1. Plate HEK293 cells in eight T175 flasks. The cell monolayer should be 70-80% confluent when they are ready for infection. (Approximately, a 70-80% confluent T175 flask contains 17-18 million cells).

2. Incubate cells for 24-48 hrs in a 37 oC incubator until they become 70-80% confluent.

3. To infect the cells, replace the medium in each flask with 10 ml of fresh growth medium that contains adenovirus. Infect cells at a multiplicity of infection (MOI) 5-10 (i.e., at 5-10 pfu/cell). For example, if the T175 flask contains ~ 18 x 10^6 cells, add 1.8 x 10^8 pfu adenovirus to get an MOI of 10 pfu/cell. Dilute the virus with media if necessary.

4. After 5 hrs of incubation at 37oC, add 5 ml more of the growth medium, to make the final volume 15ml.

5. Incubate for 3-4 days at 37 oC incubator and check for cytopathic effect.

Infected cells typically remain intact, but round up and may detach from the plate. These changes are collectively called cytopathic effect (CPE). Ideally the CPE should be complete within 72-96 hrs from the time of infection.

6. When ~80% of the cells have detached, transfer the suspension to four -50 ml tubes. Do not use trypsin. Infected cells still attached to the bottom can be dislodged into the medium by pipeting up and down.

7. Centrifuge the suspension at 1000 rpm for 5 min at room temp.

8. Collect the supernatant and store at –20 oC. (This supernatant contains viral particles, but titer may be low). Resuspend the pellet (from all 4 tubes) in a final volume of 10ml.

9. The cell pellet (in 10 ml of media) is lysed by three consecutive freeze-thaw cycles. Freeze the cells in a dry ice/ethanol bath or liquid nitrogen; thaw cells by placing the tube in a 37 oC water bath. Do not allow the suspension to reach 37 oC. Vortex cells after each thaw.

10. After the third cycle, centrifuge the cells at 1000 rpm for 5 min to pellet the debris. Discard the pellet and collect the supernatant. Aliquot and keep at –20 oC.

11. This crude lysate can be used for infecting the target cells. Also this can be further purified using the BD Ad-purification kit according to the manufacturer’s instructions.

Relative activity of Taq mutants with DNA polymerase activity after 35 min at 97°C

Taq purification method

1. Spin down cells

2. Resuspend 5 g  cells in 25 ml lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 0.1 mM PMSF (phenylmethylsulfonyl fluoride),1 mM DTT, pH 8.0)

3. Lyse cells with ultrasound.

4. Add DNase I to a final concentration of 20 mg/ml and MgCl2 to a final concentration of 4 mM.

5. incubate at 25°C for 30 min.

6. Heat incubation for 30 min at 72°C.

7. Spin (22,000 x g).

8.  Load cleared supernatant onto a 10 ml Ni-NTA superflow column (Qiagen).

9. Wash  column  with two volumes of buffer A (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0).

10. Eluate column with a volume of 100 ml of a linear gradient, starting with buffer A and ending with buffer B (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0).

11. FPLC purify using Q sepharose.

12.  Dialyze against storage buffer.

Relative activity of Taq mutants with DNA polymerase activity after 35 min at 97°C

Taq purification method

1. Spin down cells

2. Resuspend 5 g  cells in 25 ml lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 0.1 mM PMSF (phenylmethylsulfonyl fluoride),1 mM DTT, pH 8.0)

3. Lyse cells with ultrasound.

4. Add DNase I to a final concentration of 20 mg/ml and MgCl2 to a final concentration of 4 mM.

5. incubate at 25°C for 30 min.

6. Heat incubation for 30 min at 72°C.

7. Spin (22,000 x g).

8.  Load cleared supernatant onto a 10 ml Ni-NTA superflow column (Qiagen).

9. Wash  column  with two volumes of buffer A (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0).

10. Eluate column with a volume of 100 ml of a linear gradient, starting with buffer A and ending with buffer B (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0).

11. FPLC purify using Q sepharose.

12.  Dialyze against storage buffer.

Key to Genotypes

001 The Development of Lateral Flow Immunochromatographic Devices to Detect Antibodies

(Fast-Track proposals will not be accepted.)

Number of anticipated awards: 1

Budget (total costs): Phase I: $150,000 for 6 months

It is strongly suggested that proposals adhere to the above budget amounts and project periods. Proposals with budgets exceeding the above amounts and project periods may not be funded.

Background: During the past decade, several companies have developed lateral flow immunochromatographic devices to detect antibodies to individual communicable diseases. More recently, these platforms have also been adapted to detect specific antigens associated with these infections. These inexpensive point-of-care (POC) tests offer considerable advantages over conventional laboratory tests, since they can be performed in remote, peripheral settings with little or no instrumentation by primary health care workers. In addition, counseling and treatment, if appropriate, can be given at the initial consultation. They have been used successfully to screen pregnant women for HIV and syphilis to prevent vertical transmission of these infections and therefore prevent congenital disease. In addition, in areas remote from formal, organized blood banks, these and other POC tests have been used to screen potential blood donors to prevent transfusion related infections. Unfortunately these tests are usually performed as individual tests for antibodies or antigens for single infections, which results in a series of test strips being run in parallel. Each may have different flow characteristics, buffers and run times which can lead to confusion and potential inaccuracies.

Project Goal: The goal of this project is to develop a highly sensitive, highly specific, rapid and easy to use, disposable multiplex immunochromatographic screening device to detect Hepatitis BsAg and malarial antigen together with antibody to Human Immunodeficiency Virus 1 and 2 (HIV 1/2) and syphilis in a single finger-stick sample of whole blood in order to screen pregnant women to prevent vertical transmission of infection. In addition, a single device to detect Hepatitis BsAg and malarial antigen together with antibody to Hepatitis C Virus (HCV), Human Immunodeficiency Virus 1 and 2 (HIV 1/2) and syphilis in a single finger-stick sample in order to screen blood donors for transfusion- related infections in settings where conventional laboratory facilities are not available.

Vendors should have access to existing individual immununochromatographic tests to detect antibody to HIV, HCV and syphilis and tests to detect HBsAg and malaria. Likewise, vendors should be prepared to optimize their assays to detect both antibodies and specific antigens in the same cassette device on a single specimen. The contractor will be able to leverage this research and development opportunity to build capacity within the company to develop further multiplex platforms with antigen/ antibody combinations of diagnostic value. The CDC is willing to collaborate with small business by furnishing appropriate specimens to enable optimization of the multiplex platform and to facilitate both laboratory evaluations to be conducted within CDC laboratories in Atlanta and clinical evaluations in appropriate field sites in settings where these infections constitute a significant public health problem and where the tests would ultimately be used routinely.

Impact: It is anticipated that the development of these two multiplex immunochromatographic test cassettes could result in a significant reduction in rates of congenital HIV and syphilis together with other infections that can be transmitted from mother to child. In addition, make blood transfusions safer in areas where laboratory testing is either not available, or of poor quality.