bDNA Assay and HIV RNA Quantification

1. bDNA Assay and HIV RNA Quantification Molecular Diagnostics Spring 2004 Gary , Qi

2. Simple sample preparation • Centrifugation • Centrifuge whole blood to isolated plasma; 500 g for 15 min • Centrifuge plasma to pellet the virus; 23,500 g for 1 hour • Buffer contains… • Lithium laurel sulfate – detergent to dissolve cell membrane to release genomic material • Proteinase K – remove protein material and isolate RNA • Target probes • Incubate at 53ºC for 20 minutes

3. bDNA assay • Load sample to microwells coated with capture probes • Incubate at 53ºC over night and wash • Add bDNA amplifier molecules • Incubate at 53ºC and wash • Added alkaline phosphatase labeled probes • Incubate at 53ºC and wash • Add chemiluminescence substrate (dioxetane) • Measure light emission with luminometer

4. Schematic outline of bDNA Assay

5. Standard Curve • Light emission is directly proportional to the amount of viral RNA present in the plasma sample • Determine actual RNA concentration from standard curve

6. Branched DNA assay • A signal amplification assay utilizing nucleic acid hybridization • Specificity • target probes • Complementary to the RNA template • Complementary to capture probes that coated the microwells • Complementary to the bDNA amplifier molecules • Alkaline phosphatase is bound to oligonucelotide probe that is complementary to the branches on the bDNA • The alkaline phosphatase will only emit light in the presence of dioxetane substrates

7. Branched DNA assay (continue…) • Sensitivity • More than 80 different target probes available to bind with in the specific region to avoid false negative results cause by mutations • Each bDNA can bind to many enzyme linked probes • Dioxetane substrate can detect 100fg of RNA • Using signal amplification the assay can measure RNA level as low as 500 copies/mL of plasma

8. Branched DNA assay (continue…) • Controls • A known positive control, for quality of all reagents • A known negative control, for possible RNA template contamination • samples with known RNA concentration to generate the standard curve • Dynamic range: 104 to 1.6 x 106 RNA equivalent/mL of plasma

9. Current application of bDNA • Evaluate plasma viral RNA level • HIV is a RNA virus • Viral coat stability depends on viral RNA • Absolute dependency of presence of viral RNA for the infection • The assay can quantify plasma level of the virus through out all stages of the infection by measuring the level of viral RNA present

10. Stages of HIV Infection • The virus enters a period of clinical latency as infection progressed • Total viral load (i.e. the measurable quantity of virus in the body) remains in low fluctuating concentrations • Hard to measure plasma viral level and changes of the level

11. Efficiency of HIV RNA assay (bDNA) • Under most circumstances a 2.8 fold change in viral load is statistically significant when using bDNA • With special precaution, this value can be decreased to only 1.4-fold change in viral loads • Can be used to test the effective of various drugs for treatment of the HIV infection • Monitor the response of the immune system to the drug therapy and evaluate the efficiency of drug therapy • Monitor the progress of the infection • Need to increase/decrease drug dose? • A new drug needed?

12. Why is bDNA the perfect assay? • Familiar 96 well-microwells format • Easy sample preparation • No nucleic acid extraction, purification, or amplification • Does not need to worry about contamination • Chemiluminescent assay has long shelf life

13. Highly sensitive and specific • PCR-based tests use a pair of primers and a single probe • With bDNA, more than 80 nucleic acid probes target area within the highly conserved pol region of the HIV genome to avoid false negative results cause to mutations

14. Less cost and labor intensive

15. Linearity of the standard curve

16. Reproducibility • bDNA assay is highly reproducible • Good reproducibility ensures consistency

17. Bayer® System 340 (bDNA Analyzer)

18. bDNA System 340 automatic analyzer • A single instrument that successfully automates the incubation, washing, reading,and data processing • It provides precise, accurate, and reproducible quantification of HIV.

20. Compare to other HIV assays… • Quantification by PCR can monitor the therapeutic administration but the assay is labor intensive and results lacks consistency between different labs and technicians. • ELISA with p24 capture assay is easy to do and consistent but there is no 100% accurate correlation between p24 antigen level and level of viral replication present and increase in p24 is not necessarily a positive clinical outcome.

21. Perfection of bDNA assay • The assay combines all the good-qualities and diminish all those imperfect-qualities of ELISA, PCR, and evaluation of CD4 + T cell level • Viral RNA level measured by bDNA does increase with decrease in level of CD4+ T cells. • The assay easy to do and give consistent results like the ELISA • Like PCR the assay is able to demonstrate the presence of HIV replication and monitor the effects of drug therapy has on viral replication.

22. Additional application of dDNA signal amplification assay • Hepatitis C virus RNA quantitation • Hepatitis B virus RNA quantitation • Quantify insulin preRNA and mRNA in determining the stimulation of insulin synthesis by glucose • Analysis of adipose differentiation by measuring the amount of lectin, the product of the obese gene, mRNA present