An in vitro diagnostic technique, Surround optical-fiber immunoassay (SOFIA) is discussed in this article. An optical fiber assembly surrounding a platform is the major component of this system. This part captures any fluorescence emissions emitted from an entire sample. The major advantage of this technology is its high limit of detection, sensitivity, and dynamic range. The sensitivity can be measured in attogram level (10−18 g), thus it behaves as an ultrasensitive method and can be used for very low-level detection. The dynamic range of the SOFIA aids in discriminating levels of analyte in a sample over 10 orders of magnitude. This aids in accurate titering of the sample.
The above features of the SOFIA lead to its application in a broad range of diagnostics. Many numbers of studies using SOFIA was able to detect naturally occurring prions either in the blood or urine of the disease carriers. Due to its reliability, SOFIA is the go-to method for the antemortem screening test. This test can be used to detect a range of diseases like vCJD, BSE, scrapie, CWD, and other transmissible spongiform encephalopathies. SOFIA is also used in the in vitro diagnostic test for neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease.
A brief introduction of the prion disease and its existing methods of detection and also the application of SOFIA is also discussed here. Prion diseases or known as transmissible spongiform encephalopathies (TSEs) can be classified as fatal neurodegenerative disorders. This list of diseases includes bovine spongiform encephalopathy (BSE) in bovine, scrapie in sheep, chronic wasting disease (CWD) of cervids and Creutzfeldt–Jakob disease (CJD) in humans. The characteristic of these diseases is its ability to conformationally alter cellular prion protein (PrPC) into an altered form (PrPSc). The altered form is distinguished by a different altered form (PrPSc) from the original form (PrPC). The distinguishable characteristics between the two forms include aggregation, insolubility, protease digestion resistance, and a b-sheet-rich secondary structure.
The difference in protease digestion resistance is the basis of several diagnostic biochemical tests for this disease. These techniques include the pretreatment of samples with proteinase K (PK). As PrPSc is a partially digestion-resistant while PrPC is easily digested by PK, this pretreatment step results in samples rich in PrPSc then PrPC. But this test becomes not reliable after the identification PK-sensitive forms of PrPSc and also contributing to the factor that some studies suggested the highest proportion of PrPSc is sPrPSc, in brains of patients who had died of CJD.
The current set of prion detection methods include gel electrophoresis and Western immunoblot, direct-binding and sandwich ELISA, conformational-dependent immunoassay (CDI), and protein amplification. These tests are recommended in postmortem biochemical and immunological analysis only after the suspicious animals manifest one or more symptoms of the disease. But to ensure food safety these screening tests are used to all susceptible animals regardless of the presence of the symptoms. The basis of these presymptomatic animals will be the ability to differentiate PrPC from PrPSc. Some of the approaches using this test using ligands against PrPSc, spectroscopic techniques, and PrPSc amplification. The limitation of this method is the time required for maximal sensitivity and the need for PrPC as the substrate. The latest technique is developed a rapid and less sensitive seeded polymerization technique was also developed. This technique utilizes recombinant hamster PrP and detection of approximately 50 ag of PrPSc within several days.
Unlike other techniques, SOFIA has the added advantages of detection period of 24h, no use of seeded polymerization or amplification. It prevents cross-contamination. It is based on immunocapture assay inflorescence detection scheme. The detection scheme is based on laser induction and captured by laser induction.
Materials and methods
Mouse-adapted scrapie strain ME7 was injected (0.1 ml of a 10% brain homogenate) intraperitoneally (i.p.) into 7-week-old CD-1 mice. The ME7 inoculant has a titer of about 108 50% infectious doses/ml. Sham-infected, age- and sex-matched CD1 mice, normal CD1 mice, and PrPC–/— mice were used as controls. Blood was collected from individual mice by cardiac puncture with a 30-gauge needle. Plasma samples were collected after centrifugation and stored in aliquots at -80°C. All animal experiments were carried out according to institutional regulations and standards. Brains from sheep infected with scrapie and white-tailed deer infected with CWD were harvested at the time of clinical disease and frozen at−80◦C. Brains from uninfected animals were similarly harvested and frozen.
Expression and purification of recombinant protein
The coding region of the full-length deer, hamster, mouse, and sheep PrP was cloned into a pET-23 vector to produce a tag-free protein. Expression and purification were done using standard procedures.
For the preparation of 10% brain homogenates, brain tissues were prepared and consisted of homogenization in 10 volumes of ice-cold lysis buffer (10mM Tris–HCl, 150mM NaCl, 1% Igepal CA-630 (Nonidet P-40), 0.5% deoxycholate, 5mM EDTA, pH 8.0) in the presence of 1mM phenylmethylsulfonyl fluoride (PMSF) (if the homogenate was to be treated with PK, PMSF was omitted from the lysis buffer). After centrifugation at 1000×g for 10min, the supernatants were stored as individual aliquots at −80◦C.
For the capture ELISA assay, 96-well plates were coated with affinity-purified 11F12 capture monoclonal antibody (Mab) (5mg/ml) at room temperature for 2–3 h. The coated wells were blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) overnight at 4 ◦C followed by washing three times with PBS containing 0.2% Tween- 20 (PBST). The antigen was either non-PK or PK (100 mg/ml PK at 50 ◦C for 30 min) treated brain lysates to which was added a final concentration of 1% PMSF. All samples were treated with a final concentration of 1% SDS, heated at 100◦C for 10 min and centrifuged at 16,000×g for 5 min. The supernatants were serially diluted 10-fold and 100 ml was added to each well. The plates were incubated at 37◦C for 1 h. The wells were washed three times with PBST and 100 ml of the biotinylated 5D6 detector Mab (5 mg/ml) was added. After 60min the wells were washed with PBST and 100_l streptavidin conjugated to alkaline phosphatase (1:5000) was added for 60 min at 37◦C. PNPP (4-nitrophenyl phosphate disodium salt hexahydrate) substrate solution was added to each well (100 ml) and after 60min product was measured with an ELISA reader at OD405.
For laser analysis (SOFIA), incubation with the biotinylated Mab 5D6 was followed by the addition of streptavidin conjugated to Rhodamine Red X (1:1000). Following a 60min incubation at 37◦C, the wells were washed with PBST and treated with 100 ml 1N NaOH for 10 min at 100◦C and then shaken at room temperature for 5min. A 90 ml sample was placed into a 100 ml Microcap micro-capillary tube which was then inserted into a specifically designed tube sample holder for laser excitation and emission detection. Dilutions were calculated relative to the original starting brain tissue. Each value (data point) represents the mean ± standard deviation (S.D.) from multiple assays.
Analysis of various elution conditions (0.1Mglycine–HCl, pH 2.0 or 0.25N–16N NaOH for 5–60 min) indicated that incubation for 10 min with 1N NaOH was optimal for extraction of the Rhodamine Red from the antibody-antigen complex.
Ten percent brain homogenates were prepared in lysis buffer. The samples were centrifuged at low speed (2000×g for 10min). Ten microliters of the supernatants were mixed with a final of 1× sample buffer (6mM Tris, 1% SDS, 1% 2-mercaptoethanol, 5% glycerol, and 0.01% bromophenol blue, pH 6.8), heated at 100 ◦C for 4min and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (constant voltage of 100V) using 12% acrylamide gels. The resolved proteins were electroblotted to nitrocellulose membrane (40V for 18 h) and the membrane was incubated in blocking buffer (3% BSA in PBS) for 1 h at room temperature. The blot was washed in PBST and incubated with Mab 8E9 (1:5000 in PBST) for 1 h at 37◦C. Following PBST washes, the blot was immunostained with secondary antibody (1:2000 in PBST) [either goat anti-mouse IgG (Fab fragments) conjugated to alkaline phosphatase with NBT and BCIP as the substrate or horseradish peroxidase-conjugated to goat anti-mouse IgG with super signal west femto maximum sensitivity substrate (Pierce) for 1 h at room temperature. For samples that were PK-digested prior to SDS-PAGE, 40ml of the supernatants from the low-speed centrifugation was incubated with 100 mg/ml PK (final concentration) for 30min at 50◦C followed by the addition of 1% PMSF, 1× SDS-PAGE sample buffer, and heating at 100◦C for 5min.
The setup is designed around a commonly used disposable 100 ml micro-capillary as a sample holder. The sample is excited by focusing temporally modulated light from a solid state, frequency-doubled Nd:YAG laser along the axis of the capillary, with a typical power of 30mW continuous wave at a wavelength of 532 nm, which matches well with the absorption peak of Rhodamine. A fiber optic assembly was designed comprised of four linear arrays which span approximately a third of the length of the capillary and are positioned at 90◦ with respect to each other around the perimeter of the capillary. Because of the large numerical aperture (0.22, or an acceptance angle of ~23◦) of the fibers, this orientation of the fibers results in complete coverage of the samples optical radiation pattern. The light collected by the four linear arrays is ganged and focused into transfer optics in which a holographic the notch filter, and band pass filters are mounted. These are used to eliminate the scattered light from the excitation source, and band-limit the detection of the fluorescence of the reporter dye, respectively. The light is then focused back into a single, multi-mode, 400-micron optical fiber and coupled to a single low noise photo-voltaic diode detector which is mounted on a BNC connector directly on the preamplifier of the detection electronics. Detection of the signal employs a phase sensitive, or “lock-in”, detection scheme. The excitation source is modulated with an optical chopper that serves to generate the reference frequency for the detection system. The diode detector is mounted on the input of the transconductance preamplifier to reduce the total line impedance and eliminate difficulties in impedance matching of the signal at these low levels. The signal is then detected with a lock-in amplifier and data acquisition is performed through a LabView program. The program consists of an electronic strip chart which poles the lock-in amplifier for its reading in voltage, periodically displays the time history of the measurements to the operator, and stores the values with a time stamp in an ASCII file. The time constant of the lock-in amplifier should be chosen to provide a bandwidth of a few tenths of a Hertz (Hz). For our measurements, we chose a time constant of 3 s. The lock-in requires several time constants in duration to obtain a stable reading (3–30 s in our case). The values for the measurements were taken once the signal had stabilized (20–30 s) after loading a new sample. The modulation of the excitation source, and reference frequency for the lock-in detector was at a frequency of 753 Hz that was chosen to minimize environmental noise. In addition to this, filtering of the signal at line frequency and two times line frequency was performed by the lock-in amplifier. The preamplifier signal was band-pass filtered at the modulation frequency. For our samples the preamplifier sensitivity of 1 or 10 nA/V were chosen, giving an input impedance of 1MW or 10 kW, respectively. In performing the measurements we maintained a set of startup procedures which included: warm-up period of at least 15min for all electronics (the laser, lock-in amplifier, and preamplifier), assessment of dark signal levels to assure that system was properly electrically grounded, inspection of laser alignment and measurement of laser power to check for stability and output level. Measurements of baseline signal consistency were confirmed using distilled, deionized water.
Advantages of SOFIA
- The use of several PrP-specific Mabs that have a synergistic effect when used together in a capture ELISA is generally used in SOFIA.
- The hardware part of SOFIA is optimized for the total collection of light from the reporter molecule.
- The quantum efficiencies of the dyes currently used in fluorescence-based assays are near or above 90%. Rhodamine Red is used in this study because of its stability, high quantum efficiency and match to the excitation source.
- Capturing as much signal as available from a given sample is an important parameter to be considered in this technique. Thus settings for both the transconductance preamplifier and the lock-in detector becomes critical. The settings should facilitate low signal/low noise detection.
- Modulation frequency for the optical chopper is chosen considering the frequency should be incommensurate with the line frequency or other electrical sources of noise in the environment.
- Line filtering by the lock-in amplifier should be employed to increase this detection efficiency.
- Expected signal level and maximizing the preamplifier’s input impedance were the factors to be considered while choosing the sensitivity of the transconductance preamplifier
- Band-pass filter centered on the chopper frequency allows filtering of the input signal.
- Also, the sensitivity of the detection is increased with a low noise photo-voltaic detector and transconductance preamplifier.
- Noise reduction is achieved in the system by using phase sensitive detection and lock-in detection scheme. Band-limiting the noise, aids in increasing the signal to noise ratio of the detector.
- Noise reduction is calculated proportional to the square root of the ratio of the bandwidth of the detector to the bandwidth of the amplifier.
- Since SOFIA enables dilution of non-PK treated samples beyond the detection limits of PrPC false-positive results is less of an issue.
- Even at very low concentrations, fluorescent probes can be detected this property makes this to be more sensitive than the Western blot and ELISA test.
- Few modified spectroscopic methods also can be used for the detection in this purpose. The methods include fluorescence correlation spectroscopy, multispectral ultraviolet fluorescence spectroscopy, and Fourier transform infrared spectroscopy.
- The current version of SOFIA is a combination of several technologies with PrPSc detection sensitivities thus enabling a detection at a range of approximately 10 ag of PrPSc can be detected from 263K-infected hamster brains at a clinical disease. Approximately 200 PrP molecules or less than 10−4 infectious units can be detected using this method.
- SOFIA is rapid, no amplification of sample is required, and can be applied to a wide spectrum of diseases.