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Detection of HTLV-1 and -2 Antibodies using Absorbance-based ELISAダウンロード
Related Products: Synergy LX マルチモード・プレートリーダー
May 31, 2018
The human T-lymphotropic virus (HTLV) are a family of retroviruses that are known to cause adult T-cell leukemia/lymphoma and a demyelinating disease called HTLV-1 associated myelopathy.1 HTLVs are lentiviruses that infect a number of different cell types and integrate into the cellular genome as part of its growth cycle.2 HTLV testing is routinely performed in blood transfusion, tissue transplantation and clinical diagnosis and is based on the detection of HTLV-1 and -2 antibodies present in serum.3 These tests are normally done using an enzyme-linked immunosorbent assay (ELISA). Specimens with a nonreactive result from the initial ELISA are considered HTLV-negative; while specimens with a reactive ELISA result are retested in duplicate and confirmed by Western blot.
ELISA is one the most used assay formats in biomedical research. Numerous clinical, veterinary, and research assays use the specificity of antibodies to identify a diverse array of analytes from any number of different matrices. Despite the diversity of analytes, the general process of ELISA is constant. With a typical ELISA protocol, several repeat cycles of microplate washing, reagent addition and incubation are executed to elicit the chemistries and remove unbound material before data collection. In many instances, the data derived from an ELISA reaction are color absorbance. This application brief describes the use of the low cost Agilent BioTek Synergy LX multimode reader to perform detection of a human HTLV-1 and -2 ELISA assay.
Materials and methods
A multiplate HTLV-1 and -2 microelisa kit (part number 500576) was obtained from Avioq (Durham, NC). The assay was performed as described in the assay kit instructions. Briefly, controls (negative serum and positive HTLV-1 and HTLV2 serum) were diluted 1:5 with sample diluent and 100 µL pipetted into wells of the precoated ELISA plate. Samples were made by further dilution of the HTLV-1 and -2 controls with sample buffer. The plate was incubated at 37 °C for one hour, after which the plate was washed four times with 300 µL of wash buffer with a 30-second soak between wash cycles using an Agilent BioTek EL406 washer dispenser. After washing, 100 µL of EnzAbody working solution was added to all the wells. Working solution was made daily by diluting EnzAbody concentrate 1:251 with EnzAbody diluent. After EnzAbody conjugate addition the plate was incubated for one hour at 37 °C and washed again as previously described. After washing, 100 µL of TMB substrate (1:1 mixture of TMB and peroxide solutions) was added to all wells and the plate incubated in the dark at room temperature for 30 minutes. Reactions were stopped by the addition of 100 µL of 2NH2 SO4. The absorbance at 450 nm and 630 nm was determined using an Agilent BioTek Synergy LX multimode reader. Data analysis was performed using the delta OD values (630 nm reference).
Results and discussion
A comparison of absorbance for positive and negative control specimens from a clinical HTLV-1 and -2 kits show good repeatability. As demonstrated in Figure 1, separate controls for HTLV-1 or HTLV-2 prepared together, but run on separate assay plates, shows a very good correlation. All of the negative and positive controls passed the assay kit’s validation criteria (data not shown).
Figure 1. Intraplate comparison of control well absorbance values. The HTLV-1 and HTLV-2 positive and negative serum kit controls absorbance values from two separate plates are compared. Data for the negative controls represent the mean and standard deviation of eight wells from each plate, while the positive controls (HTLV-1 and -2) represent four wells on each plate.
Serial dilution of the HTLV-1 and HTLV-2 positive control serum samples demonstrates the ability of the Synergy LX to make quantitative determinations using ELISA. Measurements made on separate plates are linear with respect to dilution and repeatable (Figure 2). Note that positive controls supplied by the assay kit can be diluted approximately 30 fold before the returned absorbance values are no longer considered to be positive.
Figure 2. Comparison of HTLV-1 and HTLV-2 Positive serum sample dilutions. Dilutions of positive control samples were assayed on separate plates and the absorbance results compared. A dashed green line indicates the assay positive sample cut off. Data points represent the mean and standard deviation of four determinations.
These data indicate that the Synergy LX is capable of measuring the absorbance of routine ELISA reactions. Dilutions of known positive samples demonstrate the sensitivity of both the assay as well as the multimode reader. While many ELISA assays return a quantitative, positive/negative, determination based on a comparison between experimental and control samples, this technology can also be used to quantitate unknown determinations. The optics of the reader produce a linear absorbance response from 0 to 2 optical density (OD) units.
- Gonçalves DU, Proietti FA, Ribas JG, Araújo MG, Pinheiro SR, Guedes AC, Carneiro-Proietti AB (2010). “Epidemiology, treatment, and prevention of human T-cell leukemia virus type 1-associated diseases”. Clin. Microbiol. Rev. 23 (3): 577–89. doi:10.1128/CMR.00063-09. PMC 2901658 . PMID 20610824.
- Smith JA, Daniel R (2006). “Following the path of the virus: the exploitation of host DNA repair mechanisms by retroviruses”. ACS Chem Biol 1 (4): 217–26 doi:10.1021/cb600131q. PMID 17163676.
- Malm, K., T. Kierstadius, and S. Anderson (2010) Evaluation of a new screening assay for HTLV-1 and -2 antibodies for large scale use. J. Med. Virol. 82 (9): 1606-1611. doi. 10.1002/jmv21867.