The activity of the enzyme present on the surface of the well is 8.

0 REAGENT PREPARATION
quantitated by reaction with a suitable substrate to produce color. STOP
G. Stop Solution – 8ml/vial - Icon
One (1) vial containing a strong acid (1N HCl). Store at 2-8°C. 1. Wash Buffer
The employment of several serum references of known prostate Dilute contents of wash concentrate to 1000 ml with distilled or
specific antigen (fPSA) levels permits the construction of a dose H. Product Instructions.
deionized water in a suitable storage container. Store at room
response curve of activity and concentration. From comparison to temperature (2-30°C) for up to 60 days.
Note 1: Do not use reagents beyond the kit expiration date.
the dose response curve, an unknown specimen's activity can be 2. Working Substrate Solution – Stable for one year
Note 2: Avoid extended exposure to heat and light. Opened
correlated with fPSA concentration. Pour the contents of the amber vial labeled Solution ‘A’ into
reagents are stable for sixty (60) days when stored at
2-8°C. Kit and component stability are identified on the the clear vial labeled Solution ‘B’. Place the yellow cap on the
3.0 PRINCIPLE label. clear vial for easy identification. Mix and label accordingly.
Note 3: Above reagents are for a single 96-well microplate Store at 2 - 8°C.
Immunoenzymometric assay (TYPE 3):
The essential reagents required for an immunoenzymometric Materials Required But Not Provided: Note 1: Do not use the working substrate if it looks blue.
assay include high affinity and specificity antibodies (enzyme and 1. Pipette capable of delivering 0.50 & 0.100ml (50 & 100µl) Note 2: Do not use reagents that are contaminated or have
immobilized), with different and distinct epitope recognition, in volume with a precision of better than 1.5%. bacteria growth.
excess, and native antigen. In this procedure, the immobilization 2. Dispenser(s) for repetitive deliveries of 0.100ml (100µl) and
takes place during the assay at the surface of a microplate well 0.350ml (350µl) volumes with a precision of better than 1.5%. 9.0 TEST PROCEDURE
through the interaction of streptavidin coated on the well and 3. Microplate washers or a squeeze bottle (optional).
exogenously added biotinylated monoclonal anti-PSA antibody. 4. Microplate Reader with 450nm and 620nm wavelength Before proceeding with the assay, bring all reagents, serum
absorbance capability. reference calibrators and controls to room temperature (20-27°C).
Upon mixing monoclonal biotinylated antibody, the enzyme- 5. Absorbent Paper for blotting the microplate wells. **Test Procedure should be performed by a skilled individual
Free Prostate Specific Antigen (fPSA) labeled antibody and a serum containing the native antigen, a
6. Plastic wrap or microplate cover for incubation steps. or trained professional**
reaction results between the native antigen and the antibodies, 7. Vacuum aspirator (optional) for wash steps.
Test System without competition or steric hindrance, to form a soluble
8. Timer. 1. Format the microplates’ wells for each serum reference
sandwich complex. The interaction is illustrated by the following
Product Code: 2325-300 equation:
9. Quality control materials calibrator, control and patient specimen to be assayed in
duplicate. Replace any unused microwell strips back into
ka the aluminum bag, seal and store at 2-8°C.
5.0 PRECAUTIONS
Enz
Ab (P) + Ag fPSA + BtnAb (m) Enz
Ab (p) - Ag fPSA - Btn
Ab (m) 2. Pipette 0.050 ml (50µl) of the appropriate serum reference
1.0 INTRODUCTION calibrator, control or specimen into the assigned well.
k -a For In Vitro Diagnostic Use
Btn
Ab (m) = Biotinylated Antibody (Excess Quantity) Not for Internal or External Use in Humans or Animals 3. Add 0.100 ml (100µl) of the fPSA Enzyme Reagent to each
Intended Use: The Quantitative Determination of Free well. It is very important to dispense all reagents close to
Prostate Specific Antigen (fPSA) Concentration in Human Ag fPSA = Native Antigen (Variable Quantity)
Enz
Ab (p) = Enzyme labeled Antibody (Excess Quantity) All products that contain human serum have been found to be the bottom of the coated well.
Serum by a Microplate Enzyme Immunoassay, Colorimetric Enz non-reactive for Hepatitis B Surface Antigen, HIV 1&2 and HCV 4. Swirl the microplate gently for 20-30 seconds to mix and cover.
Ab (p) - Ag fPSA - BtnAb (m) =Antigen-Antibodies Complex
k a = Rate Constant of Association Antibodies by FDA required tests. Since no known test can offer 5. Incubate 60 minutes at room temperature (20-27°C).
2.0 SUMMARY AND EXPLANATION OF THE TEST complete assurance that infectious agents are absent, all human 6. Discard the contents of the microplate by decantation or
k -a = Rate Constant of Dissociation
serum products should be handled as potentially hazardous and aspiration. If decanting, tap and blot the plate dry with
Prostate Specific antigen (PSA) is a serine protease with Simultaneously, the complex is deposited to the well through the capable of transmitting disease. Good laboratory procedures for absorbent paper.
chymotrypsin-like activity.1,2 The protein is a single chain high affinity reaction of streptavidin and biotinylated antibody. This handling blood products can be found in the Center for Disease 7. Add 0.350ml (350µl) of wash buffer (see Reagent Preparation
glycoprotein with a molecular weight of 28.4 kDA.3 PSA derives its interaction is illustrated below: Control / National Institute of Health, "Biosafety in Microbiological Section), decant (tap and blot) or aspirate. Repeat two (2)
name from the observation that it is a normal antigen of the additional times for a total of three (3) washes. An automatic
Ab (p) - Ag fPSA - BtnAb (m) + Streptavidin C.W. ⇒ Immobilized and Biomedical Laboratories," 2nd Edition, 1988, HHS Publication
Enz
prostate, but is not found in any other normal or malignant No. (CDC) 88-8395. or manual plate washer can be used. Follow the
complex
tissue. PSA is released from the normal prostate and appears manufacturer’s instruction for proper usage. If a squeeze
Streptavidin C.W. = Streptavidin immobilized on well
at low serum concentrations in healthy men. Studies with reverse Safe Disposal of kit components must be according to local bottle is employed, fill each well by depressing the
Immobilized complex = complex bound to the solid surface
transcription-PCR have shown that PSA also is expressed at a low regulatory and statutory requirement. container (avoiding air bubbles) to dispense the wash.
concentration in peripheral blood cells and other tissues.4 High After equilibrium is attained, the antibody-bound fraction is Decant the wash and repeat two (2) additional times.
serum concentrations can be detected in patients with advanced separated from unbound antigen by decantation or aspiration. The 6.0 SPECIMEN COLLECTION AND PREPARATION 8. Add 0.100 ml (100µl) of working substrate solution to all wells
prostate cancer (PCA).5 Therefore, PSA is applied as a tumor enzyme activity in the antibody-bound fraction is directly (see Reagent Preparation Section). Always add reagents in
marker for the clinical management of PCA.6 However, proportional to the native antigen concentration. By utilizing The specimens shall be blood, serum in type and the usual the same order to minimize reaction time differences
increased PSA concentrations in serum also occur in patients with several different serum references of known antigen values, a precautions in the collection of venipuncture samples should be between wells.
benign prostate hyperplasia (BPH).7 Hence the goal is to dose response curve can be generated from which the antigen observed. For accurate comparison to established normal values, DO NOT SHAKE THE PLATE AFTER SUBTRATE ADDITION
discriminate clearly between BPH and PCA in the clinical concentration of an unknown can be ascertained. a fasting morning serum sample should be obtained. The blood 9. Incubate at room temperature for fifteen (15) minutes.
laboratory to spare the patient invasive diagnostic procedures, should be collected in a plain redtop venipuncture tube without 10. Add 0.050ml (50µl) of stop solution to each well and mix gently
such as a prostate biopsy. additives or anti-coagulants. Allow the blood to clot. Centrifuge the for 15-20 seconds. Always add reagents in the same order
4.0 REAGENTS
specimen to separate the serum from the cells. to minimize reaction time differences between wells.
In human serum, PSA occurs in two forms: free PSA (fPSA) 11. Read the absorbance in each well at 450nm (using a reference
and complexed PSA. The major form is a complex of PSA and α 1 - Materials Provided:
In patients receiving therapy with high biotin doses (i.e. wavelength of 620-630nm to minimize well imperfections) in a
antichymotrypsin (ACT). The fraction of fPSA was shown to be A. fPSA Calibrators – 1ml/vial - Icons A-F
>5mg/day), no sample should be taken until at least 8 hours microplate reader. The results should be read within thirty
substantially smaller in patients with untreated PCA than in Six (6) vials of serum references free PSA antigen at levels of
after the last biotin administration, preferably overnight to (30) minutes of adding the stop solution.
patients with BPH. Therefore, combined measurements of fPSA 0(A), 0.5(B), 1.0(C), 2.5(D), 5.0(E) and 10.0(F) ng/ml. A
ensure fasting sample.
and total PSA (tPSA) may lead to a better discrimination between preservative has been added. Store at 2-8°C.
Note: The calibrators, protein based buffered matrix, were 10.0 CALCULATION OF RESULTS
BPH and PCA Some recent studies have already shown that the Samples may be refrigerated at 2-8oC for a maximum period of
fPSA/tPSA ratio is helpful in the differential diagnosis of BPH and calibrated using a reference preparation, which was assayed
five (5) days. If the specimen(s) cannot be assayed within this A dose response curve is used to ascertain the concentration of
PCA. against the WHO 1st International Standard 96/668.
time, the sample(s) may be stored at temperatures of -20oC for up fPSA in unknown specimens.
B. fPSA Enzyme Reagent – 13 ml/vial - Icon E to 30 days. Avoid use of contaminated devices. Avoid repetitive 1. Record the absorbance obtained from the printout of the
PSA is found in benign, malignant and metastatic prostate cancer.
One (1) vial containing enzyme labeled antibody, biotinylated freezing and thawing. When assayed in duplicate, 0.100ml (100µl) microplate reader as outlined in Example 1.
Since prostate cancer is the second most prevalent form of male
specific free PSA monoclonal mouse IgG in buffer, dye, and of the specimen is required. 2. Plot the absorbance for each duplicate serum reference versus
malignancy, the detection of elevated PSA levels plays an
preservative. Store at 2-8°C. the corresponding fPSA concentration in ng/ml on linear graph
important role in the early diagnosis. Serum PSA levels have been
found to be more useful than prostatic acid phosphatase (PAP) in C. Streptavidin Coated Plate – 96 wells – Icon ⇓ 7.0 QUALITY CONTROL paper (do not average the duplicates of the serum references
One 96-well microplate coated with streptavidin and packaged before plotting).
the diagnosis and management of patients due to increased
Each laboratory should assay controls at levels in the low, normal 3. Draw the best-fit curve through the plotted points.
sensitivity.4 in an aluminum bag with a drying agent. Store at 2-8°C.
and elevated range for monitoring assay performance. These 4. To determine the concentration of fPSA for an unknown, locate
D. Wash Solution Concentrate – 20 ml/vial - Icon controls should be treated as unknowns and values determined in the average absorbance of the duplicates for each unknown
In this method, fPSA calibrator, patient specimen or control is first
One (1) vial containing a surfactant in buffered saline. A every test procedure performed. Quality control charts should be on the vertical axis of the graph, find the intersecting point on
added to a streptavidin coated well. Biotinylated monoclonal and
enzyme labeled antibodies (directed against distinct and different preservative has been added. Store at 2-8°C. (see Reagent maintained to follow the performance of the supplied reagents. the curve, and read the concentration (in ng/ml) from the
free epitopes of fPSA) are added and the reactants mixed. Preparation Section). Pertinent statistical methods should be employed to ascertain horizontal axis of the graph (the duplicates of the unknown
A
Reaction between the various fPSA antibodies and native fPSA E. Substrate A – 7ml/vial - Icon S trends. Significant deviation from established performance can may be averaged as indicated). In the following example, the
forms a sandwich complex that binds with the streptavidin coated One (1) vial containing tetramethylbenzidine (TMB) in buffer. indicate unnoticed change in experimental conditions or average absorbance (0.648) intersects the dose response
to the well. Store at 2-8°C. degradation of kit reagents. Fresh reagents should be used to curve at 2.28ng/ml fPSA concentration (See Figure 1).
B
F. Substrate B – 7ml/vial - Icon S determine the reason for the variations.
After the completion of the required incubation period, the Note: Computer data reduction software designed for ELISA
One (1) vial containing hydrogen peroxide (H 2 O 2 ) in buffer.
enzyme-fPSA antibody bound conjugate is separated from the assays may also be used for the data reduction. If such
Store at 2-8°C.
unbound enzyme-fPSA conjugate by aspiration or decantation.
 software is utilized, the validation of the software should be 10. Accurate and precise pipetting, as well as following the exact TABLE 2 Material for International Standardization of PSA
ascertained. time and temperature requirements prescribed, is essential. Within Assay Precision (Values in ng/ml) Immunoassays”, Clin Chem, 41/9, 1273-1282 (1995).
Any deviation from Monobind IFU may yield inaccurate results. Sample N X σ C.V. 9. Horton GL, Bahnson RR, Datt M, Cfhan KM, Catalona WJ and
EXAMPLE 1 11. All applicable national standards, regulations and laws, Landenson JH, ”Differences in values obtained with two
Level 1 20 0.48 0.03 5.6%
Sample Well Abs Mean Value including, but not limited to, good laboratory procedures, must Level 2 20 1.83 0.10 5.3% assays of Prostate Specific Antigen”, J Urol, 139, 762-72
I.D. Number (A) Abs (B) (ng/ml) be strictly followed to ensure compliance and proper device Level 3 20 11.35 0.47 4.2% (1988).
A1 0.019 usage. 10. Stenman UH, Leinonen J, Alfthan H, Rannikko S, Tuhkanen K
Cal A 0.021 0 12. It is important to calibrate all the equipment e.g. Pipettes, and Alfthan O, “A complex between prostate specific antigen
B1 0.022 TABLE 3
C1 0.167
Readers, Washers and/or the automated instruments used Between Assay Precision* (Values in ng/ml) and α1-anticymotrypsin is the major form of prostate specific
Cal B 0.164 0.5 with this device, and to perform routine preventative Sample N X σ C.V. antigen in serum of patients with prostate cancer:assay of
D1 0.161 maintenance. complex improves clinical sensitivity for cancer”, Cancer Res,
E1 0.300 Level 1 20 0.53 0.05 9.4%
Cal C 0.302 1.0 13. Risk Analysis, as required by CE Mark IVD Directive 98/79/EC, 51, 222-26 (1991).
F1 0.304 Level 2 20 1.93 0.14 7.2%
for this and other devices made by Monobind, can be requested
G1 0.701 Level 3 20 >11 - - Effective Date: 2019-Jul-16 Rev 5 DCO: 1353
via email from [email protected].
Cal D 0.707 2.5 *As measured in ten experiments in duplicate. MP2325 Product Code: 2325-300
H1 0.714
A2 1.353 12.2 Interpretation
Cal E 1.337 5.0 1. Measurements and interpretation of results must be 14.2 Sensitivity Size 96(A) 192(B)
B2 1.321 The theoretical sensitivity, or minimum detection limit, calculated
performed by a skilled individual or trained professional. A) 1ml set 1ml set
C2 2.286 2. Laboratory results alone are only one aspect for determining by the interpolation of the mean plus two standard deviations of
Cal F 2.300 10.0 B) 1 (13ml) 2 (13ml)
D2 2.314 20 replicates of the 0 ng/ml fPSA calibrator, is 0.008 ng/ml.

Reagent (fill)
patient care and should not be the sole basis for therapy,
E2 0.647 C) 1 plate 2 plates
Patient 0.648 2.28 particularly if the results conflict with other determinants. 14.3 Accuracy
F2 0.648 3. The reagents for the test system procedure have been D) 1 (20ml) 1 (20ml)
The fPSA AccuBind® ELISA test system was compared with a
formulated to eliminate maximal interference; however, E) 1 (7ml) 2 (7ml)
reference method. Clinical and non-clinical biological specimens
*The data presented in Example 1 and Figure 1 are for illustration potential interaction between rare serum specimens and test F) 1 (7ml) 2 (7ml)
from low, normal, and elevated concentrations were assayed. The
only and should not be used in lieu of a dose response curve reagents can cause erroneous results. Heterophilic antibodies G) 1 (8ml) 2 (8ml)
total number of such specimens was 167. The least square
prepared with each assay. often cause these interactions and have been known to be
regression equation and the correlation coefficient were computed
Figure 1 problems for all kinds of immunoassays. (Boscato LM Stuart
for the fPSA AccuBind® ELISA method in comparison with the
MC. ‘Heterophilic antibodies: a problem for all immunoassays’
reference method. The data obtained is displayed in Table 4.
Clin.Chem. 1988:3427-33). For diagnostic purposes, the
2.500 results from this assay should be used in combination with TABLE 4
clinical examination, patient history and all other clinical Least Square Correlation
2.000 findings. Method Mean Regression Analysis Coefficient
Absorbance(s)

4. For valid test results, adequate controls and other parameters Monobind (x) 1.62 x = 0.0189 + 0.9649(y) 0.957
1.500 must be within the listed ranges and assay requirements. Reference (y) 1.66
5. If test kits are altered, such as by mixing parts of different kits,
1.000
Patient which could produce false test results, or if results are Only slight amounts of bias between the fPSA AccuBind® ELISA
0.500 incorrectly interpreted, Monobind shall have no liability. test system and the reference method are indicated by the
6. If computer controlled data reduction is used to interpret the closeness of the mean values. The least square regression
0.000 results of the test, it is imperative that the predicted values for equation and correlation coefficient indicates excellent method
0 1 2 3 4 5 6 7 8 9 10 the calibrators fall within 10% of the assigned concentrations. agreement.
7. fPSA is elevated in benign prostatic hyperplasia (BPH).
fPSA Values in ng/ml Clinically an elevated fPSA value alone is not of diagnostic 14.4 Specificity:
value as a specific test for differential diagnosis of BPH. The The following substances did not interfere with the performance of
11.0 Q.C. PARAMETERS ratio of fPSA/tPSA is a better marker and should be used in fPSA determination using the fPSA AccuBind® ELISA test
conjunction with other clinical observations (DRE) and system. These substances were added to the pooled sera in
In order for the assay results to be considered valid the diagnostic procedures (prostate biopsy). concentrations 10-100 times more than normal.
following criteria should be met: 8. When the total PSA (tPSA) reads 4-10 ng/ml, the fPSA/tPSA
1. The absorbance (OD) of calibrator F should be > 1.3. ratio is useful in the differential diagnosis of BPH and PC Compound Concentration Added
2. Four out of six quality control pools should be within the (Prostate Cancer). Depending on the ratio, the probability can AFP 10 µg/ml
established ranges. be determined as follows: Atropine 100 µg/ml
Acetylsalicylic Acid 100 µg/ml
12.0 RISK ANALYSIS fPSA/tPSA Ratio Probability of Prostate Cancer Ascorbic Acid 100 µg/ml
0-10% 55% Caffeine 100 µg/ml
The MSDS and Risk Analysis Form for this product are available 10-15% 28% Dexamethasone 10 µg/ml
on request from Monobind Inc. 15- 20% 25% Flutamide 100 µg/ml
> 20% 10% hCG 100 IU/ml
12.1 Assay Performance hLH 100 IU/ml
1. It is important that the time of reaction in each well is held 13.0 EXPECTED RANGE OF VALUES Methotrexate 100 µg/ml
constant to achieve reproducible results. Prolactin 100 µg/ml
2. Pipetting of samples should not extend beyond ten (10) It is important to keep in mind that establishment of a range of TSH 100 mIU/ml
minutes to avoid assay drift. values, which can be expected to be found by a given method for
3. Highly lipemic, hemolyzed or grossly contaminated a population of "normal" persons, is dependent upon a multiplicity
specimen(s) should not be used.
15.0 REFERENCES
of factors: the specificity of the method, the population tested and
4. If more than one (1) plate is used, it is recommended to repeat the precision of the method in the hands of the analyst. For these
the dose response curve. 1. Christensson A, Laurell CB, Lilja H, Eur J Biochem, 194, 755-
reasons, each laboratory should depend upon the range of 63 (1990).
5. The addition of substrate solution initiates a kinetic reaction, expected values established by the Manufacturer only until an
which is terminated by the addition of the stop solution. 2. Watt KW, et al, Proc Nat Acad Sci USA, 83, 3166-70 (1986).
in-house range can be determined by the analysts using the 3. Chen Z, Prestiglacomo A, Stamey T, Clin Chem, 41 1273-82
Therefore, the substrate and stop solution should be added in method with a population indigenous to the area in which the
the same sequence to eliminate any time-deviation during (1995).
laboratory is located. 4. Wild D, The Immunoassay Handbook, Stockton Press, 452,
reaction.
6. Plate readers measure vertically. Do not touch the bottom of (1994).
TABLE I
5. Junker R, Brandt B, Zechel C, Assmann G, Clin Chem, 43,
the wells. Expected Values for the fPSA AccuBind® ELISA Test System
7. Failure to remove adhering solution adequately in the 1588-94 (1997).
Healthy Males < 1.3 ng/ml 6. Prestigiacomo AF, Stamey TA, “Physiological variations of
aspiration or decantation wash step(s) may result in poor
replication and spurious results. serum prostate antigen in the (4-10 ng/ml) range in male
14.0 PERFORMANCE CHARACTERISTICS volunteers”, J Urol, 155, 1977-80 (1996).
8. Use components from the same lot. No intermixing of reagents
from different batches. 7. Stamey TA, McNeal JE, Yemoto CM, Sigal BM, Johnstone IM,
14.1 Precision “Biological determinants of cancer progression in men with
9. Patient specimens with fPSA concentrations above 10 ng/ml The within and between assay precisions of the fPSA AccuBind®
may be diluted (for example 1/10 or higher) with normal female prostate cancer”, JAMA, 281, 1395-1400 (1999).
ELISA test system were determined by analyses on three different 8. Chen Z, Prestigiacomo A, Stamey T, “Purification and
serum (PSA = 0 ng/ml) and re-assayed. The sample’s levels of control sera. The number, mean value, standard
concentration is obtained by multiplying the result by the characterization of Prostate Specific Antigen (PSA)
dilution factor (10).
deviation and coefficient of variation for each of these control sera Complexed to α 1 - Anticymotrypsin: Potential reference
are presented in Table 2 and Table 3.