Substantially Equivalent 510(k) Device Information
Applicant: Biotest Diagnostics Corp.,Denville, NJ
510(k) number: BK960061
Product: Biotest DQB-ELPHA
Date: 8/4/97
Summary of Safety and Effectiveness
- Legally Marketed Device
Biotest Diagnostics claims substantial equivalence to the LYMPHOTYPE Microtrays with pre-dropped anti-HLA sera (BK830027) currently in commercial distribution by Biotest Diagnostics.
- Device Description
The Biotest DQB-ELPHA (Enzyme Linked Probe Hybridization Assay) is a DNA-hybridization test for the determination of HLA-DQB alleles. Sequence-specific oligonucleotide (SSO-) probes are used as diagnostic tools to identify polymorphic sequence motifs. The hybridization between probe and target DNA is detected by a method adapted from the protein-ELISA technique. The microtest plate format allows the use of standardized apparatuses for automation of the test procedure and data evaluation. The test, however, also can be easily performed by hand.
Starting from genomic DNA which can be readily obtained from peripheral blood, the polymorphic region in the 2nd exon of the DQB gene is amplified by means of the polymerase chain reaction (PCR1). For targeting and starting the PCR Biotin-labeled primers are used. After amplification and denaturation of the PCR products the resulting biotinylated single strand molecules are bound to Streptavidin-coated wells of a microtest plate. The wells contain FITC-labeled SSO-probes in a dried form which redissolve when the diluted solution of the PCR-products is transferred to the wells. Fixation of the PCR product to the wells and hybridization with the SSO-probes thus occur in one step. The specificity of the assay is dependent on the subsequent stringency washing step in which probes of insufficient sequence homology to the PCR-products are removed. Hybridization probes are visualized in a color reaction involving FITC-specific antibody fragments coupled to Horeseradish peroxidase (POD). The results are quantitated photometrically in an ELISA reader. The combination of DQB alleles in the test material is determined from the pattern of positive reaction either by using the Biotest HLA-DQB-typing program on s personal computer or a means of the DQB-SSO reaction scheme.
- Intended Use
The Biotest DQB-ELPHA is a enzyme linked probe hybridization assay intended for the determination of HLA-DQB alleles.
- Comparison with Predicate Device
A summary comparison of the features of the Biotest DQB-ELPHA and the LYMPHOTYPE Microtrays is provided in Table 1 below:
Table 1
Feature Comparison of DQB-ELPHA and LYMPHOCYTE Microtrays
| |
DQB-ELPHA |
LYMPHOCYTE Microtrays |
| Intended Use |
HLA Typing
(Detection of HLA Antigens) |
HLA Typing
(Detection of HLA Antigens) |
| Assay Method |
Enzyme Linked Probe Hybridization |
Lymphocytotoxicity |
| Reactive Ingredients |
Biotin-labeled primers
FITC-labeled SSO-probes |
Anti-HLA antisera |
| Specimen: Type |
Defibinated or heparinized blood |
Anticoagulated blood
(heparin, sodium citrate,EDTA) |
Min. Volume
Storage |
500 µ1
2 hours (defibrinated)/ RT
24 hours (heparinized)/ RT |
5ml
20 -25°C/3 days
-20°C/several months |
| Controls |
Positive Control (Probes E2 and H2) Inhouse panels |
HLA Positive & Negative Controls |
| Results: |
|
| Evaluation |
ELISA reader @ 450 nm |
Phase contrast microscope |
| Interpretation |
Pattern of positive reactions (computer or manual reaction scheme) |
Pattern of positive reactions (manual work sheet) |
| Kit Size |
12 analyses |
60, 72 or 120 tests |
Performance Data
The biotest DQB-ELPHA was compared to commercially available anti-HLA serological testing 312 specimens (random transplant candidates and potential donors) at two geographically distinct locations. There was 95.8% concordance (299/312) between the DQB-ELPHA results and the results obtained using the anti-HLA serological tests.
Upon further testing to resolve discordants using additional methods ( standardized inhouse DNA methods), there was 100% concordance (312/312).
Lot-to-lot reproducibility has been demonstrated in tests of a panel of 7 serologically known HLA types with at least two different lots.
1 The PCR process is covered by US patents owned by Hoffmann-La Roche Inc. The use of the PCR process requires a license. Nothing in this publication should be construed as an authorization or an implicit license to practice PCR under any patents held by H-LR Inc.
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