GENSCREEN PLUS HIV Ag-Ab

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					Genetic Systems™ HIV-1/HIV-2 PLUS O EIA

     HIV-1/HIV-2 PLUS O    HIV Subtypes and Variants
                           Description of HIV-1/HIV-2 PLUS O EIA
                              Format, components, QC criteria
                              Detection of HIV-1 (M & O), and HIV-2
                              Detection of Seroconversions
                              Specificity
                           Confirmation


                           To detect HIV Antibody Groups M & O
       HIV Subtypes and Variants
HIV-1 Subtype B is predominant in the U.S., but non-B subtypes are found in
           2% of HIV-Positive U.S. blood donors, and increasing.
(Delwart et al., AIDS Research and Human Retroviruses 19:1065-1070, 2003.)
                                BCE                    ABD
                Group
                 O?
           HIV-2
                               BO           F
          HIV-1                                       G
B                                    B                 C
                        DE                                             BE
                               HIV-1: ABDEFGHIJ…O
                                  HIV-2: ABCDEFG

    BCF                                            ABCD
                                             C

                                    DH
     HIV Subtypes and Variants
            Most Problematic for Antibody Detection

 HIV-2: HIV-2 testing mandated for blood screening
  June 1, 1992.
        1st: Genetic Systems™ HIV-1/HIV-2 EIA (viral lysate)

          2nd: Genetic Systems™ HIV-1/HIV-2 Peptide EIA
 HIV-1 Group O: FDA asked U.S. manufacturers to
  modify HIV kits (7/31/96 and 7/30/97).


    3rd: Genetic Systems™ HIV-1/HIV-2 PLUS O EIA
                      licensed 8/5/03
             Principle of the Test:
           Direct Antibody Sandwich
Immunoglobulins from the sample bind simultaneously to
   antigens (4 total) on the microplate and to similar
     HRP-conjugated antigens (5 total) in solution.

5 HRP-Antigen
  Conjugates                                  HRP
2 HIV-1 M peptides
 HIV-1 M rec. p24                                         IgG
  HIV-1 O peptide
   HIV-2 peptide


                     Rec. p24    Rec. gp160    Pep. env
                     (HIV-1 M)   (HIV-1 M)     (HIV-2)
                                  Pep env
                                 (HIV-1 O)
                            4 Plate Antigens
             Principle of the Test:
           Direct Antibody Sandwich
     Binding of IgM to the microplate is stabilized by
    multiple attachments, and the signal is amplified by
     multiple HRP-Antigen Conjugate binding sites.
                              HRP          HRP
5 HRP-Antigen
  Conjugates            HRP                      HRP

2 HIV-1 M peptides
 HIV-1 M rec. p24
                       HRP                        HRP
                                                             IgM
  HIV-1 O peptide
   HIV-2 peptide


                     Rec. p24       Rec. gp160    Pep. env
                     (HIV-1 M)      (HIV-1 M)     (HIV-2)
                                     Pep env
                                    (HIV-1 O)
                              4 Plate Antigens
Earlier IgM Detection…a Benefit of the
      Antibody Sandwich Format
                                Seroconversion Panel PRB 940
                                    Antibody Sandwich:           Indirect Antibody:
                                  HIV-1/HIV-2 PLUS O EIA      HIV-1/HIV-2 Peptide EIA
                                 Abbott HIVAb HIV-1/HIV-2
                     14.0

                     12.0
     Signal/cutoff




                     10.0
                                   ~Day 8                      ~Day 14
                      8.0

                      6.0

                      4.0

                      2.0
                                                                                               Cutoff
                      0.0
                            0         5            10           15            20          25
                                                   Time (Days)

                     HIV-1/HIV-2 Peptide EIA            GS HIV-1/HIV-2 PLUS O           Abbott HIVAb
                Kit Components
• Three kit sizes: 480/960/4800 Tests
• Direct Antibody Sandwich Format
• Unique Kit Components:
   -R1 Microwell Strip Plates (8X12)
   -R3 Specimen Diluent
   -C0 Negative Control
   -C1 HIV-1 Positive Control
   -C2 HIV-2 Positive Control
   -C3 HIV-1 Group O Positive Control
   -R4 Conjugate Concentrate (11X)
   -R5 Conjugate Diluent
• “Shared” Components: 30X Wash (R2), Substrate Buffer
  (R8), TMB Chromogen 11X (R9), Stopping Solution (R10)
          Procedure (60’ 30’ 30’)
• Add 25µl Specimen Diluent + 75µl Control or Sample
   to each well.*
• Cover and incubate 60 +/- 5 min. at 37 +/- 2ºC.
• Wash a minimum of 5 times with 30-60 second soaks.
• Add 100 µl Working Conjugate to each well.*
• Cover and incubate 30 +/- 5 min. at 37 +/- 2ºC.
• Wash a minimum of 5 times with 30-60 second soaks.
• Add 100 µl Working TMB to each well.
• Cover and incubate 30 +/- 5 min. at RT.
• Add 100 µl Stopping Solution to each well.
• Read within 30 minutes at 450nm, with the 615-630nm
  filter as a reference.
 *Optional O.D. readings may be taken to verify addition of specimen or reagent.
  Color indicates differences in the procedure from HIV-1/HIV-2 Peptide EIA.
              Procedure Monitoring


                           Before After      Optional
                                            Verification

                                            Sample
   Sample Dispensing                      OD630 > 0.150

                                            Conjugate
   Conjugate Dispensing                   OD630 > 0.100

   TMB                                   After incubation
                                            of a positive
   Stopping solution                          sample
              Quality Control:
             Validation of Results
• Kit Controls (6 wells total):
     C0 Negative Control (3 wells)
        Each value A450=0.001-0.150 (one may be
        discarded)
     C1 HIV-1 Positive Control (1 well)
        A450 >0.700
     C2 HIV-2 Positive Control (1 well)
        A450 >0.700
     C3 HIV-1 Group O Positive Control (1 well)
        A450 >0.700
• Cutoff = xNC + 0.250

        Color indicates differences in the procedure from the HIV-1/HIV-2 Peptide EIA.
               Performance Results:
             HIV-1 Group M Sensitivity
Reactivity in HIV-1 Known Positive Samples
                        Results Obtained with                           Licensed
 Genetic Systems™ HIV-1/HIV-2 PLUS O EIA                             HIV-1/HIV-2 EIA
                Group                 Number Repeatedly Reactive   Number Repeatedly Reactive
                  AIDS (N=313)                     313                       313
                                                (100.00%)                 (100.00%)
   Known HIV-1 Positive,                           490                       490
           U.S. (N=490)                         (100.00%)                 (100.00%)
   Known HIV-1 Positive,                           199                       199
      Non-U.S. (N=199*)                         (100.00%)                 (100.00%)
                           TOTAL:                  1002                      1002
                                                (100.00%)                 (100.00%)
* Australia, New S. Wales (N=36)    Nigeria (N=46)
  Central African Republic (N=40)   Sierra Leone (N=40)
  Ghana (N=5)                       Thailand (N=21)
  Kenya (N=3)                       Zimbabwe (N=8)
    Performance Results:
HIV Variant/Low Titer Samples
  BBI Performance Panels (N=130 Positives)
   •   Mixed Titer        PRB203 (N=23 Positives)
   •   Low Titer          PRB105 (N=14 Positives)
   •   African HIV Series AfrRB1 (N=46 Positives)
   •   Worldwide      WWRB301 (N=47 Positives)
       HIV-1/HIV-2 Peptide EIA   124/130 (95.4%)
  Abbott HIVAb HIV-1/HIV-2       128/130 (98.5%)
       Genetic Systems™
                                 130/130 (100.0%)
HIV-1/HIV-2 PLUS O EIA
                 Performance Results:
                   HIV-2 Sensitivity

Results Obtained from Known Positive HIV-2 Samples with
Genetic Systems™ HIV-1/HIV-2 PLUS O EIA
                                                                    Pos. by HIV-2
 Number Tested        Initially Reactive   Repeatedly Reactive      Western blot *
     302                   302                   302                    302
                        (100.00%)             (100.00%)              (100.00%)

         *HIV-2 samples were repeatedly reactive on an HIV-2 EIA, positive
  on an HIV-2 Western blot, and indeterminate or negative on an HIV-1 Western blot.
           Performance Results:
         HIV-1 Group O Sensitivity

 Results Obtained from Known Positive HIV-1 Group O
                     Samples with
   Genetic Systems™ HIV-1/HIV-2 PLUS O EIA
    Number Tested                 Initially Reactive            Repeatedly Reactive
         77*                             77                            76**
                                     (100.00%)                      (100.00%)


*Known HIV-1 Group O samples were obtained from individuals living in
Cameroon (N=70), the United States (N=2), Spain (N=2) and France (N=3).

**One initially reactive Group O specimen was not available in sufficient volume for
repeat testing.
                    Performance Results:
                      Seroconversions
                            Reactivity with HIV-1 on
             50 Commercial Seroconversion Panels
                  HIV-1/HIV-2 PLUS O HIV-1/HIV-2 PLUS O HIV-1/HIV-2 PLUS O
                      Equivalent       More Sensitive      Less Sensitive
     vs.                  12/46*                     34/46*                       0
HIV-1/HIV-2
 Peptide EIA              (26%)                      (74%)                      (0%)
     vs.                    35/50                     9/50                       6/50
Abbott HIVAb
HIV-1/HIV-2                (70%)                     (18%)                      (12%)

vs. Licensed                13/50                     37/50                       0
Western Blot               (26%)                     (74%)                      (0%)
• Four of the 50 seroconversion panels did not have test results with the licensed HIV-1/HIV-2
                      Peptide EIA and are no longer available for testing.
                      Performance Results:
                        Seroconversions
                     Detection of Seroconversion Panel BCP 9017
                         HIV-1/HIV-2 PLUS O: 21 Days Earlier
              14.0                                                                                                                        100000
                                                                                                             62790        60180
                                                                                                23590                                  25440
              12.0




                                                                                                                                                   Copies/ml (log scale)
                                                                                   11990                                                  10000
              10.0         4516
                                                                       2040
OD/Cutoff




                                                                                                                                          1000
               8.0                                          619
                                      380        404

               6.0
                                                                                                                                          100

               4.0
                                                                                                                                          10
               2.0
            cutoff
               0.0                                                                                                                        1
                       3          7         10         14         17          21           24           28           32           35

                                                                   Days
              HIV-1/HIV-2 PLUS O            Peptide EIA            Abbott HIVAb                   Abbott Prism                    Chiron HIV-1 RNA
                       Performance Results:
                        U.S. Blood Donors
          Results Obtained from Random Donors tested with
   Genetic Systems™ HIV-1/HIV-2 PLUS O EIA
                                                                    HIV-2 EIA Pos. by HIV-1
                Number                     Initially   Repeatedly   Repeatedly Western blot
Sample Type      Tested     Non-Reactive   Reactive     Reactive     Reactive     alone
   Serum           6103        6097            6            6
                                                                        0           0
 (Sites 1, 2)   (100.00%)    (99.90%)      (0.10%)      (0.10%)

   Plasma          5056        5044           12            6
                                                                        0           0
 (Sites 2, 3)   (100.00%)    (99.76%)      (0.24%)      (0.12%)

  TOTAL           11,159       11,141         18           12
                                                                        0           0
                (100.00%)    (99.84%)      (0.16%)      (0.11%)

                          Specificity 99.89%
                  (95% confidence interval 99.83-99.96)
Subtypes/Variants Tested by
    HIV-1 Western Blot
Worldwide HIV Panel WWRB302
       (members 1-13)


                              All HIV+ samples in the panel
                                    (N=28) exhibited
                               A450/A630>3.000 in testing
                                         with
                                  Genetic Systems™
                               HIV-1/HIV-2 PLUS O EIA
     Group 0




                 HIV-2



                 HIV-2
      LPC
       NC




                  Neg
       PC




       G
       G




                   G

                   G
        A


        A




                   A


                   C
                   A
                           SUMMARY
 Dissemination of HIV-1 non-B subtypes and variants is a growing
  concern in the U.S.
 Reliable detection of HIV-1 Group O or HIV-2 antibody requires the
  use of specific antigens in the test kit.
 HIV-1/HIV-2 PLUS O EIA performance was demonstrated with
  human serum, plasma, and cadaveric serum samples.
    100% detection of known HIV-1 (N=1002), HIV-2 (N=302), and
     HIV-1 group O (N=77) specimens.
    100% detection of HIV-1 low titer samples and HIV subtypes of worldwide
     origin (N=130) from four panels.
    Detection of HIV-1 seroconversion panels (N=50) better than licensed
     competitors.
    Specificity 99.89% (95% C.I. 99.83-99.96) in blood and plasma donors at
     three sites (N=11,159).
 Current confirmatory algorithms will detect most HIV-1 group O
  samples; unique banding may assist identification.

				
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