PMC:7120874 / 1825-30099 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7120874","sourcedb":"PMC","sourceid":"7120874","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7120874","text":"Introduction\nA new strain of coronavirus (CoV) caused a pandemic outbreak of severe acute respiratory syndrome (SARS) in China, Hong Kong, Singapore, Toronto, and Taiwan from 2002 to 2003, resulting in 8098 individuals being infected and 774 deaths (1). As compared to other upper viral respiratory infections (2), SARS-CoV can rapidly induce pneumonia. The accompanying adult respiratory distress syndrome can quickly progress resulting in rapid death of the patients (3,4). The most commonly used laboratory diagnostic tests for SARS-CoV-infection are reverse transcription (RT)-PCR and anti-SARS-CoV antibody serological tests (5–7), but their roles in monitoring the extent of pneumonia are rather limited. Although, serial chest radiography is helpful for monitoring the progress of disease (8,9), it is also limited by its subjectivity and variability in the interpretation of the imaging results and occasionally suboptimal quality of portable films taken in isolation ward to avoid the spread of the disease. In a recent article, the discovery of 12 up-or downregulated serum biomarkers were reported in SARS patients by a novel protein chip array profiling approach (10–14) with one biomarker appears to be useful in monitoring the extent of pneumonia (15). In this chapter, how this protein chip array profiling technique is carried out is described. The technique involves four steps with the first step being serum fractionation in ceramic ion exchange sorbent beads. This step separates serum proteins into several fractions aiming at reducing their complexity before protein chip profiling is performed. The second step involves the binding of serum protein biomarkers onto protein chip array surfaces. The third step is desorption of bound serum biomarkers by laser shots in the mass spectrometer generating a time of flight spectrum for each sample. The fourth step concerns with data acquisition, data processing and statistical analyses of the spectra in SARS patients in comparison with the control groups. These procedures will be described in full details in the following chapter and summarized in the flow chart diagram as in Fig. 1.\nFig. 1. Sample fractionation, chip binding, and reading in protein chip array profiling technique. Serum samples from SARS patients and various control groups were fractionated in Q Ceramic Hyper D F anion exchange sorbent beads. The eluted fractions were in turn spotted onto two types of protein chips, namely, copper IMAC30 Cu(II) ProteinChip array and weak cation-exchange (CM10) ProteinChip array. Each sample bound protein chip was in turn inserted into a Protein Biology System II SELDI-TOF-mass spectrometer and the protein biomarkers were ionized and desorbed by laser beam. The ions flied to the ion detector generating a biomarker peak intensity spectrum according to their mass over charge values (m/z).\n\nMaterials\n\nSARS Patients\n28 Patients with confirmed SARS-CoV infection managed in the Department of Medicine, Queen Elizabeth Hospital, Hong Kong.\n87% of the patients have abnormal chest radiographs on admission.\n13% of the patients present early thus requiring high-resolution thoracic computer tomography scanning to confirm the presence of pulmonary involvement.\n\nSerum Samples\n44 Serum samples from 24 SARS patients for differential mapping of SARS associated biomarkers.\n45 Serial serum samples from four additional SARS patients with comprehensive clinical follow-up for longitudinal correlation with clinical manifestation.\n10 Sera from 10 apparently healthy individuals (from a familial cancer screening clinic) as normal controls.\n72 Sera from 51 patients suffering from other viral or bacterial infections (influenza A virus [n = 12], influenza B virus [n = 8], respiratory adenovirus [n = 12], respiratory syncytial virus [n = 10], hepatitis B virus [n = 10], Mycobacterium tuberculosis [n = 10], other bacteria [n = 10]) as control patients for comparison with SARS patients.\n\nIon Exchange Fractionation of Serum\nBioSepra Q Ceramic Hyper D F ion exchange sorbent beads (Ciphergen Biosystems Incorporation, Fremont, CA).\n96-Well sample plates (Nalge Nunc International, Rochester, NY).\n96-Well Silent Screen filter plates with Loprodyne membrane filter (0.45 µm pore size; Nalge Nunc International).\n96-Well collection plates (Nalge Nunc International).\n50 mM Tris-HCl buffer at pH 9.0 (W1 ion exchange bead washing buffer).\n50 mM Tris-HCl buffer containing 9 mol/L urea and 20 g/L CHAPS (3,3-cholmido-propyl-dimethylammonio-1-propanesulfonate) (D1 sample denaturing buffer) at pH 9.0.\n50 mM Tris-HCl buffer containing 1 mol/L urea and 2.2 g/L CHAPS at pH 9.0 (Q1 bead equilibration buffer).\n50 mM Tris-HCl buffer containing 1 g/L N-octyl-β-D-glucopyranoside (OGP) at pH 9.0 (E1 elution buffer).\n100 mM Sodium phosphate containing 1 g/L OGP at pH 7.0 (E2 elution buffer).\n100 mM Sodium acetate containing 1 g/L OGP at pH 5.0 (E3 elution buffer).\n100 mM Sodium acetate containing 1 g/L OGP at pH 4.0 (E4 elution buffer).\n50 mM Sodium citrate containing 1 g/L OGP at pH 3.0 (E5 elution buffer).\n33.3% Isopropanol, 16.7% acetonitrile, and 0.1% trifluoroacetic acid (E6 elution buffer).\nMicromix 5-01 shaker (Euro/DPC Ltd., Gwynedd, UK).\n\nChip Pretreatment, Sample Binding, and Analysis\nCopper (II) immobilized metal affinity capture [IMAC30 Cu(II)] ProteinChip® Array (Ciphergen Biosystems Inc., Fremont, CA).\nWeak cation-exchange (CM10) ProteinChip Array (Ciphergen Biosystems, Inc.).\n96-Well ProteinChip bioprocessor (Ciphergen Biosystems, Inc.).\n100 mM Copper sulfate (CuSO4; Sigma-Adrich, St. Louis, MO).\n100 mM Sodium phosphate containing 0.5 mol/L NaCl (IMAC30 chip binding buffer).\n100 mM Sodium acetate buffer pH 4.0 (serves as both IMAC30 chip neutralizing buffer and CM10 chip binding buffer).\n50% Sinapinic acid (Ciphergen Biosystems Inc.) in 500 mL/L acetonitrile and 5 mL/L trifluoroacetic acid (Sigma-Adrich).\nProtein Biological System (PBS) IIc mass spectrometer reader (Ciphergen Biosystems).\nAll-in-1 peptide molecular mass standard (Ciphergen Biosystems Inc.).\nCiphergen ProteinChip Software 3.0.2 (Ciphergen Biosystems Inc.).\n\nMethods\n\nTreatment of SARS Patients\nPatients with fever and other symptoms of respiratory infection are initially managed with broad-spectrum antibiotics and supportive therapy.\nAfter the diagnosis of clinical SARS is made and if there is no response to antibiotic therapy, combination therapy with ribavirin and systemic steroids is initiated.\nIntravenous pulse methylprednisolone is initiated when the clinical condition, radiological presentation, or oxygen saturation status of the SARS patients further deteriorates.\nThe clinical characteristics, detailed management plan, and treatment regimen of this cohort of patients can be referred to a previous publication from our hospital (3).\n\nSerial Chest Radiographic Score\nThe extent of pneumonia in the SARS patients with longitudinal follow-up is assessed by a radiologist according to a serial chest radiographic score, modified from a score system initially proposed to assess computer tomography of the chest (16) and summarized as in Fig. 2.\nFig. 2. Assessing the extent of pneumonia by chest X-ray opacity score. The opacity in each zone (apex+upper, middle, and lower zones) from the left and right regions of the lung was scored by a “coarse semiquantitative method” with a 5 points’ scale of grades 0–4 representing involved areas of 0, 5–24, 25–49, 50–74, and 75–100%, respectively. A total score was then calculated by adding up all the grades in the six zones (Apex and upper zones were considered as one zone) to provide a 0–24 points’ scale with the higher number representing more severe pneumonic disease.\nAfter X-ray chest radiography is taken, divide the frontal chest X-ray radiograph into six lung zones, namely, left upper zone, left middle zone, left lower zone, right upper zone, right middle zone, and right lower zone.\nThe upper zone (left or right) represents area above carina (including the apex).\nThe middle zone (left or right) represents area from carina to the level of inferior pulmonary veins.\nThe lower zone (left or right) represents area from the lower margin of middle zone to the lung base.\nScore the opacity in each lung zone by a “coarse semiquantitative method” with a five points’ scale of grades 0–4.\nGrade 0 represents no opacity involved area.\nGrade 1 represents 5–24% opacity involved areas.\nGrade 2 represents 25–49% opacity involved areas.\nGrade 3 represents 50–74% opacity involved areas.\nGrade 4 represents 75–100% opacity involved areas.\nAdd the grading from each of the six lung zones to provide a 0–24 point summation scale for assessing the extent of pneumonia.\n\nSerum Preparation\n8 mL Blood is collected from each patient or control subject.\nIncubate the blood for approx 2 h at 4°C to clot the blood cells.\nSeparate the serum portion from the clotted cells by centrifugation at 230g for 15 min.\nAliquot the sera for various routine laboratory diagnostic tests.\nFreeze the remaining sera at −70°C for protein chip array profiling analysis.\n\nIon Exchange Fractionation of Serum Proteins\nOwing to the protein complexity in the serum sample, an initial fractionation in Q Ceramic Hyper D F ion exchange sorbent beads is performed to generate serum fractions for chip binding experiments (Fig. 1) (10,11).\n\nWashing of Ion Exchange Beads\nWash Q Ceramic Hyper D Fanion exchange sorbent beads three times each with five bed volumes of W1 bead washing buffer.\nDrain the washing buffer each time in vacuum after washing.\nKeep beads in 50% suspension in W1 buffer at RT.\n\nDenaturation of Serum Proteins\nThaw serum from −70°C freezer immediately before serum fractionation.\nAliquot 20 µL of serum to each well in a 96-well sample plate.\nAdd 30 µL of D1 denaturing buffer to the well containing the serum to denature the serum proteins.\nShake the sample plate on a Micromix 5-01 shaker vigorously (in an amplitude of 7 and form of 20 Hg) for 20 min at 4°C to mix the serum with the D1 buffer well.\n\nEqualibration of Ion Exchange Beads and Binding of Serum Samples\nAdd 180 µL of 50% suspension of ion exchange beads to each well of a 96-well Silent Screen filter plate and drain off the W1 bead-washing buffer by vacuum.\nWash the ion exchange beads in each well three more times by adding 200 µL of Q1 bead equilibration buffer to the beads and draining off the buffer by vacuum.\nAfter the last vacuum drain, transfer 50 µL of the denatured serum sample from the well in the sample plate to the corresponding well in the filter plate containing the equilibrated ion exchange beads.\nAdd 50 µL of Q1 buffer to the well in the sample plate where serum was originally placed and rinse the residual serum by pipetting up and down five times.\nTransfer the 50 µL of the serum rinse to the well in the filter plate containing the mixture of denatured serum sample and ion exchange beads.\nShake the filter plate vigorously on the Micromix shaker (in an amplitude of 7 and form of 20 Hg) for 30 min at 4°C to mix the serum and beads well.\n\nElution of Serum Fractions\nAfter shaking, place a 96-well F1 collection plate under the filter plate and centrifuge at 1600g for 1 min to collect the flow-through fraction in the wells of the F1 collection plate.\nAdd 100 µL of E1 elution buffer (at pH 9.0) to each well in the filter plate containing the serum bound beads and shake vigorously again on orbital shaker (speed and time as before) for 10 min at RT.\nCollect the eluate in the same collection plate F1 by centrifugation at 1600g for 1 min again.\nThis represents fraction 1, which contains both the flow-through fraction at pH 9.0 and the pH 9.0 eluate.\nAdd 100 µL of E2 elution buffer (at pH 7.0) to each well in the filter plate and again shake at the same speed and time.\nCollect the eluate in another new F2 collection plate by centrifugation as before.\nAdd 100 µL of E2 elution buffer again to each filter well and elute a second time by shaking and centrifugation as before onto F2 collection plate again.\nThis represents fraction 2, which contains the pH 7.0 eluate.\nElute the serum protein bound beads similarly in turn with E3, E4, and E5 elution buffers as above resulting in fractions 3, 4, and 5 containing pH 5.0, pH 4.0, and pH 3.0 eluates, respectively, in three new F3, F4, and F5 collection plates.\nFinal elution is achieved by adding E6 organic solvent elution buffer and centrifugation at 2000g for 5 min giving rise to fraction 6 containing the organic solvent eluate in collection plate F6.\nFreeze fractions 1–6 in collection plates E1–6 at −70°C until the chip binding protocol proceeds.\n\nChip Pretreatment\nThe ProteinChip Array Profiling technique is also called surface enhanced laser desorption and ionization time of flight mass spectrometry (SELDI-TOFMS, Ciphergen Biosystems Inc., Fremont, CA). The chip array used is a 10 mm wide × 80 mm long metal chip having eight 2-mm spots with a specific chromatographic surface for binding of biomarkers of interest (see Fig. 1). For IMAC30 Cu(II) ProteinChip Array, a pretreatment procedure is required for loading copper ions onto the chip for binding protein biomarkers with affinity to copper ions. This metal loading step is, however, not required for CM10 ProteinChip Array, which is impregnated with carboxylate ions as weak cation exchanger for biomarker binding. CM10 chips only requires buffer washing instead. Both types of chips are tested to be ideal for binding serum proteins/peptides for the study.\n\nIMAC30 Chip Pretreatment\nAssemble the 96-well bioprocessor for chip pretreatment by placing 12 strips of protein chips in the base clamp assembly and then putting a 96-hole rubber gasket sheet and a 96-well bioprocessor reservoir on top of the chips.\nClamp this sandwich tight with the 96 incubation wells in the reservoir placed directly on top of the 96 chip spots.\nAdd 50 µL of 100 mM CuSO4 solution into each well in the reservoir making direct contact with each IMAC30 chip spot.\nGet rid of air bubbles if present.\nShake the chips in the bioprocessor for 5 min at the same speed and time as before in the orbital shaker at RT to allow copper ions from CuSO4 solution to bind to the chip surface.\nPipet away the CuSO4 solution.\nRinse the chip spot in each well with 100 µL of Milli-Q grade of water and shake again under the same condition for 1 min at RT.\nRinse each well with 100 µL of sodium acetate buffer at pH 4.0 (IMAC30 chip neutralizing buffer) for 5 min with shaking at RT.\nRemove the neutralizing buffer after shaking.\nRinse the well again with 100 µL of Milli-Q water by shaking for 1 min at room temperature.\nAdd 150 µL of 100 mMsodium phosphate buffer containing 0.5 mol/L NaCl (IMAC30 chip binding buffer) onto each well and shake for 5 min at RT to wash the chip spot.\nRemove the buffer after shaking.\nWash with the same IMAC30 chip-binding buffer one more time.\nRemove the buffer after washing.\nProceed immediately to Subheading 3.6.1. for sample binding without letting the chip surface to dry.\n\nCM10 Chip Pretreatment\nAfter assembling the CM10 chips onto the 96-well bioprocessor (see steps 1–2 in Subheading 3.5.1.), add 150 µL of 100 mM sodium acetate buffer at pH 4. (CM10 chip binding buffer) onto each reservoir well and shake vigorously on micromix shaker again as before for 5 min at RT to wash the chip spot.\nRemove the buffer from the well.\nWash with the same chip-binding buffer one more time.\nRemove the buffer.\nProceed immediately to Subheading 3.6.2. for sample binding without letting the chip surface to dry.\n\nSerum Fraction Binding on Chips\n\nIMAC30 Chips\nAdd 80 µL of the IMAC30 chip-binding buffer into each well in the bioprocessor set up containing the IMAC30 chips.\nAdd 20 µL of each ion exchange bead eluate (serum fractions 1–6) to the well for sample binding.\nMix well by shaking for 30 min at RT.\nRemove the serum fractions.\nWash the chip spot in the well by adding 150 µL of IMAC30 chip-binding buffer onto each well and shake for 5 min at RT.\nRemove buffer.\nRepeat washing (steps 5 and 6) once more.\nRinse with water two times by adding 200 µL of Milli-Q water to each well and discard immediately.\nRemove the chips from bioprocessor and air-dry the chips for 5 min.\nAdd 1 µL of 50% sinapinic acid (which is an energy absorbing molecule solution) to each chip spot.\nAir-dry the chips for about 10 min.\nAdd 1 µL of 50% sinapinic acid and air-dry again.\nThe chip is ready to be read in the PBS IIc mass spectrometer reader.\nRemember to process at least one reference control serum concurrently with the patients’ samples on each chip for quality control of chip-to-chip variability.\n\nCM10 Chips\nAdd 90 µL of 100 mM sodium acetate buffer at pH 4.0 (CM10 chip binding buffer) into each well in the bioprocessor set up containing the CM10 chips.\nAdd 10 µL of each ion exchange bead eluate (sample fractions 1–6) to the well for sample binding.\nMix well by shaking for 30 min at RT.\nRemove the sample fractions.\nWash the chip spot in the well by adding 150 µL of CM10 chip binding buffer again into each well and shake for 5 min at RT.\nRemove buffer.\nRepeat washing (steps 5 and 6) once more.\nRinse with water two times by adding 200 µL of Milli-Q water to each well and discard immediately.\nRemove the chips from bioprocessor and air-dry the chips for 5 min.\nAdd 1 µL of 50% sinapinic acid to each chip spot.\nAir-dry the chip for around 10 min.\nAdd 1 µL of 50% sinapinic acid and air-dry again.\nThe chip is ready to be read in the PBS IIc mass spectrometer reader.\nProcess at least one reference control serum concurrently with the patients’ samples on each chip for quality control of chip-to-chip variability as before.\n\nChip Reading and Data Acquisition\nThe PBS IIc SELDI-TOF mass spectrometer reader is a laser desorption ionization time of flight mass spectrometer equipped with a pulsed ultraviolet nitrogen laser source. When the laser activates the bound serum biomarkers on the chip surface, the biomarkers become desorbed and ionized. Ionized molecules fly along the mass spectrometer to the ion detector in a time of flight manner according to their mass over charge ratio (m/z). When the ion signal is detected in the ion detector, signal processing is accomplished by high-speed analog-to-digital converter linking to a computer. Detected protein biomarkers are displayed in spectral, map or gel view formats by the Ciphergen ProteinChip software 3.0.2. The following steps show how the chips are read in the mass spectrometer.\nClick the “Sample Exchange Dialog” button from the manu bar of the Ciphergen ProteinChip software 3.0.2 in the PBS IIc SELDI-TOF mass spectrometer reader.\nClick “Open Lid” button in the “Sample Exchange Dialog” box to open the lid of the chip chamber.\nPlace the sample treated chips into the slot of the chip chamber.\nClick “Close Lid” button.\nThe chip is automatically inserted into the chip chamber.\nIn the “Sample Exchange Dialog” box, input the serial number, chip type, and chip format.\nClick “Chip” button from the “Sample Exchange Dialog” box.\nInput sample name and sample group.\nClick “OK” button.\nClick “Chip Protocol” button from the menu bar.\nInput spectrum tag (which is the sample name) and the following spot protocols: Starting laser intensity to 165.\nStarting detector sensitivity to 9.\nHighest mass of detection to 200,000 Da.\nOptimal mass range of detection from m/z of 2000 to 20,000 (signals from m/z of 0–2000 are not analyzed as artifacts can be produced by energy-absorbing molecules or other chemical contaminants at this mass range).\nFocus lag time at 800 ns.\nMass deflector to Auto.\nData acquisition method to SELDI quantitation.\n338 laser shots per sample.\nClick “Start Running” button from the menu bar.\nBy the time 338 laser shots are fired, the collected data is automatically saved.\nIt is important to first carry out external calibration of the equipment using the All-In-1 Peptide molecular mass standard (according to the instruction sheet from the manufacturer) to ensure mass accuracy of the spectra before running the samples.\nCarry out baseline subtraction and calibration for the spectra.\nGenerate labeled peak groups (clusters) across multiple spectra by the Biomarker Wizard mode.\nCompare peak groups detected in the SARS patients with those of the controls by nonparametric two sample Mann-Whitney U-test in the Biomarker Wizard mode.\nBiomarker Wizard operates in two passes, with the first pass uses low sensitivity settings to detect obvious and well-defined peaks and the second pass uses higher sensitivity settings to search for smaller peaks.\nThis will identify both strong and weak peaks that are significantly increased or decreased in the patients vs the controls.\n\nData Analyses\nAs an example, Fig. 3 illustrates a comparison of multiple biomarkers in serum fraction 1 (pH 9.0 eluate) of SARS patients vs all the other controls in a gel view format in the m/z range of 2000–15,000.\nHighlighted in three boxes (A, B, and C) with arrows are biomarkers at m/z of 11,695, 9159, and 7784, respectively with biomarker at 11,695 being significantly increased in the SARS patients vs the controls and biomarkers 9159 and 7784 being significantly decreased.\nNine biomarker were found within m/z range of 4900–15,000 with highly significant increase in their normalized peak intensities in the SARS patients vs those of the controls and three biomarkers with highly significant decrease (Table 1).\nSeparating the controls into individual groups, a highly significant increase of biomarker at 11,695 is also observed in SARS patients vs each control group (Fig. 4).\nAs pneumonia is a frequent life threatening clinical manifestation in the SARS patients, we further monitor the biomarker—11,695 level in a SARS patient longitudinally (Fig. 5) and correlate the biomarker level with the extent of pneumonia by chest X-ray opacity score (Fig. 2).\nFigure 5 illustrates, in this patient, an elevation of the biomarker—11,695 level preceding the development of pneumonia at the onset of disease (as indicated by an increasing opacity score) followed by a rapid drop of the biomarker level down to the background on recovery from the illness after stringent antiviral and steroid treatments (seeNote 1 for details of clinical presentation of the patients).\nOn the other hand, Fig. 6 shows low biomarker—11,695 level during the whole monitoring period in another SARS patient who has low X-ray scores and whose clinical course is relatively mild (see Note 2).\nBy means of peptide mapping and tandem MS/MS mass spectrometry, this biomarker is identified to be Serum Amyloid A protein (SAA), which is an acute phase reactant frequently and rapidly induced in abundance at pneumonia (see ref. 15 for the protein ID results and ref. 17–20 for the techniques adopted).\nThis serial study demonstrates the potential of the biomarkers discovered by protein chip array profiling in monitoring the disease manifestation in SARS patients.\nFig. 3. Gel view of partial protein chip array profiling results in SARS patients’ sera vs control infection groups’ sera. Part of the profiling results of fraction 1 from the ion exchange serum eluate in SARS patients vs the control patients in m/z range from 2000 to 15,000 was illustrated. HBV, hepatitis B virus; TB, M. tuberculosis; RSV, respiratory syncytial virus. The three arrows showed three clusters of biomarkers, A–C at m/z of 11695, 9159, and 7784, respectively, with cluster A being significantly increased in SARS patients and the last two clusters (B and C) significantly decreased.\nTable 1 Serum Biomarkers Significantly Increased or Decreased in SARS Patients vs Seven Control Groups of Patients With Other Infections and Healthy Individuals\nBiomarker number Mass over charge (m/z) Differential expression p-valuea\n1 4922 Increased 1.1 × 10−4\n2 5104 Increased 7.1 × 10−8\n3 5215 Increased 6.4 × 10−4\n4 5833 Increased 3.1 × 10−9\n5 7784 Decreased 4.9 × 10−8\n6 8416 Decreased 1.6 × 10−7\n7 9159 Decreased 1 × 10−10\n8 10,867 Increased 3.6 × 10−7\n9 11,508 Increased 1.3 × 10−5\n10 11,695 Increased 5.4 × 10−6\n11 11,871 Increased 8.4 × 10−4\n12 14,715 Increased 3.9 × 10−9\nap-values were obtained in Mann-Whitney U-test by comparing the normalized peaki intensities of the biomarkers in 44 sera from 24 SARS patients vs those in 72 sera from 51 control patients with various viral or bacterial infections (see Materials for individual control group) plus 10 sera from 10 apparently healthy individuals.\nFig. 4. Comparison of normalized peak intensities of the biomarker at m/z of 11,695 in sera from SARS patients and control groups. The normalized peak intensities of this marker in SARS patients’ sera were compared with those of the other seven control infection groups and the normal individuals by Mann-Whitney U-test. A p-value of 0.05 or less demonstrated a statistically significant difference between the two groups. Adeno, adenovirus; Bact, bacterial culture positive; HBV, hepatitis B virus; Influ A, influenza A virus; Influ B, influenza B virus; Normal, apparently normal subjects; RSV, respiratory syncytial virus; TB, M. tuberculosis.\nFig. 5. Correlation of the level of serum biomarker—11,695 with the extent of pneumonia in a SARS patient under clinical follow-up. Longitudinal follow- up of the clinical profile of this patient was illustrated in the top panel. Monitoring of the biomarker—11,695 level and chest X-ray opacity score (an indicator of the extent o pneumonia) was shown in the lower left panel. The lower right panel illustrates the biomarker—11,695 in a gel view measured at different time points. CoV IgG \u003c25 and 100, Serum SARS-Coronavirus IgG antibody titers of \u003c1/25 and 1/100; CXR, Lung consolidation as shown by chest X-ray imaging; FU outpatient, During follow-up as outpatient; ICU, Admitted to Intensive care unit; i.v. Ig, Intravenous injection of immune globulin; MP, Daily treatment by 500 mg × 2 of methylprednisolone; NP Swab CoV −ve, RT-PCR negative for SARS Coronavirus in nasopharyngeal swab; Rectal Swab CoV +ve, RT-PCR positive for SARS Coronavirus in rectal swab; Ribavirin, Daily treatment by 400 mg Ribavirin; Stool CoV +ve, RT-PCR positive for SARS Coronavirus in the stool; Throat Swab CoV +ve, RT PCR positive for SARS Coronavirus in throat swab.\nFig. 6. Correlation of the level of serum biomarker—11,695 with the extent of pneumonia in the second SARS patient under clinical follow-up. Longitudinal follow-up of the clinical profile of this patient was illustrated in the top panel. Monitoring of the biomarker—11,695 level and chest X-ray opacity score was shown in the lower panel. Annotations were similar to those as tabulated in Fig. 5. Antibiotic, Treatment with conventional antibiotics; CoV IgG \u003c10 and 160, Serum SARS-Coronavirus IgG antibody titers of \u003c1/10 and 1/160; NP Swab CoV −ve, RT-PCR negative for SARS Coronavirus in nasopharyngeal swab; Rectal Swab CoV −ve, RT-PCR negative for SARS Coronavirus in rectal swab; Throat Swab CoV −ve, RT PCR negative for SARS Coronavirus in throat swab.\n\nNotes\nClinical and protein chip monitoring of the first SARS patient (see Fig. 5): one day after SARS-CoV infection was diagnosed, the radiographic score of this patient was increased from 6 to a peak value of \u003e16 and then dropped to 12, 9, and finally to 4 demonstrating a progressive recovery. The biomarker—11,695 level by protein chip array profiling study showed an elevation that peaked earlier than the radiographic score but it then gradually subsided along with the score to a nadir when the patient was discharged.\nClinical and protein chip monitoring of the second SARS patient (see Fig. 6): the second patient had all the typical clinical symptoms of SARS-CoV infection with left upper lobe consolidation in chest radiographs. Despite negative SARS-CoV RT-PCR, titer elevation in paired anti-SARS CoV serum antibody tests performed in two laboratories confirmed the viral infection. He was managed as SARS on the next day. The clinical course was uneventful and uncomplicated. His pneumonia had never been extensive and hence his radiographic score showed just a low level of lung involvement from May 8th to 15th and then totally subsided from May 16th onwards. Biomarker—11,695 level was essentially low throughout the monitoring 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