Name: the &alpha;-test: rapid cell-free cd4 enumeration using whole saliva
Uploaded: 2012-05-16T13:00:00
Description: Watch this Scientific Journal Video about The ÃŽÂ±-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva at JoVE.com

अनुसंधान हैरी विंस्टन रिसर्च फाउंडेशन द्वारा समर्थित किया गया.

1. ताजा कार्य समाधान तैयार <ol><li> टीबीएस: 0.05 एम Tris खारा पीएच 7.8 (टीबीएस) के बफर. 60 मिलीलीटर टीबीएस / microplate तैयार. </li><li> पीपीई: सुअर अग्नाशय इलास्टेज, टाइप 1 (पीपीई) के निलंबन के रूप में प्रदान की जाती है. अच्...

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Undiluted सीरम मंझला 36 माइक्रोन α 1 PI और 3.6 α 2 (α 2 एम) macroglobulin माइक्रोन एक 10 गुना 8 अंतर. दोनों प्रोटीन पीपीई के लिए बाध्य करने के लिए प्रतिस्पर्धा. इन दो विशेषताओं, एकाग्रता और पीपीई आत्मीयता, के लिए एक ?...

<table border="1"><tbody><tr><td> अभिकर्मक का नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td></tr><tr><td> Sialogogue, 0.05M साइट्रिक एसिड </td><td> Eda Sugarfree कैंडीज, 4900 रियर N.20th, स्ट्रीट, फिलाडेल्फिया, फिलीस्तीनी अथॉरिटी 19,144 </td><td> चीनी, नींबू बूँदें </td></tr><tr><td> सुअर का अग्नाशय इलास्टेज, टाइप 1 (ईसी 3.4.21.36) </td><td> सिग्मा Aldrich, सेंट लुइस, MO 63,103 </td><td> E1250 </td></tr><tr><td> Succinyl - एल - आला - एल आला - एल आला - पी nitroanilide </td><td> सिग्मा Aldrich </td><td> S4760 </td></tr><tr><td> Coomassie खूब R-250 ब्लू </td><td> सिग्मा Aldrich </td><td> B7920 </td></tr><tr><td> टिशू कल्चर का इलाज, 96 अच्छी तरह से microplates </td><td> Becton डिकिंसन, फ्रेंकलिन झील, एनजे 07417-1886 </td><td> 35-3072 </td></tr><tr><td> 405 एनएम और 595 एनएम के लिए फिल्टर के साथ microplate रीडर </td><td> MTX लैब सिस्टम, 8456 टाइको रोड, Building डी, वियना, वर्जीनिया 22,182 </td><td> Dynex P97277 </td></tr></tbody></table>

the &alpha;-test: rapid cell-free cd4 enumeration using whole saliva

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 &alpha;1proteinase inhibitor (&alpha;1PI, &alpha;1antitrypsin, SerpinA1)3. In HIV-1 disease, &alpha;1PI is inactivated due to disease processes 4. In the early asymptomatic categories of HIV-1 disease, active &alpha;1PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 &mu;M, 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 &gt;220 CD4 cells/&mu;l, CD4 counts were correlated with serum levels of active &alpha;1PI (r2=0.93, p&lt;0.0001, n=26) and inactive &alpha;1PI (r2=0.91, p&lt;0.0001, n=26) 5. Administration of &alpha;1PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting &alpha;1PI 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 &alpha;1PI 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 &alpha;1PI 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 &alpha;1PI 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 &alpha;1PI in saliva. The resulting inhibition of PPE by active &alpha;1PI can be measured by adding the PPE substrate SA3NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/&mu;l), the concentration of &alpha;1PI 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 &alpha;1PI in saliva is calculated as a ratio to saliva protein content and is termed the &alpha;1PI Index. Results presented herein demonstrate that the &alpha;1PI Index provides an accurate and precise physiologic method for calculating CD4 counts.

Watch this Scientific Journal Video about The ÃŽÂ±-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva at JoVE.com

The ÃƒÅ½Ã‚Â±-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

एक सीडी 4 गणना विधि, α परीक्षण, वर्णित है जो पूरे लार का उपयोग करने के लिए तेजी से और सही CD4 गिनती प्रदान. α परीक्षण लागत पैसे और मोनोक्लोनल एंटीबॉडी, उपकरण, प्रशीतन, नमूनों के परिवहन, के रूप में के रूप में अच्छी तरह से संग्रह और रक्त की हैंडलिंग के रूप में तकनीकी प्रशिक्षण, महंगा अभिकर्मकों के लिए की आवश्यकता eliminates.

α परीक्षण: रैपिड सेल मुक्त सीडी 4 पूरे लार का प्रयोग गणन

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 ...

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The &alpha;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

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