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This Package Insert is provided for product evaluation purposes only and is
not intended to be used in place of the Package Insert shipped with the product.

RETRO-TEK
HIV-1 ICx/CRx Kit
Reagents for Immune Complex Dissociation
and Reactivity Confirmation

FOR RESEARCH USE ONLY. NOT FOR in vitro DIAGNOSTIC OR CLINICAL USE.

ZMC Catalog # : 0801096
   
INTENDED
USE
  The RETRO-TEK HIV-1 ICx/CRx Kit is supplied for research purposes only. It is not intended for use in the diagnosis or prognosis of disease, or for screening, and may not be used as a confirmatory test in diagnostic situations.

HIV-1 p24 antigen may be detectable at the onset of an infection with the Human Immunodeficiency Virus Type 1 (HIV-1), prior to seroconversion, and late in infection as the disease progresses to AIDS. However, during the ARC (AIDS Related Complex) phase of the disease, p24 antigen assays have had limited utility, since the majority of patients produce both anti-p24 antibody and p24 antigen and form immune complexes1. These immune complexes may either interfere with or prevent the measurement of p24 antigen by conventional immunoassays2,3.

The RETRO-TEK HIV-1 ICx/CRxKit complements the RETRO-TEK HIV-1 p24 Antigen ELISA (ZMC Catalog #  0801111). The ICx/CRx Kit contains two sets of reagents, each of which may provide supplemental information that enhances the detection p24 antigen in serum or plasma:
 
  • The Immune Complex Dissociation (ICX) Reagents may identify serum or plasma specimens in which p24 antigen is masked by antibody.
  • The Confirmation Reagents (CRX) may confirm the reactivity of positive specimens.
   
REAGENTS   Materials Supplied:
 
  • ICx Acid, 25 ml: Contains glycine-HCl. (Reagent A)
  • ICx Base, 25 ml: Contains tris-HCl and sodium azide. (Reagent B)
  • ICx Positive Control, 1 ml: Contains human source material with p24 immune complexes (heat inactivated), and sodium azide.(Reagent C)
  • ICx/CRxNegative 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)
  • CRx 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)
     
  • CRx 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 - 8C. 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 37C 1C
     
  • Validated microplate reader
     
  • Timer
  • 1% sodium hypochlorite as disinfectant. May be prepared from household bleach.
     
  • Distilled or deionized water
   
PRECAUTIONS   FOR RESEARCH USE ONLY. Not For in vitro Diagnostic Use.
  • 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, ICx/CRx Negative Control, CRx Control Reagent, and CRx 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.
   
1. IMMUNE
COMPLEX
DISSOCIATION

 
  TEST PROCEDURE

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 l of ICx Positive Control and 75 l of ICx Acid into the appropriate tubes and mix. Prepare ICx/CRx Negative Control and all serum or plasma specimens in the same manner.

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

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

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

Step 5: Pipet 25 l 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

 

ICx ACID REAGENT,
CONTROLS AND SPECIMENS

INCUBATE 1 HOUR AT 37C 1C

PIPET ICx BASE REAGENT

PIPET LYSING BUFFER

PROCEED WITH RETRO-TEK
HIV-1 p24 ANTIGEN ELISA

   
2.CONFIRMATION
OF p24 ANTIGEN
POSITIVE
SPECIMENS BY
NEUTRALIZATION
  PRINCIPLE OF TEST

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 CRx Neutralization and the CRx 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 CRx Neutralization Reagent; conversely, no antigen-antibody complexing will occur in the sample incubated with the CRx 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 ICx treatment, proceed with Steps 1 to 4 on this page. For specimens initially reactive after ICx treatment, proceed with Steps 1 to 6 on Page 6.

   
Test Procedure
Without ICx
Treatment
 

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 l of the HIV-1 p24 Antigen Standard into 975 l of Assay Diluent (62.5 pg/ml final concentration) from the RETRO-TEK HIV-1 p24 Antigen ELISA.

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

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

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)

Reagents (l)
 
Tube
Label
AgPC D Specimen LB E F  
PCC 225 0 0 25 30 0 I
PCN 225 0 0 25 0 30 N
NCC 0 225 0 25 30 0 C
NCN 0 225 0 25 0 30 U
S1C 0 0 225 25 30 0 B
S1N 0 0 225 25 0 30 A
S2C 0 0 225 25 30 0 T
S2N 0 0 225 25 0 30 E
etc.              
Legend
Reagent AgPC   Antigen Positive Control prepared in Step 1
Reagent D   ICx/CRx Negative Control
Reagent LB   Lysing Buffer
Reagent E   CRx Control Reagent
Reagent F   CRx Neutralization Reagent


NEUTRALIZATION
PROCEDURAL FLOW CHART
Without ICx Treatment


PREPARE SPECIMENS, CONTROL
AND REAGENTS

PIPET SPECIMENS, CONTROLS
AND REAGENTS

INCUBATE 1 HOUR AT 37C 1C

PROCEED WITH RETRO-TEK
HIV-1 p24 ANTIGEN ELISA

   
TEST
PROCEDURE
WITH ICx
TREATMENT
  Allow all reagents to reach room temperature before use. For the following procedure, consult Table 2 for the appropriate test tube labels, reagents and volumes.

Step 1: Pipet 75 l of ICx Positive Control (Reagent C), 75 l of ICx/CRx Negative Control (Reagent D), or 75 l of each specimen into their respective test tubes. Pipet 75 l of ICx Acid (Reagent A) into their respective test tubes and mix.

Step 2: Incubate for 1 hr. at 37C (1C).

Step 3: Pipet 75 l of ICx Base (Reagent B) into each test tube and mix.

Step 4: Pipet 25 l of Lysing Buffer (Reagent LB) from the RETRO-TEK HIV-1 p24 Antigen ELISA into each test tube and mix.

Step 5: Pipet 10 l of the CRx Control Reagent (Reagent E) or 10 l of the CRx Neutralization Reagent (Reagent F) into their respective tubes and mix.

Step 6: Incubate for 1 hr. at 37C (1C).

Step 7: 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 2
Preparation of Specimens and Controls (with Immune Complex Dissociation)


Reagent (l)
Tube
Label
C D Specimen A   B LB E F  
PCC 75 0 0 75 I 75 25 10 0 I
PCN 75 0 0 75 N 75 25 0 10 N
NCC 0 75 0 75 C 75 25 10 0 C
NCN 0 75 0 75 U 75 25 0 10 U
S1C 0 0 75 75 B 75 25 10 0 B
S1N 0 0 75 75 A 75 25 0 10 A
S2C 0 0 75 75 T 75 25 10 0 T
S2N 0 0 75 75 E 75 25 0 10 E
etc.                    
Legend
Reagent C ICx Postive Control
Reagent D ICxCRx Negative Control
Reagent LB Lysing Buffer
Reagent A ICx Acid
Reagent B ICx Base
Reagent E CRx Control Reagent
Reagent F CRx Neutrilization Reagent

 

NEUTRALIZATION
PROCEDURAL FLOW CHART

With ICx Treatment
 

PREPARE SPECIMENS, CONTROL
AND REAGENTS

PIPET SPECIMENS, CONTROLS,
AND REAGENT A

INCUBATE 1 HOUR AT 37C 1C

INCUBATE 1 HOUR AT 37C 1C

PROCEED WITH RETRO-TEK
HIV-1 p24 ANTIGEN ELISA

   
REFERENCES  
  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.
   

REV 08/00

 

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Email: info@gentaur.com
 

International 

+32 (0) 16 58 90 45

+32 (0) 16 50 90 45

France

01 43 25 01 50

01 43 25 01 60

Italy

02 36 00 65 93

02 36 00 65 94

Germany

0241 6085 13140

0241 6085 33033

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Last modified: feb-07