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

Summary

Une méthode de numération des CD4, l'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'une formation technique, des réactifs coûteux tels que les anticorps monoclonaux, l'instrumentation, de réfrigération, le transport des échantillons, ainsi que de collecte et de traitement du sang.

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 (HLE_CS) and the HLE_CS-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 categories^{4, 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

1. Préparer une solution fraîche Solutions de travail SCT: 0,05 M Tris Buffered Saline, pH 7,8 (TBS). Préparer 60 ml TBS / microplaque. EPI: l'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). SA 3 NA: succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA 3 NA). Préparer 0,1 M…

Discussion

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'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 …

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