α-テスト：全唾液を用いた迅速な無細胞CD4列挙
Summary
CD4列挙法、α-テストは、迅速かつ正確なCD4 + Tリンパ球数を提供するために唾液を使用していますが記載されている。 α-テストコストのペニーと技術トレーニングなど、モノクローナル抗体、計測機器、冷凍、サンプルの輸送と同様に、血液の収集と処理などの高価な試薬の必要がなくなります。
Abstract
There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time and temperature restrictions, technical sophistication, and cost of reagents, in particular monoclonal antibodies to measure CD4 on blood cells, the only currently acceptable method. A commonly used cost-saving and time-saving laboratory strategy is to calculate, rather than measure certain blood values. For example, LDL levels are calculated using the measured levels of total cholesterol, HDL, and triglycerides1. Thus, identification of cell-free correlates that directly regulate the number of CD4+ T cells could provide an accurate method for calculating CD4 counts due to the physiological relevance of the correlates.
The number of stem cells that enter blood and are destined to become circulating CD4+ T cells is determined by the chemokine CXCL12 and its receptor CXCR4 due to their influence on locomotion2. The process of stem cell locomotion into blood is additionally regulated by cell surface human leukocyte elastase (HLECS) and the HLECS-reactive active α1proteinase inhibitor (α1PI, α1antitrypsin, SerpinA1)3. In HIV-1 disease, α1PI is inactivated due to disease processes 4. In the early asymptomatic categories of HIV-1 disease, active α1PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 μM, and to achieve normal levels during the symptomatic categories4, 5. This pattern has been attributed to immune inactivation, not to insufficient synthesis, proteolytic inactivation, or oxygenation. We observed that in HIV-1 subjects with >220 CD4 cells/μl, CD4 counts were correlated with serum levels of active α1PI (r2=0.93, p<0.0001, n=26) and inactive α1PI (r2=0.91, p<0.0001, n=26) 5. Administration of α1PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting α1PI participates in regulating the number of CD4+ T cells in blood 3.
With stimulation, whole saliva contains sufficient serous exudate (plasma containing proteinaceous material that passes through blood vessel walls into saliva) to allow measurement of active α1PI and the correlation of this measurement is evidence that it is an accurate method for calculating CD4 counts. Briefly, sialogogues such as chewing gum or citric acid stimulate the exudation of serum into whole mouth saliva. After stimulating serum exudation, the activity of serum α1PI in saliva is measured by its capacity to inhibit elastase activity. Porcine pancreatic elastase (PPE) is a readily available inexpensive source of elastase. PPE binds to α1PI forming a one-to-one complex that prevents PPE from cleaving its specific substrates, one of which is the colorimetric peptide, succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA3NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active α1PI in saliva. The resulting inhibition of PPE by active α1PI can be measured by adding the PPE substrate SA3NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/μl), the concentration of α1PI in saliva is related to the concentration of serum in saliva, not to volume of saliva since volume can vary considerably during the day and person to person6. However, virtually all the protein in saliva is due to serum content, and the protein content of saliva is measurable7. Thus, active α1PI in saliva is calculated as a ratio to saliva protein content and is termed the α1PI Index. Results presented herein demonstrate that the α1PI Index provides an accurate and precise physiologic method for calculating CD4 counts.
Protocol
Discussion
原液血清は、中央値36μMを含むα1 PIおよび3.6μMα2マクログロブリン（α2 M）の10倍の差が8。両方のタンパク質は、PPEへの結合について競合します。これら二つの特性、濃度、PPE親和性は、特にM 8α2、競合の存在下で正常な血清中のα1 PIを測定することができる方法を開発するために利用されています。このメソッドは、0.3％の血清中で?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
研究では、ハリーウィンストン研究財団によってサポートされていました。
Materials
| Name of the reagent | Company | Catalogue number |
| Sialogogue, 0.05M citric acid | Eda’s Sugarfree Candies, 4900 Rear N.20th Street, Philadelphia, PA 19144 | Sugar-Free Lemon dropS |
| Porcine pancreatic elastase, type 1 (EC 3.4.21.36) | Sigma-Aldrich, St. Louis, MO 63103 | E1250 |
| Succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide | Sigma-Aldrich | S4760 |
| Coomassie Brilliant Blue R-250 | Sigma-Aldrich | B7920 |
| Tissue culture-treated, 96 well microplates | Becton Dickinson, Franklin Lakes, NJ 07417-1886 | 35-3072 |
| Microplate Reader with filters for 405 nm and 595 nm | MTX Lab Systems, 8456 Tyco Road, Building D, Vienna, Virginia 22182 | Dynex P97277 |
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