There is an urgent need for affordable CD four enumeration to monitor HIV disease in resource limited regions. This video demonstrates a low cost point of care screening test in which CD four counts are determined based on the presence of the proteinase inhibitor Alpha one PI in saliva. To do this, the subject or patient is first given a lemon drop to stimulate exudation of serous fluid into the saliva.
The saliva is collected in a specimen cup and transferred to a 96 well assay plate. Then a saturating amount of the proteinase porcine pancreatic elastase is added to the saliva where it binds and inhibits alpha one pi. Next colormetric elastase substrate is added.
Any remaining unbound elastase cleaves the substrate resulting in a color change from clear to yellow. Kumasi blue is added to the wells to stain total protein and color metric measurements are taken using a microplate reader. Ultimately, the ratio of alpha one PI to total protein is determined and used to accurately assess CD four counts based on an index value.
There are several advantages of this technique over the existing method, which is flow cytometry. The assay can be done in the field without collecting blood, which is traumatizing and expensive without using monoclonal antibodies, which are expensive without technical education and without the need for refrigeration or sample transport. This method overcomes a critical barrier to AIDS intervention and should have a major impact on the cost and health outcomes of patients and the frequency of HIV transmission, all of which have a major impact on the global economy.
This method can provide insight into how Alpha and PI regulates CD four T cell number via adherence and migration, but it can also be applied to other systems such as how other proteins inhibitors regulate the adherence and cell migration of other cell types. We first had the idea for this method when we discovered that Alpha one PI was so low in HIV infected patients, that it actually limits of how many CD four T cells make it into blood. Begin this protocol by preparing the needed working solutions for each microplate that will be used.
Prepare 60 milliliters of 0.05 molar tris, buffered saline or TBS at a pH of 7.8. Also, for each microplate, prepare six milliliters of porcine pancreatic elastase type one or PPE. Shake the PPE suspension well and then dilute it in TBS to a concentration of approximately 0.01 units per milliliter.
Next, prepare a 0.1 molar stock solution of the color metric substrate SA three NA.In DMSO, this stock solution should either be used immediately or stored at minus 20 degrees Celsius if the stock will be used immediately. Prepare six milliliters of a working solution for each microplate by diluting it one in 100 in TBS. Finally, prepare a stock solution of 10%kumasi brilliant blue R two 50 into TBS and either use it immediately or store at minus 20 degrees Celsius if it'll be used immediately.
Prepare six milliliters for each microplate by diluting the stock one in 1000 in TBS. When handling saliva, it is important to take appropriate precautions such as wearing gloves and a lab coat with the research subject or patient in the standing position. Place a SGO in the mouth to stimulate serum exudation.
The role of the HIV patient in this demonstration is being played by a research assistant. A sugar-free lemon drop is convenient for HIV one patients who are frequently edentulous and unable to chew gum swallow as needed for the first three minutes and collect whole mouth saliva into a specimen cup for the next three minutes. This will provide approximately two milliliters of saliva.
If the saliva will not be used immediately aquatic and store it at minus 80 degrees Celsius on the day of processing th the specimen at 37 degrees Celsius. Once thawed, the saliva can be stored at two to four degrees Celsius for up to three days. Each sample and control will be plated in triplicate in a 96 well flat bottom plate to set up the assay at 130 microliters of TBS to each of the sample wells and control wells.
Then avoiding mucin, which is viscous and does not contain serum exudate. Transfer 20 microliters of saliva to each of three test wells and three negative control wells. Then add 20 microliters of TBS to positive control wells mixed by pipetting.
At this step, it is important to avoid vortexing making bubble when you pipetting the saliva and avoiding pipetting the solid particles and musing. Next, shake the PO sign pancreatic elastase or PPE working solution. Well to mix then add 50 microliters to of the sample and positive control wells to the negative control wells.
Add 50 microliters of TBS. It is not necessary to mix since the volume added is large enough to allow even distribution into the saliva mixture. Incubate the plate for 10 minutes of room temperature.
Following the incubation, add 50 microliters of the elastase substrate SA three NA working solution to each. Well then incubate for an additional 45 minutes at 23 degrees Celsius. After 45 minutes of passed, add 50 microliters of Kumasi blue working solution to each.
Well then read the absorbance in a microplate reader at 405 nanometers to detect SA three NA cleavage and 595 nanometers to detect kumasi blue, which represents total protein content. To calculate the alpha one PI index, first, use the following formula to obtain the normalized absorbent for each wavelength by dividing the difference between the specimen average and the negative control average by the difference between the positive control average and the negative control average. Then to get the alpha one PI index, divide the normalized absorbance at 405 nanometers by the normalized absorbance at 595 nanometers as shown here to determine whether the alpha test might be suitable for use as a point of care screening test to monitor CD four counts in endemic resource limited regions.
Stimulated saliva was collected from 20 female and 11 male HIV one subjects attending clinic for routine care in Cameroon, consistent with preliminary observations during development of the alpha test using saliva collected in New York City, the alpha one PI index and saliva collected in Cameroon correlated with CD four counts with an R squared value of 0.91 and a P value of less than 0.0001. The relationship was defined by a three parameter sigmoidal curve, which is consistent with the dose response relationship. The 95%confidence and prediction intervals are depicted with blue and red lines respectively.
In a previous study, we determined that CD four positive T cells exhibit sinusoidal cycling with periodicity 23 plus minus 3.5 days in HIV one subjects and in subjects with the inherited version of alpha one PI deficiency, PIZZ depicted Here is a representative example of computer generated sign curve analysis of CD four cycling in a non H HIV healthy control exhibiting 27 day periodicity computer generated sign curve analysis of the CD four positive T-cell. Changes in six subjects yielded values for peak-to-peak amplitude and axis of oscillation as would be expected. The axis of oscillation was correlated with amplitude with an R-squared value of 0.96 and a p value of 0.009.
In patients with low CD four positive T-cell counts, the cyclic changes in CD four T-cell count were small and in patients with high CD four positive T-cell counts. Cyclic changes were large because the regression line depicted here estimates the axis of oscillation and the axis of oscillation is correlated with amplitude. The amplitude can be calculated for the regression line to estimate how far above and below the regression line CD four positive T cells would be expected to vary due to cycling overlaying the amplitude calculated and the confidence interval, it was found that amplitude indicated by the green line lies within the 95%confidence interval indicated by the blue line.
Once master, this technique can be done in 90 minutes from the time of saliva collection to getting CD four cell count number to the patient. This procedure is intended to be used as a screening test to monitor CD four counts. If a patient shows major changes in CD four counts, then that patient should be followed up using flow cytometry to make sure that the measurements of CD four counts are valid and to assist in treatment decisions.
In the next stage of development, the Microplate assay will be developed into dipstick technology, which will allow for tests to be performed without an instrument or electricity. It is unlikely for HIV to be transmitted from saliva, but HIV patients often have other infectious diseases such as tuberculosis and hepatitis C, so don't forget that working with saliva like working with blood can be extremely hazardous due to unknown infectious particles. Precautions should always be taken such as wearing gloves and other personal protective equipment while performing this procedure.
