• The Immune Complex Dissociation (IC_X) Reagents may identify serum or plasma specimens in which p24 antigen is masked by antibody.
• The Confirmation Reagents (CR_X) may confirm the reactivity of positive specimens.

• IC_x Acid, 25 ml: Contains glycine-HCl. (Reagent A)
• IC_x Base, 25 ml: Contains tris-HCl and sodium azide. (Reagent B)
• IC_x Positive Control, 1 ml: Contains human source material with p24 immune complexes (heat inactivated), and sodium azide.(Reagent C)
• IC_x/CR_xNegative Control, 1 ml: Contains human source material non-reactive for antibodies to HIV-1 and non-reactive for HIV-1 p24 antigen, and sodium azide.(Reagent D)
• CR_x Control Reagent, 1ml: Contains human source material non-reactive for antibodies to HIV-1 and non-reactive for HIV-1 p24 antigen, Triton X-100^®, and sodium azide.(Reagent E)
   
• CR_x Neutralization Reagent, 1 ml: Contains human source material with antibodies to HIV-1 (heat inactivated), Triton X-100^®, and sodium azide. Non-reactive for HIV-1 p24 antigen.
  
  Triton X-100^® is a registered trademark of Rohm and Haas
  
  Storage:
  
  Store Kit at 2° - 8°C. DO NOT FREEZE. When stored properly, the Kit is stable until the expiration date indicated on the box label.

Materials Required But Not Supplied:
 

• Lysing Buffer, HIV-1 p24 Antigen Standard, and Assay Diluent from RETRO-TEK HIV-1 p24 Antigen ELISA
   
• RETRO-TEK HIV-1 p24 Antigen ELISA
   
• Disposable gloves
   
• Validated adjustable micropipettes, single and multichannel
   
• Test tubes and racks for preparing specimen and control dilutions
   
• Graduated cylinders and assorted beakers
   
• Validated automatic microplate washer or manual vacuum aspiration equipment
   
• Validated incubator for 37°C ±1°C
   
• Validated microplate reader
   
• Timer
• 1% sodium hypochlorite as disinfectant. May be prepared from household bleach.
   
• Distilled or deionized water

• Prior to performing the assay, carefully read all instructions.
• Use universal precautions when handling kit components and test specimens.*
• To avoid cross-contamination, use separate pipet tips for each specimen.
• Do not interchange components of this kit with any other kits or reagents.
• Disposal: When testing potentially infectious human specimens, adhere to all applicable local, state and federal regulations regarding the disposal of biohazardous material.
• Human source material used in the manufacture of the HIV-1 p24 Detector Antibody, ICx Positive Control, IC_x/CR_x Negative Control, CR_x Control Reagent, and CR_x Neutralization Reagent has been tested and found negative for Hepatitis B surface antigen. The viral lysate used to prepare the HIV-1 p24 Antigen Standard has been inactivated by chemical disruption and heating. Handle these reagents as if capable of transmitting infectious agents.
  
  * from MMWR, June 24, 1988, Vol. 37, No. 24, pp. 377-382, 387-388.

Allow all reagents to reach room temperature before use. Label test tubes to be used for preparation of Positive Control, Negative Control, and specimens.

Step 1: Pipet 75 µl of IC_x Positive Control and 75 µl of IC_x Acid into the appropriate tubes and mix. Prepare IC_x/CR_x Negative Control and all serum or plasma specimens in the same manner.

Step 2: Incubate for 1 hour at 37°C (±1°C).

Step 3: During incubation, prepare all reagents necessary to perform the RETRO-TEK HIV-1 p24 Antigen ELISA.

Step 4: Pipet 75 µl of IC_x Base reagent into each sample or control and mix.

Step 5: Pipet 25 µl of Lysing Buffer into each test tube and mix.

Step 6: Immediately assay each specimen using the RETRO-TEK HIV-1 p24 Antigen ELISA as described on Page 4 of the p24 ELISA Product Insert. Omit Step 1 since Lysing Buffer has already been added.
 

IMMUNE COMPLEX DISSOCIATION
PROCEDURAL FLOW CHART

IC_x ACID REAGENT,
CONTROLS AND SPECIMENS

INCUBATE 1 HOUR AT 37°C ± 1°C

PIPET IC_x BASE REAGENT

PIPET LYSING BUFFER

PROCEED WITH RETRO-TEK
HIV-1 p24 ANTIGEN ELISA

Samples found reactive in the RETRO-TEK HIV-1 p24 Antigen ELISA should be confirmed by neutralization. Each reactive sample is incubated separately with the CR_x Neutralization and the CR_x Control reagents prior to analysis in the RETRO-TEK HIV-1 p24 Antigen ELISA. During the incubation, p24 antigen present in the sample will complex with the anti-p24 antibody contained in the CR_x Neutralization Reagent; conversely, no antigen-antibody complexing will occur in the sample incubated with the CR_x Control Reagent. When tested in the ELISA, non-complexed antigen in the sample will be captured by the anti-p24 coated microplate; complexed antigen will not be captured by the anti-p24 coated microplate, resulting in reduction of the optical density.

In order to perform confirmation testing, samples must have a concentration of p24 antigen less than 125 pg/ml. Specimens containing more than 125 pg/ml must be diluted to a concentration of approximately 50 to 125 pg/ml prior to testing.

For confirmation of p24 antigen for specimens initially reactive without IC_x treatment, proceed with Steps 1 to 4 on this page. For specimens initially reactive after IC_x treatment, proceed with Steps 1 to 6 on Page 6.

Allow all reagents to reach room temperature before use. For the following procedure, consult Table 1 for the appropriate test tube labels, reagents and volumes.

Step 1: Prepare the Antigen Positive Control (AgPC) by pipetting 25 µl of the HIV-1 p24 Antigen Standard into 975 µl of Assay Diluent (62.5 pg/ml final concentration) from the RETRO-TEK HIV-1 p24 Antigen ELISA.

Step 2: Pipet 225 µl of Antigen Positive Control (AgPC), 225 µl of IC_x/CR_x Negative Control (Reagent D), or 225 µl of each specimen into their respective test tubes. Pipet 25 µl of Lysing Buffer from the RETRO-TEK HIV-1 p24 Antigen ELISA into each test tube. Pipet 30 µl of the CR_x Control Reagent (Reagent E) or 30 µl of the CR_x Neutralization Reagent (Reagent F) into their respective test tubes and mix.

Step 3: Incubate for 1 hour at 37°C (±1°C).

Step 4: Immediately assay each specimen using the RETRO-TEK HIV-1 p24 Antigen ELISA as described on Page 4 of the p24 ELISA Product Insert. Omit Step 1 since Lysing Buffer has already been added.

Information regarding test validity requirements, calculations and interpretations of results is contained in the p24 ELISA Product Insert.

TABLE 1
Preparation of Specimens and Controls (without Immune Complex Dissociation)

NEUTRALIZATION
PROCEDURAL FLOW CHART
Without IC_x Treatment


PREPARE SPECIMENS, CONTROL
AND REAGENTS

PIPET SPECIMENS, CONTROLS
AND REAGENTS

INCUBATE 1 HOUR AT 37°C ± 1°C

PROCEED WITH RETRO-TEK
HIV-1 p24 ANTIGEN ELISA

TABLE 2
Preparation of Specimens and Controls (with Immune Complex Dissociation)

NEUTRALIZATION
PROCEDURAL FLOW CHART
With IC_x Treatment
 

PREPARE SPECIMENS, CONTROL
AND REAGENTS

PIPET SPECIMENS, CONTROLS,
AND REAGENT A

INCUBATE 1 HOUR AT 37°C ± 1°C

INCUBATE 1 HOUR AT 37°C ± 1°C

PROCEED WITH RETRO-TEK
HIV-1 p24 ANTIGEN ELISA

1. Morrow, WJW et al. 1986. Circulating immune complexes in patients with acquired immune deficiency syndrome contain the AIDS-associated retrovirus. Clin Immunol Immunopathol. 40: 515-524.
2. Nishanian, P et al. 1990. A simple method for improved assay demonstrates that HIV p24 antigen is present as immune complexes in most sera from HIV-infected individuals. J Inf Dis. 162: 21-28.
3. Bollinger, RC, Jr. et al. 1992. Acid dissociation increases the sensitivity of p24 antigen detection for the evaluation of antiviral therapy and disease progression in asymptomatic human immunodeficiency virus-infected persons. J Inf Dis. 165: 913-916.