OFFICE OF THE DEPUTY DIRECTOR

VIROLOGY

NATIONAL INSTITUTE FOR COMMUNICABLE DISEASES

1 Modderfontein Road, Sandringham, 2031

Tel: +27 (0)11 386-6328 Fax: +27 (0)11 386-6411

Validation of the Q – Preven HIV 1 + 2 DBS Immunoenzyme Kit

Summary:

The Q-Preven HIV 1+2 DBS kit is an enzyme immunosorbent assay (ELISA) for the detection of

antibodies against HIV 1, HIV 2 and HIV 1 subtype O in Dried Blood Spot Specimens (DBS). The

kit consists of the following items: Dried Blood Spot Collection Kit which consists of the filter paper

and lancet; and the Test Kit which consists of ELISA Microplate and ELISA reagents required to

conduct an ELISA test. Supplies with the kit are negative and positive dried blood spot controls.

The following steps were completed during the validation:

Replicate testing using Kit Controls:

The negative DBS control and the positive DBS control from the Q-Preven test kit was tested 8

times in the same run and the results analyzed. All the controls passed validation and all controls

run as samples were within acceptable ranges i.e. the negative controls were negative and the

positive controls were positive.

Precision and Accuracy Testing:

Three negative and three positive controls taken from the Q-Preven test kit were tested over a

period of five consecutive days. The same operator tested the controls over the five day period and

the same plate washer, plate incubator and plate reader was used during the testing. The results of

the precision and accuracy testing are listed in Table 1 below:

Table 1

ID Day 1 -

Ratio

Day 2 -

Ratio

Day 3 -

Ratio

Day 4 -

Ratio

Day 5 -

Ratio

MEAN SD %CV

Negative

control

0.317 0.332 0.347 0.450 0.365 0.3622 0.0522 14.41%

Negative

control

0.328 0.328 0.325 0.399 0.355 0.347 0.0315 9.08%

Negative

control

0.257 0.328 0.332 0.383 0.329 0.3258 0.0449 13.78%

Positive

control

16.845 22.969 21.487 15.192 16.589 16.6164 3.3971 18.25%

Positive

control

18.506 22.969 17.776 19.038 19.579 19.5736 2.0116 10.28%

Positive

control

19.642 22.969 19.949 19.038 19.579 20.2354 1.563 7.72%

The %CV is detailed in the kit insert as follows for Inter Assay Reproducibility:

Negative 7.3%

Positive 3.5%

The %CV is detailed in the kit insert as follows for Inter Assay Repetitively:

Negative 17.3%

Positive 9.5%

The %CV obtained for all the negative samples should be between 7.3% and 17.3%. All three

negative control %CV was less than 17.3% and is acceptable. The OD value between each

negative sample over the 5 days was <30%, therefore Accuracy between each negative control

reading was good.

The %CV obtained for all the positive samples should be between 3.5% and 9.5%. Two of the three

positive control %CV was out of the acceptable range. The OD value between each positive sample

over the 5 days was <30%, therefore Accuracy between each positive control reading was good.

Sensitivity and Specificity testing:

50 known negative DBS samples and 42 known positive DBS samples were randomly selected and

tested on the Q-Preven assay. The result of the samples on the Q-Preven assay is 100% in

concordance with the initial NICD laboratory results. The sensitivity and specificity of the Q-Preven

Assay is listed below.

Sensitivity: 100%

Specificity: 100%

Negative Predictive Value: 100%

Positive Predictive Value: 100%

Replicate Testing - New Controls

Additional controls were tested.The lot numbers received were 2028200180 and 2028300180 for the

negative and the positive controls respectively. The negative and the positive controls were run 8

times in the same run and the results analyzed. All controls passed validation and all the controls

run as samples were within acceptable ranges i.e. the negative controls were negative and the

positive controls were positive.

The test method, calculation procedure, specifications of Validity and Interpretation of results was

taken from the Q-Preven Package insert Revision 3 – 28/12/2011

Limitations of Validation:

Specimens known to be positive or negative based on NICD test results were used.

There was no direct comparison testing between plasma/serum and dried blood spot testing to

assess performance of the kit.

Limited numbers of specimens were used for sensitivity and specificity to demonstrate that the

assay performed as expected.

General comment

The package insert although readable requires additional editing to ensure that there is no ambiguity

in terms of performing the assay.

Testing and Excel Spreadsheets done by: Ushmita Patel

Date: 28 December 2011

Report compiled by: Beverley Singh and Ushmita Patel

Date: 11 January 2012

Report Reviewed and Approved By: Prof Adrian J Puren

Date: 11 January 2012

Signature:

The report is not an endorsement or otherwise of the product.