ROLE OF hsCRP IN PREDICTING CARDIO VASCULAR

DISEASES IN SOFT WARE PROFESSIONAL AND

DEVELOPMENT OF HIGH SENSITIVE CRP KIT

Thesis submitted to

BHARATHIDASAN UNIVERSITY

For the award of the degree of

DOCTOR OF PHILOSOPHY

IN

MICROBIOLOGY

BY Mr.P.K.SOJU, M.Sc.,

[Ref. No.6037/Ph.D-1/Micro/F.T./April 2009]

Guided By Dr.A.PANNEERSELVAM, M.Sc.,M.Phil.,B.Ed.Ph.D

P.G. AND RESEARCH DEPARTMENT OF BOTANY AND

MICROBIOLOGY

A.V.V.M. SRI PUSHPAM COLLEGE (AUTONOMOUS),

(AFFILIATED TO BHARATHIDASAN UNIVERSITY)

POONDI-613 503, THANJAVUR DISTRICT

TAMIL NADU (INDIA)

August- 2012

P.G. AND RESEARCH DEPARTMENT OF BOTANY AND

MICROBIOLOGY

A.V.V.M. SRI PUSHPAM COLLEGE (AUTONOMOUS) POONDI-613 503, THANJAVUR DISTRICT,

TAMIL NADU, INDIA.

[Affiliated to Bharathidasan University, Tiruchirapalli]

Dr. A. Panneerselvam Associate Professor Date :

CERTIFICATE

This is to certify that the thesis entitled “Role of hsCRP in Predicting

Cardio Vascular Diseases in Software Professional and Development of

High sensitive CRP Kit” submitted to Bharathidasan University,

Tiruchirapalli, for the award of the degree of Doctor of Philosophy in

Microbiology, embodies the result of the bonafide research work carried out

by Mr. P.K. SOJU, under my guidance and supervision in the P.G. and

Research Department of Botany and Microbiology, A.V.V.M. Sri Pushpam

College (Autonomous), Poondi, Thanjavur district, Tamil Nadu, India.

I further certify that no part of this thesis has been submitted

anywhere else for the award of any degree, diploma, associateship,

fellowship or other similar titles to any candidate.

Signature of Research Adviser

(Dr. A. Panneerselvam)

DECLARATION

I do hereby declare that this work has been originally

carried out by me under the supervision of Dr. A.

Panneerselvam Department of Botany and Microbiology,

A.V.V.M. Sri Pushpam College (Autonomous), Poondi,

Thanjavur district, Tamil Nadu, affiliated to Bharathidasan

University, Tiruchirapalli – 620 024 and this work has not

been submitted elsewhere for any other degree.

ugust, 2012, Signature of the

Candidate

Poondi-613 503. (P.K.SOJU)

4

ACKNOWLEDGEMENT

I am duty bound to record my sincere thanks with gratitude to

Dr. A.PANNEERSELVAM, M.Sc., M.Phil., BEd. Ph.D., Associate Professor,

Department of Botany and Microbiology, A.V.V.M. Sri Pushpam College

(Autonomous), Poondi, for suggesting the problem, supervising the work at

every stage, evincing enthusiasm and keen interest at the progress of my

work, guiding me at every step, offering valuable assistance at critical stages

of my progress and giving final shape to my research work. I profoundly

thank and express my deepest sense of gratitude to my teacher, philosopher

and guide.

My profound thanks are due to the Secretary and Correspondent,

Shreeman K. Thulasiah Vandayar, A.V.V.M. Sri Pushpam College

(Autonomous), Poondi, who gave a chance for doing research.

I honestly thank my Doctoral Committee Members

Dr.R. Saravanamuthu, M.Sc., M.Phil., Ph.D., Head, Department of Botany,

A.V.C. College (Autonomous), Mayiladuthurai, Nagapattinam district and Dr.

N. Thajuddin, Associate Professor, Department of Microbiology,

Bharathidasan University, Tiruchirapalli, for their judicious suggestions on the

subject matter of this investigation.

I owe my special thanks to Dr. R.Rajendran, M.A., M.Phil., Ph.D.,

Principal, A.V.V.M. Sri Pushpam College (Autonomous), Poondi for admitting

me to the Ph.D. programme.

5

I am thankful to Mr.U.Balasubiramanian, M.Sc., M.Phil., Dean,

Faculty of Science, A.V.V.M. Sri Pushpam College (Autonomous), Poondi, for

according permission to utilize the facilities available in the department.

I wish to render my most grateful thanks to

Dr. S. Jeyachandran, M.Sc., M.Phil., Ph.D., Co-Ordinator, Department of

Botany and Microbiology, A.V.V.M. Sri Pushpam College (Autonomous),

Poondi, for his valuable suggestions.

I thank Dr. S. Kulothungan, Dr. T. Kumar, Dr. C. Chandran,

Dr. V. Ambikapathy, Dr. P. Pandiyan, Dr. S. Murugesan,

Mrs. P. Vanathi, Dr. G. Senthilkumar, Dr. Madanraj, Dr. P. Kavitha,

Dr. K. Kanimozhi, Dr. G. Kanimozhi, Mr. V. Baskar and other faculty

members, both teaching and non-teaching, Department of Botany and

Microbiology, A.V.V.M. Sri Pushpam College (Autonomous), Poondi, for their co-

operation to complete the work successfully.

I am extremely grateful to Dr. M.Prabakaran Managing Director, Gowri

Biotech, Thanjavur, Research Scholars and Office Staffs for their moral

support, and continuous help to complete my research successfully.

I express my deep sense of gratitude to my wife Dr.S.Dhiva and other

family members for their kind understanding and constant encouragement to

complete my study successfully.

I wish to record my heartful thanks to M/S KR Printers, Thanjavur for

their timely help in typing the manuscript.

P.K.Soju

6

CONTENTS

Chapter

No Title

Page

No

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 17

3 MATERIALS AND METHODS 29

4 RESULTS 47

5 DISCUSSION 99

6 CONCLUSION 108

7 BIBLIOGRAPHY 111

8 PAPERS PUBLISHED

7

1.INTRODUCTION

1.1. What is CRP?

1.2. Chemical and Physical characters

1.3. What is hsCRP

1.4 Factors affecting hsCRP levels

1.4.1. Chemical and Physical characters of hsCRP

1.4.2. Role of hsCRP in predicting Coronary Heart Disease

1.5 Isolation of hsCRP

1.6 Purification and Characterization of hsCRP

1.7. Preparation of a diagnostic kit

1.8. Difference in the values of CRP

1.9. Scope of the Research work

2. REVIEW OF LITERATURE

3 MATERIALS AND METHOD

3.1 Sample Collection

3.1.1. Sample Processing

3.2. Screening for alcoholism

3.3. Screening for smokers

3.4. Screening of the Samples (Biochemistry)

3.4.1. Analysis of Serum CRP

3.4.2. Analysis of Serum hsCRP

3.4.3 Analysis of Total cholesterol

3.4.4 Analysis of HDL-Cholesterol

3.4.5 Analysis of LDL-cholesterol

3.4.6 Analysis of Triglycerides

3.4.6 Analysis of Very Low Density Lipids

3.5.Isolation of hsCRP from serum of both Males and Females

3.5.1 Sample Preparation

3.5.2 Preparation of p-Diazonium Phenyl-phosphorylchloine

(DPPC) resin column

8

3.5.3 Purification of CRP

3.6. Characterization of hsCRP

3.6.1 Single radial immuno diffusion

3.6.2. Analysis of Molecular weight by SDS-PAGE

3.6.2.1 Preparation of the Buffer

3.6.2.1. A. Preparation of 5x Sample Buffer

3.6.2.1. B. Preparation of 1x Running Buffer

3.6.2.2. Preparation of the Gel

3.6.2.2. a. Preparation of 1x Running Gel Solution

3.6.2.2. B. Stacking Gel Solution

3.6.2.3. Preparing the Sample

3.6.2.4. Running the gel

3.7 Animal Inoculation Studies

3.7.1. Inoculation of the rabbit

3.7.2 Bleeding

3.7.3 Purification of anti hsCRP by Affinity chromatography:-

3.7.3.1. Sample Preparation

3.7.3.2. Preparation of p-Diazonium Phenyl-phosphorylchloine

(DPPC)

3.7.3.3. Purification of Anti hsCRP

3.7.4. Characterization of Anti-hsCRP

3.7.4.1. Single radial Immunodiffusion

3.8. Preparation of Slide Agglutination Kit

3.8.1 Preparation of Anti serum or IgG

3.8.2. Selection of Latex Particles and coating

3.8.3. Determining Antibody and Latex Quantities

3.8.4. Protocol for Latex coating

3.8.5. Evaluation of the optimal amount of Antibody required for

latex coating

3.8.6. Latex agglutination assay

3.8.7. Stability Studies of Slide agglutination kit

9

3.8.8. Comparative evaluation Studies of New Agglutination kit

with Commercial Brand

3.9. Preparation of an ELISA Kit

3.9.1. Coating of antihsCRP to PVC micro titer plate

3.9.2. Blocking of the free spaces

3.9.3. Performing the ELISA test

3.9.3.1. Preparation of Calibration Curve for ELISA testing

3.9.4. Precision and Linearity study of newly developed ELISA kit

3.9.5. Stability Studies of ELISA Kit

3.9.6. Comparative evaluation Studies of New ELISA kit with

Commercial Brand

4. RESULTS AND DISCUSSION

4.1 Sample Collection

4.1.1. Sample Processing

4.2. Screening for alcoholism in Females below 30 years

4.3. Screening for smokers in Females below 30 years

4.4. Screening of the Samples (Biochemistry)

4.4.1. Analysis of Serum CRP in Females below 30 years

4.4.2. Analysis of Serum hsCRP in Females below 30 years

4.4.3 Analysis of Total cholesterol in Females below 30 years

4.4.4 Analysis of HDL-Cholesterol in Females below 30 years

4.4.5 Analysis of LDL-cholesterol in Females below 30 years

4.4.6 Analysis of Triglycerides in Females below 30 years

4.4.6 Analysis of VLDL in Females below 30 years

4.4.7. Analysis of Serum CRP in Males below 30 years

4.4.8. Analysis of Serum hsCRP in Males below 30 years

4.4.9. Analysis Total cholesterol in Males below 30 years

4.4.10. Analysis of HDL-Cholesterol in Males below 30 years

4.4.11. Analysis of LDL-cholesterol in Males below 30 years

4.4.12. Analysis of Triglycerides in Males below 30 years

4.4.13. Analysis of VLDL in Males below 30 years

4.4.14. Analysis of Alcoholism in Males below 30 years

10

4.4.15. Analysis of Smoking in Males below 30 years

4.4.16. Analysis of Serum CRP in Males between 30 and 45 years

4.4.17. Analysis of Serum hsCRP in Males between 30 and

45 years

4.4.18. Analysis Total cholesterol in Males between 30 and

45 years

4.4.19. Analysis HDL cholesterol in Males between 30 and

45 years

4.4.20. Analysis LDL cholesterol in Males between 30 and

45 years

4.4.21. Analysis VLDL cholesterol in Males between 30 and

45 years

4.4.22. Analysis Triglycerides in Males between 30 and 45 years

4.4.23. Analysis of Alcoholism in Males between 30 and 45 years

4.4.24. Analysis of Smoking in Males between 30 and 45 years

4.5. Isolation of hsCRP from serum of both Males and Females:

4.5.1 Sample Preparation

4.5.2 Isolation of hsCRP from serum of both Males and Females

Affinity chromatography

4.6. Characterization of hsCRP

4.6.1 Single radial immuno diffusion

4.6.2.Analysis of Molecular weight by SDS-PAGE

4.7 Animal Inoculation Studies

4.7.1. Inoculation of the rabbit

4.7.2 Bleeding

4.7.3 Purification of anti hsCRP by Affinity chromatography:-

4.7.3.1. Sample Preparation

4.7.3.2. Isolation using DPCC column

4.7.3.3. Purification of Anti hsCRP

4.7.4. Characterization of Anti-hsCRP

4.7.4.1. Single radial Immunodiffusion

4.8. Preparation of Slide Agglutination Kit

4.8.1 Preparation of Anti serum or IgG

11

4.8.2. Latex agglutination assay

4.8.3. Stability Studies of Slide agglutination kit

4.8.4 Repeatability study of New Kit

4.8.5 Evaluation of Turbidometric hsCRP kits Commercially

Available

4.9. Preparation of an ELISA Kit

4.9.1 Preparation of Calibration Curve for ELISA testing

4.9.2. Results of Precision and Linearity study of newly developed

ELISA kit

5. DISCUSSION

6. CONCLUSION

7. BIBLIOGRAPHY

8. PAPERS PUBLISHED

12

INTRODUCTION