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

Research was supported by the Harry Winston Research Foundation.

1. Prepare Fresh Working Solutions
<ol>
	<li>TBS: 0.05 M Tris Buffered Saline, pH 7.8 (TBS). Prepare 60 ml TBS/microplate.</li>
	<li>PPE: Porcine pancreatic elastase, type 1 (PPE) is provided as a suspension. Shake well and dilute PPE in TBS to approximately 0.01 U/ml. Prepare 6 ml working solution/microplate).</li>
	<li>SA3NA: Succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA3NA). Prepare 0.1 M stock SA3NA solution in dimethyl sulfoxide (D...

<ol>
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L., Patel, H., Arnold, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self+antigen+prognostic+for+human+immunodeficiency+virus+disease+progression.">Self antigen prognostic for human immunodeficiency virus disease progression.</a> Clin. Diagn. Lab. Immunol. 8, 937-942 (2001).</li><li>Bristow, C. L., Babayeva, M. A., LaBrunda, M., Mullen, M. P., Winston, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;1Proteinase+Inhibitor+regulates+CD4++lymphocyte+levels+and+is+rate+limiting+in+HIV-1+disease.">&#945;1Proteinase Inhibitor regulates CD4+ lymphocyte levels and is rate limiting in HIV-1 disease.</a> PLoS One. , (2011).</li><li>de Almeida, P. D. V., Gregio, A. M. T., Machado, M. A. N., de Lima, A. A. S., Azevedo, L. 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Oral Biol. 40, 1151-1155 (1995).</li><li>Shak, S., Capon, D. J., Hellmiss, R., Marsters, S. A., Baker, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+human+DNase+I+reduces+the+viscosity+of+cystic+fibrosis+sputum.">Recombinant human DNase I reduces the viscosity of cystic fibrosis sputum.</a> PNAS. 87, 9188-9192 (1990).</li><li>LeVeque, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multicenter,+randomized,+double-blind,+placebo-controlled,+dose-+titration+study+of+oral+pilocarpine+for+treatment+of+radiation-induced+xerostomia+in+head+and+neck+cancer+patients.">A multicenter, randomized, double-blind, placebo-controlled, dose- titration study of oral pilocarpine for treatment of radiation-induced xerostomia in head and neck cancer patients.</a> J. Clin. 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Undiluted serum contains median 36 &mu;M &alpha;1PI and 3.6 &mu;M &alpha;2macroglobulin (&alpha;2M) a 10-fold difference 8. Both proteins compete for binding to PPE. These two characteristics, concentration and PPE affinity, have been exploited to develop a method that is capable of specifically measuring &alpha;1PI in normal serum in the presence of competing &alpha;2M 8. This method can detect as little as 50 nM &mu;1PI in 0.3% ser...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Sialogogue, 0.05M citric acid</td>
 <td>Eda's Sugarfree Candies, 4900 Rear N.20th Street, Philadelphia, PA 19144</td>
 <td>Sugar-Free Lemon dropS </td>
 </tr>
 <tr>
 <td>Porcine pancreatic elastase, type 1 (EC 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>Coomassie Brilliant Blue R-250</td>
 <td>Sigma-Aldrich</td>
 <td>B7920</td>
 </tr>
 <tr>
 <td>Tissue culture-treated, 96 well microplates</td>
 <td>Becton Dickinson, Franklin Lakes, NJ 07417-1886</td>
 <td>35-3072</td>
 </tr>
 <tr>
 <td>Microplate Reader with filters for 405 nm and 595 nm</td>
 <td>MTX Lab Systems, 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 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

A CD4 enumeration method, the &#x03B1;-test, is described which uses whole saliva to provide rapid and accurate CD4 counts. The &alpha;-test costs pennies and eliminates the need for technical training, costly reagents such as monoclonal antibodies, instrumentation, refrigeration, transport of samples, as well as collection and handling of blood.

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 ...

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