The α-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

Summary

A CD4 enumeration method, the α-test, is described which uses whole saliva to provide rapid and accurate CD4 counts. The α-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.

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. Prepare Fresh Working Solutions

1. TBS: 0.05 M Tris Buffered Saline, pH 7.8 (TBS). Prepare 60 ml TBS/microplate.
2. 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).
3. SA³NA: Succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA³NA). Prepare 0.1 M stock SA³NA solution in dimethyl sulfoxide (DMSO) and use immediately or store at -20 °C. Prepare 6ml working solution/microplate by diluting stock SA³NA solution 1/100 in TBS.
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Discussion

Undiluted serum contains median 36 μM α₁PI and 3.6 μM α₂macroglobulin (α₂M) 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 α₁PI in normal serum in the presence of competing α₂M ⁸. This method can detect as little as 50 nM μ₁PI in 0.3% ser.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 405nm and 595nm	MTX Lab Systems, 8456 Tyco Road, Building D, Vienna, Virginia 22182	Dynex P97277