTNESS
Serotype A Robustness Run-4
96
64 64
4
0
20
40
60
80
100
120
400:1 200:1 100:1 50:1
E:T Ratio
Titre
E-Effector, T-Target
Graph 4.3
Graph 4.4
Table 4.3
Table 4.4

20. 4 ROBUSTNESS
Calculation:
 The results for robustness are plotted with the Dilution for HL-60 cells (i.e.
400:1, 200:1,100:1 and 50:1) on the (EFFECTOR TO TARGET) X-axis
and the OPA titre obtained for the same on the Y-axis.
Result:
 It was observed that the HL-60 ratio till 400:1 , 200:1 and 100:1 showed
no reduction in the titre whereas the ratio of 50:1 shows a dip in the titer
values.

21. 5 SPECIFICITY
Graph 5.1
Graph 5.2
Table 5.1
Table 5.2

22. 5 SPECIFICITY
Calculation: The result for specificity plotted as on Y axis the no of
colonies formed and on X axis different serum dilution, trendlines
indicate results for the FOUR different treatments.
Result :
Specificity (competitive inhibition) –The result shows homologous
PnPs inhibited ≥80% of Opsonophagocytic activity and
heterologous PnPs or no polysaccharide inhibited ≤20% of
opsonophagocytic activity. The data obtained indicate the assays for
A Serotype specific.

23. 6 Conclusion
 To Establish a more reliable and robust assay certain
parameters need to be studied according to the ICH guidelines
and After validating the serotype “A” of S. pneumoniae on
various parameters like Precision, linearity, Accuracy,
Robustness and Specificity. we can concluded that the
validation of Opsonophgocytic killing assay for S.
pneumoniae is successfully done and the outcomes from the
assessment of these parameters is going to help us for
understand and to develop new and improved vaccines for
various dreadful diseases caused by serotype “A” of S.
pneumoniae.

24. 7 References
1. www.who.int/mediacentre/factssheets/f5331/en/index.html
2. www.ariatlas.org/treatingpneumonia/html
3. www.pdrhealth.org/ari/pneumonia infection/html
4. http://en.wikipedia.org/wiki/pneumococcal vaccines
5. http://wwwumm.edu/patended/articles/htm
6.www.who.int/mediacentre/factssheets/f5331/en/index.htm
7.www.pdrhealth.org/ari/pneumoniainfection/html
8.http://en.wikipedia.org/wiki/Pneumococca.
9.www.ariatlas.org/treatingpneumonia/htm7.
10.www.ariatlas.org/treatingpneumonia/htm
11.http://en.wikipedia.org/wiki/Pneumococcal.vaccines.
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