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 Î±-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva at JoVE.com

La recherche a été soutenue par la Fondation de recherche Harry Winston.

1. Préparer une solution fraîche Solutions de travail <ol><li> SCT: 0,05 M Tris Buffered Saline, pH 7,8 (TBS). Préparer 60 ml TBS / microplaque. </li><li> EPI: l&#39;élastase pancréatique porcine, de type 1 (PPE) est fourni sous forme de suspension. Bien agiter et diluer EPI SCT à environ 0,01 U / ml. Préparer 6 ml de solution de travail / microplaque). </li><li> SA 3 NA: succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA 3 NA). Préparer 0,1 M...

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Sérum non dilué contient médiane 36 uM α 1 PI et 3,6 uM α 2-macroglobuline (α 2 M) une différence de 10 fois 8. Les deux protéines compétition pour la liaison aux EPI. Ces deux caractéristiques, la concentration et l&#39;affinité EPI, ont été exploitées pour développer une méthode qui est capable de mesurer précisément α 1 PI dans le sérum normal en présence de concurrence α 2 M 8. Cette méthode permet de détecter aussi ...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Sialogogue, 0,05 M d&#39;acide citrique </td><td> Bonbons sans sucre eda, 4900, rue arrière N.20th, Philadelphia, PA 19144 </td><td> Gouttes de citron sans sucre </td></tr><tr><td> Élastase pancréatique porcine, de type 1 (CE 3.4.21.36) </td><td> Sigma-Aldrich, St. Louis, MO 63103 </td><td> E1250 </td></tr><tr><td> Succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide </td><td> Sigma-Aldrich </td><td> S4760 </td></tr><tr><td> Bleu de Coomassie R-250 </td><td> Sigma-Aldrich </td><td> B7920 </td></tr><tr><td> La culture de tissus traités, 96 microplaques ainsi </td><td> Becton Dickinson, Franklin Lakes, NJ 07417-1886 </td><td> 35-3072 </td></tr><tr><td> Lecteur de microplaques avec des filtres pour 405 nm et 595 nm </td><td> MTX Systems Lab, 8456 Tyco Road, Building D, Vienna, Virginia 22182 </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 Î±-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva at JoVE.com

The &#945;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

Une méthode de numération des CD4, l&#39;α-test, est décrite qui utilise la salive ensemble, de fournir rapides et précises des taux de CD4. Les sous-α essai coûts et élimine la nécessité d&#39;une formation technique, des réactifs coûteux tels que les anticorps monoclonaux, l&#39;instrumentation, de réfrigération, le transport des échantillons, ainsi que de collecte et de traitement du sang.

Le α-test: Rapid portable sans énumération des CD4 utilisant la salive entier

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 ...

Research

JoVE Journal

Medicine

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

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

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Mesure précise et simple de la cytokine pro-inflammatoire IL-1β en utilisant un dosage de sang total Stimulation

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