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)

INTRODUCTIONStreptococcus 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.

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

Incubation in RT 20 min

Streptococcus pneumoniae

Serum

C1q

+

Baby rabbit sera

HL-60 cells

Plated

Incubation at 37oC for 45 min

INTRODUCTIONOpsonophagocytic 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.

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 .

2.AIM AND OBJECTIVEAIM : 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.

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.

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

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

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

1:8A 20 µl serum(for dilution) , 20 µl bacteria, 10 µl complement, 40 µl HL-60

cellsC’ Ctrl

C’ Ctrl

1:16 BAll wells finally contain

10 µl of serum diluted in OPB20 µl of bacterial suspension

10 µl complement source40 µl of HL-60 cells

1:32 C

1:64D

1:128 E

1:256 F

1:512 G

1:1024 H

Fig 4 Plate Format For OPA:

Fig 4 Plate Format For OPA

Results and Discussion

1 PRECISION

GRAPH 1.1 PRECISION

64

128

256

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.

2.AccuracyTable 2.1 Table 2.2

NS-Negative sera

Table 2.3 Table 2.4

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

3 LINEARITYTable 3.1

Table 3.2

Graph 3.1

Graph 3.2

3 LINEARITYTable 3.3

Serotype A Linearity Run-4

3

56.0

5

y = -0.9415x + 6.0585R2 = 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

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.

4 ROBUSTNESSTable 4.1

E-Effector, T-Target

Table 4.2

Graph 4.1

Graph 4.2

4 ROBUSTNESS

Serotype A Robustness Run-4

96

64 64

40

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

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.

5 SPECIFICITYGraph 5.1

Graph 5.2

Table 5.1

Table 5.2

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.

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.

7 References

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key to pneumococcal disease. Lancet Infect Dis 4: 144-54.14.Collins, S. J. 1987. The HL-60 promyelocytic leukemia cell line: proliferation, differentiation,

and cellular oncogene expression. Blood 70:1233– 1244.

7 References

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