Sera can be inactivated but this is not a requirement for optimal test performance.  Inactivate as follows: 1.   Loosen caps of serum containers. 2.   Heat sera to 56°C for 30 minutes in water bath. 3.   Allow sera to cool before retightening caps. 4.   Sera can be stored frozen until analysis. We recommend that the sera should not undergo repeated freeze-thaw cycles prior to testing.

1.     DILUTED WASH BUFFER a)    Dilute 1 volume of WASH BUFFER CONCENTRATE (20X) with 19 volumes of reagent grade         water. Mix well. 2.     BLOTTING BUFFER a)    BLOTTING BUFFER should be prepared fresh prior to use. b)     Dilute 1 volume of STOCK BUFFER CONCENTRATE (10X) with 9 volumes of reagent grade         water. Mix well. c)     Add 1g of BLOTTING POWDER to every 20 ml of DILUTED STOCK BUFFER prepared in step         2(b) above. Mix well. 3.     WORKING CONJUGATE SOLUTION a)    Prepare WORKING CONJUGATE SOLUTION by diluting CONJUGATE 1:1000 in BLOTTING         BUFFER, for example, 5 ul CONJUGATE to 5 ml BLOTTING BUFFER. b)     WORKING CONJUGATE SOLUTION should be prepared fresh prior to use. 4.     SUBSTRATE SOLUTION (ready to use) a)    Dispense directly the required volumes from the bottle. Use a clean pipette. Cap         tightly after use.

NOTE:      Aspirate all used chemicals and reagents into trap containing sodium                 hypochlorite. Using forceps, carefully remove required number of STRIPS from the tube and place numbered side up into each well. Include strips for Strong Reactive, Weak Reactive and Non-Reactive controls.   Add 2 ml of DILUTED WASH BUFFER to each well.   Incubate the strips for at least 5 minutes at room temperature (25±3°C) on a rocking platform. Remove buffer by aspiration.   Add 2ml of BLOTTING BUFFER to each well followed by 20ul each of sera or controls to appropriate wells.   Cover the tray with the cover provided and incubate for 1 hour at room temperature (25 ± 3°C) on the rocking platform.   Carefully uncover the tray to avoid splashing or mixing of samples. Aspirate the mixture from the wells. Change aspirator tips between the samples to avoid cross contamination.   Wash each strip 3 times with 2 ml of DILUTED WASH BUFFER, allowing 5 minutes soak on the rocking platform between each wash.   Add 2 ml of WORKING CONJUGATE SOLUTION to each well. Cover tray and incubate for 1 hour at room temperature (25±3°C) on a rocking platform.   Aspirate CONJUGATE from the wells. Wash as in step 7.   Add 2 ml of SUBSTRATE SOLUTION to each well. Cover tray and incubate for 15 minutes on the rocking platform.   Aspirate the SUBSTRATE and rinse the strips several times with reagent grade water to stop the reaction.    Using forceps, gently remove strips onto the paper towels. Cover with paper towels and dry.   Mount strips on worksheet (non-absorbent white paper). Do not apply adhesive tape over the developed bands. Observe the bands and grade the results. For storage, keep the strips in the dark.

We recommend that the Non-Reactive, Strong Reactive and Weak Reactive controls be run with every assay regardless of the number of samples tested.  1.     NON-REACTIVE CONTROL No HIV-1 specific bands should be observed on the Non-Reactive control strips. The band for the serum control should be visible (Fig 1c). 2.     STRONG REACTIVE CONTROL All relevant molecular weight bands must be evident. Figure 1a provides a guide to the relative positioning of bands visualised with the Genelabs Diagnostics HIV-1 Blot 1.3 and permits identification of bands observed for the STRONG REACTIVE CONTROL. The bands are p17, p24, p31, gp41, p51, p55, p66 and gp120/gp160. Other bands associated with core antigens (ie. p39, p42) may also be visible. Be careful not to misinterpret these as gp41. The envelope antigens, gp41, gp120/gp160 appear as diffuse bands as they are typical of glycoproteins. The serum control band will be visible. 3.      WEAK REACTIVE CONTROL          Weak bands at p24 and gp120/160 should appear . Some additional weak bands may or may          not be present. The serum control band will be visible (Fig 1b).

The presence or absence of antibodies to HIV-1 in a sample is determined by comparing each nitrocellulose strip to the assay control strips tested with the NON-REACTIVE, STRONG REACTIVE and WEAK REACTIVE controls. The Figure 1a on Page 11 is suggested as an aid to identify the various bands which develop on the strip reacted with the STRONG REACTIVE Control. Molecular Weight   Gene Antigen Description gp 160 ENV Polymeric form of gp41 Broad diffuse  glycoprotein gp120 ENV Outermembrane Diffuse glycoprotein p66 POL Reverse Transcriptase Discreet band p55 GAG Precursor protein Discreet band p51 POL Reverse Transcriptase Discreet band just below p55 gp41 ENV Transmembrane Diffuse glycoprotein p39 GAG Frogment of p55 Discreet band p31 POL Endonuclease Doublet p24 GAG Core Protein Broad band p17 GAG Core Protein Broad band Some of the different antigens mentioned in the Table above are derived from the same precursor protein and may have overlapping epitopes. This should be considered when interpreting the pattern, for example:- 1. It is unlikely to detect gp41 in the absence of gp160 because the gp160 is the polymeric form of gp41 and the concentration of gp160 is higher than gp41 on the HIV-1 Blot 1.3. 2. The p55 band is generally detected when there is strong reactivity to p24 and/or p17. The bands seen as p42 and p39 are both GAG fragments and should not be interpreted as gp41 (ENV). 3. The POL bands p66, p51 and p31 are generally detected simultaneously. However the sensitivity of p66 and p31 are greater than p51. 4. HIV-2 cross reactivity is variable but typically shows reactivity with GAG and/or POL antigens. However, there can be cross reactivity with the gp160 band in some cases, but rarely with gp41. 5. There is also a high molecular weight band around 160KD that is presumed to be a GAG-POL precursor protein. This is seen with some high titered HIV-2 or Indeterminate (GAG Reactive Only) sera but the band pattern is a sharp discreet band which is different from the diffuse band of ENV gp160.

Deviation from the recommended procedure may lead to aberrant results

The manufacturer makes no warranty other than that the test kit will function as a Research Use Only assay within the specifications and limitations described in the Product Instruction Manual when used in accordance with the instructions contained therein. The manufacturer disclaims any warranty, express or implied, including such express or implied warranty with respect to merchantability, fitness for use or implied utility for any purpose. The manufacturer is limited to either replacement of the product or refund of the purchase price of the product. The manufacturer shall not be liable to the purchaser or third parties for any damage, injury or economic loss howsoever caused by the product in the use or in the application thereof.

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