TEST FOR DIFFERENT IMMUNOLOGICAL PRINCIPLES

Mary Jesreth V. Bayas

Biological Science Department, College of Science and Computer Graduate Studies,

De La Salle Univeristy – Dasmariñas, Dasmariñas City, Cavite Philippines.

OVERVIEW
In the field of medicine, different techniques were applied in order to achieve certain results all for the
benefit of preserving life. The immune system of the body processes different chemical reactions to
maintain homeostasis, either detecting infections or resolving problems before it reaches its chronic stages.
Identifying the source of infection or the level of severity of the condition a significant variable to be able to
provide the appropriate treatment. Thus, different tests were established based on the nature and condition
of the disease. The following samples of immunological testes namely testing on the concentration of
creatinine and phosphorous are applicable for identifying the agent of the illness. Given also are the test
for diagnosing herpes and detection of anti-hepatitis B core antibody are useful for preventing the
prevalence of these common health problems. Using the prescribed reagents and equipment and strict
monitoring of the processes will be able to save lives and prevent further complications upon early
detection.

FOLLOWING IMMUNOLOGICAL PROTOCOLS

Immunological laboratory tests are available in different laboratories depending on its purpose of diagnosis.
There are techniques that have become important tools in order to identify imbalances in terms of our
immune system or impurities leading to the identification of the antigen and production of antibodies. Such
methods are specific for the production of antibodies for their target proteins, detecting the presence of
antigen or of the pathogen in a specific specimen. Following proper principles, required reagents and
procedures are necessary to carry out results and maintain homeostasis.

Enumerated below are some immunological protocols used in laboratories to test the presence of different
compounds that may cause infections, diseases or negative effects to living organisms.
This paper aims to present different immunological protocols with their principles and guided laboratory
procedure. It will also provide guides on the test results and reporting systems and other methods to for
quality control.

The following are the laboratory procedures included in this paper:

• Creatinine Concentration
• Phosphorus Concentration
• Detection of Hepatitis B Core Antibody
• Characterization of Herpes Virus
 CREATININE CONCENTRATION

Determining the level of creatinine concentration present in serum, plasma or urine is an important
tool in diagnosing and treating renal diseases. Following the method on the DxC800 is IDMS
Standardized. A precise volume of sample is introduced into a reaction cup containing an alkaline picrate
solution. Absorbance readings are taken at 520 nm between 19 and 25 seconds after sample injection.
Creatinine from the sample combines with the reagent to produce a red color complex. The absorbance
rate has been shown to be a direct measure of the concentration of the creatinine in the sample. It is
important to all serums are potentially positive for infectious agents thus universal precautions are
required.

SPECIMEN COLLECTION, STORAGE, AND HANDLING PROCEDURES

a. Interferences:
1) No interference from hemolysis or lipemia.
2) No interference from <20 mg/dL bilirubin.
b. Separated serum or plasma should not remain at +15°C to +30°C longer than 8 hours. If assays
are not completed within 8 hours, serum or plasma should be stored at +2°C to +8°C. If assays are
not completed within 48 hours, or the separated sample is to be stored beyond 48 hours, samples
should be frozen at -15°C to -20°C. Frozen samples should be thawed only once. Analyte
deterioration may occur in samples that are repeatedly frozen and thawed.
c. Fasting is not required.
d. A minimum of 0.6 mL serum is needed for the Multi-Analyte Panel.
e. Sample volume for individual test is 30 μl added to 570 μl reagent.
f. Sample is run singly as part of Multi-analyte Biochemistry Panel.

EQUIPMENT AND INSTRUMENTATION, MATERIALS, REAGENT PREPARATION, CALIBRATORS

(STANDARDS), AND CONTROLS

a. Instrumentation: Beckman Coulter UniCel® DxC800 Synchron Clinical System

b. Materials
1) Beckman Micro Tube (Part #448774)
2) S/P Plastic Transfer Pipet (Cat. #P5214-10)
3) S/P Brand Accutube Flange Caps (Cat. #T1226-37)
c. Reagent Preparation: Synchron Creatinine Reagent Kit (Part #472525).
1) Prepare Creatinine Reagent by pouring Picric Acid Solution into Alkaline Buffer Solution. Replace
cap and mix at least 10 times by gentle inversion.
2) Unopened Creatinine Reagent Kit is stable until expiration date when stored at room temperature.
3) The combined Creatinine Reagent is stable on the instrument for 30 days from the date of preparation.
4) Do not freeze or refrigerate. If precipitate has formed in the bottom from cold
temperatures during shipping, place reagent in warm water bath to dissolve, then mix well before using.
5) Combined reagent works best if left to stand for ≥24 hours after preparation.
6) Avoid contact of reagent with skin, eyes, or clothing. In case of spill, flush with large amounts of water.
 d. Standards Preparation: None required.
1) Beckman Synchron Aqua Calibrator 1 and 2 (Part #471288 and 471291).
e. Control Material
1) Bio-Rad Liquid Unassayed Multiqual level 1 and 3 (Part# 697 and #699).
- Thaw bottle of control and mix very well.
- Thawed control is stable 7 days. Mix well prior to each use.

OPERATING PROCEDURE:

Specimens arrive refrigerated. Specimens are kept refrigerated until ready to transfer to Micro tubes.
Capped Micro tubes are kept refrigerated until ready to put on instrument. Specimen vials are returned to
container and refrigerated after transfer of aliquot and double checking of pour off tubes. Specimen vial
container is placed in -70°C Freezer after testing is complete. Micro tubes are refrigerated, and then
frozen after analysis.

a. Preliminaries
1) Enter test in L.I.S. as a part of a panel according to procedure listed in this
b. Sample Preparation
1) Procedure for labeling Micro tube (CX tube) and transferring serum
c. Operation
1) Refer to Operation Procedures for programming controls/patients and loading
sectors/racks in the Beckman Coulter Synchrony UniCel DxC 600/800 System Instructions For Use
(IFU) manual or DxC800 and DxC600 Operating Procedure.

d. Recording of Data
Microsoft Excel software on a PC and our Laboratory Information Systems (L.I.S.) are used to manage
the data. The test is analyzed on a Beckman Coulter UniCel® DxC800 Synchron Clinical System. The
DxC800 is interfaced to the Laboratory Information Systems (L.I.S.) with a bidirectional interface. After
tests are completed, the results will go to the L.I.S. Host Computer Interface to be verified by qualified
analyst.
b. Reflex testing is set up in the L.I.S. to order a repeat of any critical result, to verify abnormal values.
c. Statistical evaluation of the runs is accomplished with Microsoft Excel software on a PC

1) Operator will review and verify results in the L.I.S. The L.I.S. reorders tests to verify any critical results.
2) These results are stored in the L.I.S. along with the original results. Original values are used when
repeat results match the original within 3 CV.
3) Project supervisor will export data from the L.I.S. into an Excel file. The data is copied in
into another Excel file for further evaluation.
4) An Excel spreadsheet printout of the results for each container ID is made and comments
noted.
5) Project supervisor reviews the results. If problems noted with results or QC, Project
 Supervisor investigates and discusses issues if necessary with Laboratory Director. Repeat samples if
necessary.
6) Daily log sheets are completed, and any problems or issues noted.
e. Replacement and Periodic Maintenance of Key Components
f. Calculations: Synchron Systems perform all calculations internally to produce the final reported result.
The system will calculate the final result for sample dilutions made by the operator when the dilution factor
is entered into the system during sample programming

ANALYTICAL RANGE:

1) 0.1 - 25 mg/dL
2) Samples out of analytical range high should be diluted with saline or deionized water and reanalyzed.
Enter dilution factor at sample information screen or multiply printout by dilution factor to obtain the final
result.
3) Limits of detection (LOD) are established by Beckman Coulter and linearity data verifies the reportable
range. Detection of results below the reportable range is not relevant and formal limit of detection study
is unnecessary.
4) Sensitivity is defined as the lowest measurable concentration which can be distinguished from zero
with 95% confidence. Sensitivity for creatinine determination is 0.1 mg/dL.
6) 0 is not a reportable value.

REFERENCE RANGES (NORMAL VALUES)

Values greater than 6.0mg/dL are called for CLS patients. For this study we early report results if
Creatinine is greater than 1.7 mg/dL.
 PHOSPOROUS CONCENTRATION IN SERUM

In diagnosing and treating kidney diseases and disorders with the involvement of the parathyroid gland,
one laboratory test to check is using a time-rated method in determining phosphorus concentration in
serum, plasma or urine. The reaction of phosphorus and ammonium molybdate forms a colored
phosphomolybdate complex in an acidic solution. The concentration of the compound depends on the
changes in absorbance at 365 nm at fixed time interval. The concentration and the changing level of
absorbance is observed to be directly proportional. Similar to any test, safety precautions is strictly
required to prevent infection.

SPECIMEN COLLECTION, STORAGE, AND HANDLING PROCEDURES

A. Interferences:
(1) No interference from <30 mg/dL bilirubin.
(2) No interference from <2+ hemolysis.

B. Separated serum or plasma should not remain at +15 to +30°C longer than 8 hours. If assays are not
completed within 8 hours, serum or plasma should be stored at +2 to +8°C. If assays are not completed
within 48 hours, or the separated sample is to be stored beyond 48 hours, samples should be frozen at –
15 to –20°C. Frozen samples should be thawed only once. Analyte deterioration may occur in samples
that are repeatedly frozen and thawed.
C. Fasting is not required.
D. A minimum of 0.6 mL serum is needed for the Multi-Analyte Panel.
E. Sample volume for individual test is 8 μl added to 570 μl of reagent.
F. Sample is run singly as part of the Multi-analyte Biochemistry Panel.

EQUIPMENT AND INSTRUMENTATION, MATERIALS, REAGENT PREPARATION, CALIBRATORS

(STANDARDS), AND CONTROLS

A. Instrumentation: Beckman Synchron LX20

B. Materials
Beckman Synchron CX Micro Sample Tube (Part #448774)
S/P Plastic Transfer Pipette (Cat. #P5214-10)
S/P Brand Accutube Flange Caps (Cat. #T1226-37)

C. Reagent Preparation:

Synchron LX Phosphorus (PHOSm) Reagent (Part #467868)

1. Pour a 200 mL bottle of molybdate reagent into the 1800 mL bottle of diluent. Recap and mix at least
10 times. (Technical tip: let prepared reagent stand at least 2 hours prior to loading on LX).
2. Unopened reagent when stored at room temperature will remain stable until the expiration date.
3. Combined reagent is stable for 30 days unless the expiration date is exceeded.
4. Do not freeze or refrigerate.
5. If reagent is frozen in transit, thaw completely, warm to room temperature and mix thoroughly by
inverting at least 10 times.
6. Irritating to skin and eyes. Avoid contact with reagent. In case of contact with eyes, rinse immediately
with plenty of water and seek medical advice.
D. Standards Preparation: no preparation required.

Synchron LX Aqua Calibrators 1, 2 (Part #471288 and #471291).

E. Control Material
(1) Bio-Rad Liquid Unassayed Multiqual (Cat. #697, 699).
- In use from August 24, 2002
- Thaw new bottle weekly. Mix very well, using rocker prior to use.
- Thawed control is stable 7 days. Mix well prior to each use.

REPORTABLE RANGE OF RESULTS

A. Analytical Range:

0.5–12.0 mg/dL

a. Samples out of analytical range high should be diluted with saline and reanalyzed. Enter dilution
factor at sample information screen or multiply printout by dilution factor to obtain the final result.

b. If phosphorus result is <1.6 mg/dL and patient does not have a known monoclonal gammopathy,
result may be low due to non-fasting.

c. Any out of range low result (provided there is no known monoclonal gammopathy) should be
reported as “<0.5 mg/dL”.

2. Limits of detection (LOD) are established by Beckman-Coulter and linearity data verifies the
reportable range. Detection of results below the reportable range is not relevant and formal limit of
detection study is unnecessary.

3. Sensitivity is defined as the lowest measurable concentration which can be distinguished from zero
with 95% confidence. Sensitivity for the phosphorus determination is 0.5 mg/dL.

4. 0 is not a reportable value.

REFERENCE RANGES (NORMAL VALUES)

 HERPES TEST USING SOLID-PHASE ENZYMATIC IMMUNODOT ASSAY

The process of solid-phase enzymatic immunodot assays detects antibodies reactive to antigens such as
a viral glycoprotein specific for herpes simplex virus type 2 (HSV-2) (designated gG-2), and a glycoprotein
specific for herpes simplex virus type 1 (HSV-1) (designated gG-1). Purifying the glycoproteins uses
monoclonal antibodies and affinity chromatography. The process is required to provide antigens for type-
specific herpes serological assays. gG-1 or gG2 is absorbed to the center of a nitrocellulose disk, the rest
of the disk surface is coatd with bovine serum albumin (BSA) to avoid adsorption of non-specific protein.
Identification of bound antibodies are processed by sequential treatment with peroxidase-conjugated goat-
anti-human IgG and the enzyme substrate (H2O2 with chromogen 4-chloro-1-naphthol). A discoloration of
blue dot at the center of the disk will show a positive result. Serum reactive to an immunodot charged with
gG-1 indicates previous and probable latent HSV-1 infection. Latent HSV-1 infection is associated with
upper body infection. Serum reactive with gG-2 indicates previous and probable latent HSV-2 infection
which is a common infection to genitalia and sexually transmitted. Perinatal transmissions are often
observed with fatal diseases in newborns. Detection using assays for antibodies against herpes viruses
are important for clinical and epidemiological significance. It is also important to process tests with safety
precautions to avoid contact. Thus, decontamination is required using autoclaving at 250°F, 19 pounds
pressure, for 1 hour.

SPECIMEN COLLECTION, STORAGE, AND HANDLING PROCEDURES

A. No special instructions such as fasting or special diets are necessary. Blood is collected in a red-top
Vacutainer tube by standard venipuncture procedures.
B. Specimens for HSV-1 and HSV-2 analysis should be fresh or frozen serum.
C. A 0.5-mL sample of serum is preferable. The minimum sample volume required for analysis is 50 μL.
Specimens are rejected if insufficient quantity is available for analysis.
D. The appropriate amount of serum is dispensed into a Nalgene cryovial or other plastic screw-capped
vial labeled with the participant's ID.
E. Specimens collected in the field should be frozen, then shipped on dry ice by overnight mail. Once
received, specimens are stored at ≤–20°C until analyzed. Portions of the specimen that remain after
analytical aliquots are withdrawn should be refrozen at ≤–20°C. Samples thawed and refrozen several
times are not compromised, but multiple brief freeze/thaw cycles should be avoided.

EQUIPMENT AND INSTRUMENTATION, MATERIALS, REAGENT PREPARATION, CALIBRATORS

(STANDARDS), AND CONTROLS

Instrumentation
(1) Multiple-head 96-hole puncher
(2) TekPro rotating platform
(3) Manual ELISA washer

Other Materials
(1) Nitrocellulose membrane sheets
(2) Polyvinyl chloride plates, 96-well
(3) Microsyringe fitted with a repeating dispenser
(4) Bovine serum albumin (BSA)
(5) Hydrogen peroxide, H2O2, 30%
(6) 4-chloro-1-napthol, C10H7CIO
(7) Methanol, CH3OH
 (8) Horseradish peroxidase-conjugated goat anti-human IgG
(9) 0.55% Triton X-100 in Tris-buffered saline.
(10) Antigens, gG-1 and gG-2
(11) Tris-HCl
(12) Trizma base
(13) Sodium chloride (NaCl)
(14) Distilled water
(15) In-house HSV-1 and HSV-2 positive and negative control serum

Reagent Preparation

(1) gG-1 and gG-2 Antigens

The gG-1 and gG-2 antigens have been prepared by affinity chromatography using specific
monoclonal antibodies (H1379-2 and H1206), respectively. The purified materials are diluted 1:64
in Tris-buffered saline (pH 7.2) before they are used.
(2) Conjugate solution
Horseradish peroxidase-conjugated goat anti-human IgG. Dilute 1:1000 in phosphate-buffered
saline (pH 7.2) containing 3% bovine serum albumin and 1% goat serum.
(3) Buffer solution
Tris-buffered saline (pH 7.2) containing 3 g/dL of bovine serum albumin.
(4) Substrate solution
6 mg of 4-chloro-1-napthol (C10H7CIO) dissolved in 2 mL methanol mixed with 10 mL of TBS and 5
μL of 30% (v/v) hydrogen peroxide(H2O2).

(5) Tris-buffered saline (TBS), pH 7.2

Dissolve 6.6 g of Tris-HCl, 1.0 g of Trizma base, and 11.6 g of NaCl and bring to volume with 1000
mL distilled water in a 1-L flask.

Preparation of Quality Control Materials

In-house HSV-1, HSV-2, and negative control serum pools were prepared at Emory University.
High-titered serum samples from patients with primary HSV-1 infection were pooled and then diluted to
be used as HSV-1 positive controls. Serum samples from convalescent patients with primary HSV-2
infections were pooled, diluted and used as HSV-2 positive controls. Both positive pools are mono-
specific, i.e. they do not cross-react with the other virus type. Serum samples from healthy donors,
nonreactive to both HSV types in the screening ELISA, were pooled, diluted, and used as negative
controls. The dilution scheme for controls is shown in Table 1.
PROCEDURE OPERATING INSTRUCTIONS; CALCULATIONS; INTERPRETATION OF RESULTS

A. Preliminaries
(1) Prepare dilutions of controls, conjugate, buffer, substrate, and antigens.
(2) Assay one negative and two positive controls in duplicate for each virus type with each run of
specimens.
(3) Ensure that all disks and plates are subjected to the same process and incubation times.
(4) Once the assay has been started, complete all subsequent steps without interruption and within
the recommended time limits.

B. Sample Preparation
(1) Bring serum specimens to 20–25°C.
(2) Mix serum samples gently before testing to eliminate stratification which may occur when
serum is frozen or stored at 4°C for extended periods.
(3) Identify the reaction tray wells for each specimen or control.
(4) Dilute test serum initially 1:10 in 0.55% Triton X-100 in TBS. After the incubation at room
temperature, further dilute with Tris-buffered saline (pH 7.2) containing 3% bovine serum
albumin, to a final serum dilution of 1:50.

C. Instrument Setup
There is no instrument required for this solid-phase enzymatic immunodot assay. Purified
HSV-1 or HSV-2 antigens are immobilized on a small disk and incubated with test serum. A
positive reaction is demonstrated by the appearance of a bluish-purple dot at the center of the
disk.

D. Operation of Assay Procedure

HSV-1 and HSV-2 assays are run simultaneously in separate wells. Half of each plate is
precoated with gG-1 antigen for HSV-1; the other half of the plate is precoated with gG-2 antigen
for HSV-2.
(1) Prepare and deposit small disks of nitrocellulose membrane directly in the 96-well polyvinyl
chloride plates with a 96-hole punch.
(2) Wash nitrocellulose disks in each well once with distilled water. Dry the discs completely at
20–25°C.
(3) Onto the center of each disk, deliver 1 μL of appropriately diluted antigen with a microsyringe
fitted with a repeating dispenser.
(4) After drying the disks at 20–25°C overnight, wash them twice with TBS for 10 min each.
(5) Add 100 μL of buffer containing 3% BSA to each well and incubate at 20–25°C for 30 min on
a rotating platform.
(6) Remove the buffer by suction.
(7) Add 100 μL of diluted serum or control to duplicate wells and incubate at 20–25°C overnight
on a rotating platform.
(8) Remove the serum from each well by suction using the manual washer. Add 100 μL of TBS
and incubate at 20–25°C for 10 min on rotator. Remove the TBS by suction. Repeat this
procedure two times. Add 100 μL of buffer (3% BSA) to each well and incubate for 30 min.
(9) Remove the buffer by suction. Add 100 μL diluted conjugate solution to each well and incubate
at 20–25°C for 2 hours on a rotating platform.
(10) Remove the conjugate by suction. Add 100 μL TBS to each well and incubate at 20–25°C for
10 min on rotator. Remove TBS by suction. Repeat this procedure two times.
(11) Remove the TBS by suction. Add 100 μL of freshly prepared substrate solution to each well.
(12) After 15 min, stop the reaction by removing the substrate and washing the plate twice with
distilled water.
 (13) Dry the plates overnight at room temperature in the dark. Examine the disks for color
development. A positive reaction is demonstrated by the appearance of a bluish-purple dot at
the center of the disk.

E. Recording of Data

(1) Quality Control Data

Positive and negative controls are determined to be valid or invalid. Results of each dilution of assay
controls are recorded in standard forms as the test results are read by the investigators. The sample
data are then entered into the computer database.

(2) Analytical Results

Results of each assay sample are recorded in standard forms as the test results are read by the
investigators. The sample data are then entered into the computer database.

F. Replacement and Periodic Maintenance of Key Components

(1) Monitor and document the refrigerator temperature, freezer temperature, and room temperature
on a weekly basis.
(2) Pipettors
All micropipettors that are used in testing clinical specimens should be checked for calibration every
six months. Pipettors that do not conform to specifications should be autoclaved and sent out for
recalibration in accordance with the manufacturer's recommendations. Calibration records should be
kept for each pipettor by serial number.

G. Special Procedure Notes - Emory University

(1) With the availability of mouse monoclonal antibodies, it has become possible to purify HSV-2
proteins that fail to express type-common antigenic determinants detectable in serological assays.
(2) The use of gG-2, purified from extracts of HSV-2-infected cells, led to the development of an
assay of high sensitivity, specificity, and reproducibility.
(3) This immunodot assay is suitable for large numbers of serum samples because it requires a small
amount of purified glycoprotein.
(4) Purified gG-2 retains antigenicity at ≤–70°C for over 5 years if stored in glass (but not plastic)
ampules.
(5) BSA from different sources could cause significant reductions in the sensitivity of the
(6) gG-2 assay. This problem is overcome by testing different batches of BSA from several sources
and choosing a large stock of the best batch.

REPORTABLE RANGE OF RESULTS

Final reports express results as positive or negative for the presence of anti-HSV-1 or HSV-2
antibody in the sample. Standard recording keeping involves using the mainframe computer, floppy
disks, and the hard copy results themselves to track specimens. Records are maintained indefinitely.
Only numerical identifiers (e.g., case ID numbers) should be used. All personal identifiers should be
available only to the medical supervisor or project coordinator to safeguard confidentiality. Qualitative
assays are qualitative assays with a positive, negative or borderline/indeterminate result. The
absorbance or reactivity values of specimens are compared with a cutoff value that is a ratio of the
negative control mean and the positive control mean. Since the controls are read as cutoff values,
plots of these values are not generated for quality control purposes.
 Hepatitis B Core Antibody Test using ORTHO HBc ELISA Test System 480 Test Kits

To detect the total antibody against Hepatitis B virus core antigen (Anti-HBc) in human serum or plasma,
screening blood and blood products uses ORTHO HBc ELISA Test System. The qualitative enzyme-linked
immunosorbent assay is a significant test in diagnosing hepatitis B virus infection. Past or recent infections
can be detected using serological markers. Enzyme-linked immunosorbent assay (ELISA) can detect
antibodies to specific antigen. In the phase of HBV infection, anti-HBc appears after the production of
hepatitis B surface antigen and continues until HBsAg clearance. Anti-HBs is delayed when the HBsAg has
cleared, thus anti-HBc will be applicable to recent HBV infection and will react with the serological tests
such as ELISA. Using test kits for ELISA use components derived from human serum or plasma, thus
precautionary measures are strictly applied.

SPECIMEN COLLECTION, STORAGE, AND HANDLING PROCEDURES

A. No special instructions such as fasting or special diets are required. Diurnal variation is not a major
B. consideration.
C. Specimens may be serum, recalcified plasma, or plasma. Serum specimens may be collected using
regular red-top or serum-separator Vacutainers.
D. Required sample volume is 10 :L for the assay; 1.0 mL will permit repeat analyses as well as other testing.
E. Specimens should be stored in plastic vials and sealed tightly to prevent desiccation of the sample.
F. Serum or plasma samples are collected aseptically to minimize hemolysis and bacterial contamination.
G. Samples are stored in labeled 2 mL Nalgene cryovials or equivalent.
H. Serum is best stored frozen, and freeze/thaw cycles should be kept to a minimum. Store samples at 4-
8EC for no more than 5 days.
I. For storage >5 days, samples are held at -20EC. Samples held in long-term storage at -20EC are
indexed in the database for easy retrieval.
J. Specimens are rejected if contaminated, hemolyzed, or stored improperly. However, rejection is done
only after consultation with NCHS.
K. Avoid multiple freeze/thaw cycles.
L. Do not use heat-activated specimens.
M. Performance has not been established for cadaver specimens or body fluids other than serum or plasma
(such as urine, saliva or pleural fluid.)

SPECIMEN COLLECTION, STORAGE, AND HANDLING PROCEDURES

A. Instrumentation
(1) Multichannel aspirator-washer device capable of dispensing and aspirating 300 :l to 800 :l per well.
(2) Dual wavelength microwell reader capable of reading at 490 nm or 492 nm with a reference filter of 620
nm or 630 nm. Linearity of the microwell reader must range from at least 0 to 2.5 absorbance units.
Adjustable multichannel micropipette capable of delivering 50 μl and 200 μl with at least ± 5% accuracy
or equivalent reagent dispenser.
(3) Fixed or adjustable single-channel micropipette capable of delivering 10 μl with at least ± 10%
accuracy or equivalent sample dilutor.

B. Materials
(1) Deionized water
(2) 5 :l and 250 :l disposable pipette tips or equivalent.
(3) Multichannel micropipette reservoir or equivalent reagent container.
(4) Protective gloves, Tronex or Flexam, small/medium/large
(5) 2 mL cryovials, cat. no. 5000-0020
(6) Cryovial boxes, cat. no. 5026-0909
(7) 1.5 mL microtubes
(8) 50 mL-polypropylene tubes
(9) ORTHO HBc ELISA Test System 480 Test Kit
(10) 4N sulfuric acid (H2SO4)
(11) 20X Wash Buffer Concentrate Phosphate buffer with sodium chloride and detergent. Preservative: 2%
2-chloroacetamide.

C. Reagents

ORTHO HBc ELISA Test System 480 Test Kits contain the following reagent
prepared by the manufacturer. Volumes listed are for 480 tests.

(1) Hepatitis B Virus Core Antigen (HBcAg) (Recombinant)

Coated Microwell Plates 5 Plates. Each plate has 8 strips of 12 wells in each holder.
(2) Antibody Conjugate (Murine Monoclonal)
1 bottle (125 mL). Mixture of anti-human IgG and anti-human IgM conjugated to horseradish peroxidase
with protein stabilizers. Preservative: 0.02% thimerosal.
(3) Specimen diluent
1 bottle (150 mL). Phosphate-buffered saline with protein stabilizers and detergents. Preservative: 0.02%
thimerosal.
(4) OPD Tablets
30 Tablets. o-phenylenediamine·2HCl.
(5) Substrate buffer
1 bottle (190 mL). Citrate-phosphate buffer with 0.02% hydrogen peroxide. Preservative: 0.01% thimerosal.
(6) Positive control (Human)
1 vial (1.0 mL). Source: Human serum or plasma containing anti-HBc and non-reactive for HBsAg and
antibody to human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2). Preservative: 0.02%
thimerosal.
(7) Negative control (Human)
2 vials (1.0 mL each). Source: Human serum non-reactive for anti-HBc, HBsAg and antibody to human
immunodeficiency virus type 1 (HIV-2) and type 2 (HIV-2). Preservative: 0.02% thimerosal.
(8) Plate sealers
21, disposable.

D. Reagent Preparation

(1) Preparation of Wash Buffer (1X): Mix 50 ml of 20X Wash Buffer Concentrate with 950 ml of
distilled or deionized water. Wash Buffer (1X) is stable for 30 days when stored at room
temperature. For longer storage (up to 60 days), keep at 2 to 80EC. Record the date the Wash
Buffer (1X) is prepared and the expiration date on the container. Discard Wash Buffer (1X) if
visibly contaminated.
(2) Preparation of Substrate Solution: Clean glass or plastic vessels must be used. Prior to the end of
the second incubation, transfer a sufficient amount of Substrate Buffer to a container and protect
the contents from light. Completely dissolve the appropriate number of OPD tables in Substrate
Buffer prior to use.
Each Microwell plate requires at least 20 mLof Substrate Solution. More Substrate Solution may
be needed depending upon the reagent dispenser used. Below are guidelines for general use.

The Substrate Solution is stable for 60 minutes after the addition of OPD tablets when held at room
temperature in the dark and should be colorless to very pale yellow when used.

PROCEDURE OPERATING INSTRUCTIONS; CALCULATIONS; INTERPRETATION OF RESULTS

A. Preliminaries
(1) Prior to the beginning of the procedure, bring kit components to room temperature (15 to 30oC). Invert
liquid reagents gently several times but avoid foaming. Check the incubator temperature; maintain at
37oC ± 1oC

(2) Determine the total number of wells needed for the assay. In additional to specimens, one substrate
blank, three negative controls and two positive controls will be included on each plate or partial plate. If
the entire strip is not needed, an appropriate number of wells can be broken off. Unused wells should
be stored at 2 to 8oC in the supplied foil pouch, tightly sealed with desiccant and used within 14 days of
opening the foil pouch. Record the date the pouch is opened and the expiration date of the unused
wells on the pouch.

Performing the test on less than a full plate is permitted as long as the following conditions are met:
• Microwell strips from different plates can be mixed to assemble full or partial plates as long as
they are from the same lot, within the open pouch expiration date and have come from plates
that have previously demonstrated proper response to kit controls.
• When assembling a plate that contains strips from newly opened, previously untested plate,
one of these strips should be placed at the beginning of the plate and received the full
complement of kit controls.

(3) Once the assay has been started, complete all subsequent steps without interruption and within the
recommended time limits.

B. Sample Preparation

(1) Allow the serum specimens to come to 20-25EC. Serum and plasma samples may stratify when frozen
or stored at 4-8C for extended periods. Mix the specimens gently before testing.
(2) Assemble the microwell strips into the microwell strip holder, if necessary. Microwell strips must be
level in the microwell strip holder. For incomplete plates, add black or uncoated microwell strips.
(3) Prepare a record (plate map) identifying the placement of the controls and specimens in the microwells.
(4) Arrange the assay control wells so that well 1A is the substrate blank. From well 1A, arrange all
controls in a horizontal or vertical configuration as follows: Configuration is dependent upon software.
Operation of the Assay Procedure

(1) Pipette 200 :L of Specimen diluent to all wells of the microtiter plate except well 1A.
(2) Pipette 10 :L of controls or serum samples into the appropriate wells.
(3) Apply cover seal. Incubate at 37oC ± 1oC for 1-hour ± 5 minutes.
(4) Place the microtiter plate on the AutoWash and wash all the wells five times with Wash Buffer (1X).
(5) Add 200 :L of Antibody Conjugate to all wells except 1A.
(6) Apply cover seal. Incubate at 37oC ± 1oC for 1-hour ± 5 minutes.
(7) Prepare sufficient Substrate Solution to fill the control and test wells. Allow time for the OPD tablets
to dissolve completely.
(8) Place the microtiter plate on the AutoWash and wash all the wells five times with Wash Buffer (1X).
(9) Add 200 :L of Substrate Solution to all the wells including 1A.
(10) Apply cover seal. Incubate at room temperature for 30 minutes ± 1 minute in the dark.
(11) Add 50 :L of 4N sulfuric acid (H2SO4) to all wells including 1A.
(12) Read the reaction at 492 nm in the Ortho AutoReader II spectrophotometer.
(13) Retest positive samples in duplicate using this procedure.
(14) Specimens may remain at 20-25EC during preparation and testing for 4 hours.

Recording of Data
(1) Quality Control Data
Multiple positive and negative controls are averaged by the reading instrument and are determined
to be valid or invalid. Raw data are transcribed manually from the instrument readout sheet into a
computerized database.
(2) Analytical Results
Antibody to Hepatitis B Virus Core Antigen in Serum -- NHANES 2003-2004
For ELISA, raw data are expressed as absorbance value. Raw data are transcribed manually from
the instrument readout sheet into a computerized database.

Calculations
(1) The cutoff calculation is done by the reading instrument and by the data management software which
uses the following formula:
Cutoff = NCx + 0.400
(2) Calculate the negative control mean absorbance (NCx) by determining the mean of the negative
controls.

REFERENCE RANGES (NORMAL VALUES)

• Normal human serum is negative for hepatitis B core antigen.

• Samples generating values ±10% of the cutoff are repeated

Given above are examples of immunological tests used to diagnose, characterize and help in treating
common health problems based on the results of each tests. However, such tests can be varied or
manipulated depending on the condition or given samples to test. Immunological test can be universal such
as it aims to solve global health problems and prevent the development of the condition to be untreatable.
However, due changing conditions and unhealthy and unregulated human activities, such tests may not be
applicable at this period. Thus, emergence of new diseases needs to have a simultaneous establishment
of new techniques and protocols.
References:

N. Wellinghausen, M. Abele-Horn, O. Donoso Mantke, M. Enders, V. Fingerle, B. Gärtner, J. Hagedorn,

H.F. Rabenau, I. Reiter-Owona, K. Tintelnot, M. Weig, H. Zeichhardt, K.-P. Hunfeld. Immunological
Methods for the Detection of Infectious Diseases. Germany

Debebe, S. (2004). Immunology and Serology. Ethiopia Public Health Training Initiative, The Carter Center.
Ethiopia Ministry of Health. Ethiopia Ministry of Education.

Kuhnert, W. (2008). Antibody to Hepatitis B Virus Core Antigen in Serum: ORTHO HBc ELISA Test System
480 Test Kits. Hepatitis Branch. Division of Viral Hepatitis. National Center for Infectious Diseases

Gerety RJ. Hepatitis B core antigen and antibody (HBcAg/anti-HBc). In: Gerety RJ, ed. Hepatitis B. New
York: Academic Press, 1985:27-43

Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA): quantitative assay of

immunoglobuilin G. Immunochem 1971;8:871-874.

HSV-1 and HSV-2 in Serum (1999). Herpes in Serum: Solid-Phase Enzymatic Immunodot Assay. Emory
University. The Centers for Disease Control and Prevention. USA.

Lee FK, Pereira L, Griffin C. A novel glycoprotein for detection of herpes simplex virus type 1-specific
antibodies. J Virol Meth. 1986;14:111–118.

Creatinine in Refrigerated Serum ( 2011). Collaborative Laboratory Services, L.L.C

Beckman Coulter Synchron Clinical Systems Chemistry Information Manual, 2007.