antigen and the antibody, forming an antibody-antigen complex. 2-8°C.

Kit and component stability are identified on the

The interaction is illustrated by the following equation: label. Note1: Do not use the working substrate if it looks blue.
ka Note 3: Above reagents are for a single 96-well microplate Note 2: Do not use reagents that are contaminated or have
bacteria growth.
Ag (CA19-9) + BtnAb (m) Ag (CA19-9) - BtnAb (m) 4.1 Required But Not Provided:
k -a 1. Pipette capable of delivering 0.025 & 0.050ml (25 & 50µl) 9.0 TEST PROCEDURE
Btn
Ab (m) = Biotinylated Monoclonal Antibody (Excess Quantity) volumes with a precision of better than 1.5%.
Ag (CA19-9) = Native Antigen (Variable Quantity) 2. Dispenser(s) for repetitive deliveries of 0.100 & 0.350ml (100 & Before proceeding with the assay, bring all reagents, serum
Btn
Ag (CA19-9) - Ab (m) = Antigen-antibody complex (Variable 350µl) volumes with a precision of better than 1.5%. reference calibrators and controls to room temperature (20 -
Quantity) 3. Microplate washers or a squeeze bottle (optional). 27°C).
k a = Rate Constant of Association 4. Microplate Reader with 450nm and 620nm wavelength **Test procedure should be performed by a skilled individual
k -a = Rate Constant of Disassociation absorbance capability. or trained professional**
5. Absorbent Paper for blotting the microplate wells.
Simultaneously, the complex is deposited to the well through the 6. Plastic wrap or microplate cover for incubation steps. 1. Format the microplates’ wells for each serum reference
high affinity reaction of streptavidin and biotinylated antibody. This 7. Vacuum aspirator (optional) for wash steps. calibrator, control and patient specimen to be assayed in
interaction is illustrated below: 8. Timer. duplicate. Replace any unused microwell strips back into
Ag (CA19-9) - BtnAb (m) + Streptavidin CW ⇒ Immobilized complex (IC) 9. Quality control materials. the aluminum bag, seal and store at 2-8°C.
Streptavidin CW = Streptavidin immobilized on well 2. Pipette 0.025ml (25µl) of the appropriate serum reference
Immobilized complex (IC) = Ag-Ab bound to the well 5.0 PRECAUTIONS calibrator, control or specimen into the assigned well.
3. Add 0.100ml (100µl) of the biotinylated labeled antibody to
After a suitable incubation period, the antibody-antigen bound For In Vitro Diagnostic Use each well. It is very important to dispense all reagents
fraction is separated from unbound antigen by decantation or
Cancer Antigen 19-9 (CA 19-9) Not for Internal or External Use in Humans or Animals close to the bottom of the coated well.
aspiration. Another antibody (directed at a different epitope)
4. Swirl the microplate gently for 20-30 seconds to mix and cover.
Test System labeled with an enzyme is added. Another interaction occurs to All products that contain human serum have been found to be 5. Incubate 60 minutes at room temperature.
form an enzyme labeled antibody-antigen-biotinylated-antibody non-reactive for Hepatitis B Surface Antigen, HIV 1&2 and HCV
Product Code: 3925-300 complex on the surface of the wells. Excess enzyme is washed off Antibodies by FDA licensed reagents. Since no known test can
6. Discard the contents of the microplate by decantation or
aspiration. If decanting, tap and blot the plate dry with
via a wash step. A suitable substrate is added to produce color offer complete assurance that infectious agents are absent, all absorbent paper.
measurable with the use of a microplate spectrophotometer. The human serum products should be handled as potentially
1.0 INTRODUCTION 7. Add 0.350ml (350µl) of wash buffer (see “Reagent
enzyme activity on the well is directly proportional to the native hazardous and capable of transmitting disease. Good laboratory Preparation“), decant (tap and blot) or aspirate. Repeat two (2)
free antigen concentration. By utilizing several different serum procedures for handling blood products can be found in the
Intended Use: The Quantitative Determination of Cancer additional times for a total of three (3) washes. An automatic
references of known antigen concentration, a dose response Center for Disease Control / National Institute of Health,
Antigen 19-9 (CA 19-9) Concentration in Human Serum by a or manual plate washer can be used. Follow the
curve can be generated from which the antigen concentration of "Biosafety in Microbiological and Biomedical Laboratories," 2nd
Microplate Enzyme Immunoassay, Colorimetric manufacturer’s instruction for proper usage. If a squeeze
an unknown can be ascertained. Edition, 1988, HHS Publication No. (CDC) 88-8395. bottle is employed, fill each well by depressing the
kb
2.0 SUMMARY AND EXPLANATION OF THE TEST container (avoiding air bubbles) to dispense the wash.
Safe disposal of kit components must be according to local Decant the wash and repeat two (2) additional times.
(IC) + EnzAb (x-CA19-9) Enz
Ab (x-CA19-9) - IC regulatory and statutory requirement.
A mucin type Sialyl Lewis Antigens group of glycoproteins (SLA) k -b 8. Add 0.100ml (100µl) of the CA19-9 Enzyme Reagent labeled
such as CA 19-9, 19-5 have been recognized as circulating
Enz
Ab (x-CA19-9) = Enzyme labeled Antibody (Excess Quantity) antibody to each well.
Enz 6.0 SPECIMEN COLLECTION AND PREPARATION DO NOT SHAKE THE PLATE AFTER ENZYME ADDITION
cancer associated antigens for gastrointestinal cancer. The Ab (x-CA19-9) - IC = Antigen-Antibodies Complex
discovery of a monoclonal antibody clone (1116NS 19-9), which k b = Rate Constant of Association 9. Cover and incubate 60 minutes at room temperature.
The specimens shall be blood serum and the usual precautions in 10. Discard the contents of the microplate by decantation or
exhibited selective reactivity with human gastrointestinal k -b = Rate Constant of Dissociation the collection of venipuncture samples should be observed. For
carcinomas through the recognition of a carbohydrate determinant aspiration. If decanting, blot the plate dry with absorbent
accurate comparison to established normal values, a fasting paper.
(CA 19-9) defined as a sialyl lacto-N-flucopenrose II, resulted in 4.0 REAGENTS morning serum sample should be obtained. The blood should be
the successful purification and thus, determination of human 11. Add 0.350ml (350µl) of wash buffer (see Reagent Preparation
collected in a plain redtop venipuncture tube without additives or Section), decant (tap and blot) or aspirate. Repeat two (2)
gastrointestinal tumor associated glycoprotein antigen expressing Materials Provided: anti-coagulants. Allow the blood to clot for samples. Centrifuge the
CA 19-9 from colorectal carcinoma cell lines. Recently, reports A. CA 19-9 Calibrators – 1ml/vial - Icons A-F additional times for a total of three (3) washes. An automatic
specimen to separate the serum from the cells. or manual plate washer can be used. Follow the
indicate that serum CA 19-9 level is frequently elevated in the Six (6) vials of human serum based reference calibrators at
circulation of patients with various gastrointestinal malignancies, concentrations of 0 (A), 10 (B), 50 (C), 100 (D), 250 (E) and In patients receiving therapy with high biotin doses (i.e. manufacturer’s instruction for proper usage. If a squeeze
500 (F) U/ml. A preservative has been added. Store at 2-8°C. bottle is employed, fill each well by depressing the
such as pancreatic, colorectal, gastric and hepatic carcinomas. >5mg/day), no sample should be taken until at least 8 hours
Together with CEA, elevated CA 19-9 is suggestive of gallbladder Note: The standards, human serum based, were made using a after the last biotin administration, preferably overnight to container (avoiding air bubbles) to dispense the wash.
>99% pure affinity purified preparation of CA 19-9. The Decant the wash and repeat two (2) additional times.
disease. The tumor associated antigen may also be associated in ensure fasting sample.
some malignant conditions. Research studies demonstrate that preparation was calibrated against Centocor CA 19-9 IRMA 12. Add 0.100 ml (100µl) of working substrate solution to all wells
test. Samples may be refrigerated at 2-8°C for a maximum period of (see Reagent Preparation Section). Always add reagents in
serum CA 19-9 values may have utility in monitoring subjects with
the above mentioned diagnosed malignancies. B. CA 19-9 Biotin Reagent – 13ml/vial ∇ five (5) days. If the specimen(s) cannot be assayed within this the same order to minimize reaction time.
One (1) vial of Anti-Human CA19-9 (MoAb)-Biotin reagent in a time, the sample(s) may be stored at temperatures of -20°C for up DO NOT SHAKE THE PLATE AFTER SUBSTRATE ADDITION
In this method, CA 19-9 calibrator, patient specimen or control is protein-stabilized matrix. A preservative has been added. to 30 days. Avoid use of contaminated devices. Avoid repetitive 13. Incubate at room temperature for fifteen (15) minutes.
first added to a streptavidin coated well. Biotinylated monoclonal freezing and thawing. When assayed in duplicate, 0.050ml (50µl) 14. Add 0.050ml (50µl) of stop solution to each well and gently mix
Store at 2-8°C.
antibody (specific for CA 19-9) is added and the reactants mixed. of the specimen is required. for 15-20 seconds.
C. CA 19-9 Enzyme Reagent – 13ml/vial - Icon E 15. Read the absorbance in each well at 450nm (using a reference
Reaction between the CA 19-9 antibodies and native CA 19-9
One (1) vial of Anti-Human CA19-9-HRP conjugate in a wavelength of 620-630nm to minimize well imperfections) in a
forms complex that binds with the streptavidin coated to the well. 7.0 QUALITY CONTROL
protein-stabilized matrix. A preservative has been added. microplate reader. The results should be read within thirty
The excess serum proteins are washed away via a wash step.
Another enzyme labeled monoclonal antibody specific to CA 19-9 Store at 2-8°C. Each laboratory should assay controls at levels in the low, normal (30) minutes of adding the stop solution.
is added to the wells. The enzyme labeled antibody binds to the D. Streptavidin Plate – 96 wells – Icon ⇓ and elevated range for monitoring assay performance. These
CA 19-9 already immobilized on the well through its binding with One 96-well microplate coated with streptavidin and packaged controls should be treated as unknowns and values determined in 10.0 CALCULATION OF RESULTS
the biotinylated monoclonal antibody. Excess enzyme is washed in an aluminum bag with a drying agent. Store at 2-8°C. every test procedure performed. Quality control charts should be
off via a wash step. A color is generated by the addition of a maintained to follow the performance of the supplied reagents. A dose response curve is used to ascertain the concentration of
E. Wash Solution Concentrate – 20ml/vial - Icon
substrate. The intensity of the color generation is directly Pertinent statistical methods should be employed to ascertain CA19-9 in unknown specimens.
One (1) vial containing a surfactant in buffered saline. A
proportional to the concentration of the CA 19-9 in the sample. trends. Significant deviation from established performance can 1. Record the absorbance obtained from the printout of the
preservative has been added. Store at 2-8°C.
indicate unnoticed change in experimental conditions or microplate reader as outlined in Example 1.
F. Substrate A – 7ml/vial - Icon SA
3.0 PRINCIPLE degradation of kit reagents. Fresh reagents should be used to 2. Plot the absorbance for each duplicate serum reference versus
One (1) vial containing tetramethylbenzidine (TMB) in acetate
determine the reason for the variations. the corresponding CA 19-9 concentration in U/ml on linear
buffer. Store at 2-8°C. See “Reagent Preparation.” graph paper (do not average the duplicates of the serum
Immunoenzymometric sequential assay (TYPE 4): G. Substrate B – 7ml/vial - Icon S
B
The essential reagents required for an immunoenzymometric 8.0 REAGENT PREPARATION references before plotting).
One (1) vial containing hydrogen peroxide (H 2 O 2 ) in acetate 3. Draw the best-fit curve through the plotted points.
assay include high affinity and specificity antibodies (enzyme and
buffer. Store at 2-8°C. See “Reagent Preparation.” 4. To determine the concentration of CA 19-9 for an unknown,
immobilized), with different and distinct epitope recognition, in 1. Wash Buffer
excess, and native antigen. In this procedure, the immobilization STOP
Dilute contents of wash solution to 1000ml with distilled or locate the average absorbance of the duplicates for each
H. Stop Solution – 8ml/vial - Icon
takes place during the assay at the surface of a microplate well deionized water in a suitable storage container. Store diluted unknown on the vertical axis of the graph, find the intersecting
One (1) vial containing a strong acid (1N HCl). Store at 2-8°C.
through the interaction of streptavidin coated on the well and buffer at 2-30°C for up to 60 days. point on the curve, and read the concentration (in U/ml) from
I. Product Instructions.
exogenously added biotinylated monoclonal anti-CA19-9 antibody. 2. Working Substrate Solution – Stable for one (1) year the horizontal axis of the graph (the duplicates of the unknown
Note 1: Do not use reagents beyond the kit expiration date. Pour the contents of vial labeled Solution ‘A’ into the vial may be averaged as indicated). In the following example, the
Upon mixing monoclonal biotinylated antibody, and a serum Note 2: Avoid extended exposure to heat and light. Opened labeled Solution ‘B’. Place the yellow cap on the mixed average absorbance (1.004) intersects the dose response
containing the native antigen, reaction results between the native reagents are stable for sixty (60) days when stored at reagent for easy identification. Mix and label accordingly. Store curve at 82.9U/ml CA 19-9 concentration (See Figure 1).
at 2-8°C.
Note: Computer data reduction software designed for ELISA concentration is obtained by multiplying the result by the of patients with ovarian cancer”, Curr Opin Gynecol, 9, 8-13
assay may also be used for the data reduction. If such dilution factor (10). (1997).
software is utilized, the validation of the software should be 10. Accurate and precise pipetting, as well as following the exact TABLE 2 Revision: 4 Date: 2019-Jul-16 DCO: 1353
ascertained. time and temperature requirements prescribed are essential. Within Assay Precision (Values in U/ml) MP3925 Product Code: 3925-300
Any deviation from Monobind’s IFU may yield inaccurate Sample N X σ C.V.
EXAMPLE 1 results. Size 96(A) 192(B)
Level 1 20 3.1 0.22 7.1%
Sample Well Mean Value 11. All applicable national standards, regulations and laws, Level 2 20 28.0 1.42 5.0% A) 1ml set 1ml set
Abs (A)
I.D. Number Abs (B) (U/ml) including, but not limited to, good laboratory procedures, must Level 3 20 161.2 4.21 2.6% B) 1 (13ml) 2 (13ml)

Reagent (fill)
A1 0.013 be strictly followed to ensure compliance and proper device C) 1 (13ml) 2 (13ml)
Cal A 0.014 0 usage.
B1 0.014 TABLE 3 D) 1 plate 2 plates
C1 0.210 12. It is important to calibrate all the equipment e.g. Pipettes, Between Assay Precision* (Values in U/ml)
Cal B 0.208 10 Readers, Washers and/or the automated instruments used E) 1 (20ml) 1 (20ml)
D1 0.212 Sample N X σ C.V.
with this device, and to perform routine preventative F) 1 (7ml) 2 (7ml)
E1 0.754 Level 1 10 3.7 0.34 9.2%
Cal C 0.708 50 maintenance. G) 1 (7ml) 2 (7ml)
F1 0.662 Level 2 10 25.3 1.81 7.1%
13. Risk Analysis - as required by CE Mark IVD Directive 98/79/EC H) 1 (8ml) 2 (8ml)
G1 1.128 Level 3 10 154.0 5.11 3.4%
Cal D 1.140 100 - for this and other devices, made by Monobind, can be
H1 1.152 requested via email from [email protected]. *As measured in ten experiments in duplicate.
A2 1.850
Cal E 1.805 250 12.2 Interpretation 14.2 Sensitivity
B2 1.760 The CA 19-9 AccuBind® ELISA test system has a sensitivity of
C2 2.310 1. Measurements and interpretation of results must be
Cal F 2.355 500 performed by a skilled individual or trained professional. 1.0 U/ml. The sensitivity was ascertained by determining the
D2 2.400 variability of the ‘0’ calibrator and using the 2σ (95% certainty)
2. Laboratory results alone are only one aspect for determining
A3 1.009 patient care and should not be the sole basis for therapy, statistic to calculate the minimum dose.
Patient 1.004 82.9
B3 0.999 particularly if the results conflict with other determinants.
14.3 Accuracy
3. The reagents for the test system procedure have been
The CA 19-9 AccuBind® ELISA test system was compared with a
Figure 1 formulated to eliminate maximal interference; however,
reference method. Biological specimens from low, normal, and
potential interaction between rare serum specimens and test
2.500 elevated concentrations were assayed. The total number of such
reagents can cause erroneous results. Heterophilic antibodies
specimens was 136. The least square regression equation and
often cause these interactions and have been known to be
2.000 the correlation coefficient were computed for the CA 19-9 in
problems for all kinds of immunoassays. (Boscato LM Stuart
comparison with the reference method. The data obtained is
Absorbance(s)

MC. ‘Heterophilic antibodies: a problem for all immunoassays’

1.500 displayed in Table 4.
Clin.Chem. 1988:3427-33). For diagnostic purposes, the
1.000 Patient results from this assay should be used in combination with TABLE 4
clinical examination, patient history and all other clinical
Method Mean
Least Square Correlation
0.500 findings.
Regression Analysis Coefficient
4. For valid test results, adequate controls and other parameters
0.000 must be within the listed ranges and assay requirements. This Method (X) 18.62 x = 1.4577+0.8837(y) 0.955
0 50 100 150 200 250 300 350 400 450 500 5. If test kits are altered, such as by mixing parts of different kits, Reference (Y) 19.43
which could produce false test results, or if results are
CA 19-9l Values in U/ml
incorrectly interpreted, Monobind shall have no liability. 14.4 Specificity
6. If computer controlled data reduction is used to interpret the In order to test the specificity of the antibody pair used massive
*The data presented in Example 1 and Figure 1 is for illustration results of the test, it is imperative that the predicted values for concentrations of possible cross-reactants were added to known
only and should not be used in lieu of a dose response curve the calibrators fall within 10% of the assigned concentrations. serum pools and assayed in parallel with the base sera. No cross
prepared with each assay. 7. CA 19-9 has a low clinical sensitivity and specificity as a tumor reaction was found. Percent cross-reactions for some of these
marker. Clinically an elevated CA 19-9 value alone is not of additions are listed below in Table 5.
11.0 Q.C. PARAMETERS diagnostic value as a test for cancer and should only be
TABLE 5
used in conjunction with other clinical manifestations
In order for the assay results to be considered valid the (observations) and diagnostic parameters. Analyte Concentration Percent (%)
following criteria should be met: Cross Reaction
1. The absorbance (OD) of calibrator F should be > 1.3 13.0 EXPECTED RANGES OF VALUES CA 19-9 - 100
2. Four out of six quality control pools should be within the CA 125 10000 U/ml 0.001
established ranges. The serum CA 19-9 is elevated in 1% of normal healthy women, CA 15-3 1000 U/ml ND*
3% of normal healthy women with benign ovarian diseases, 6% of PSA 5000 ng/ml ND*
12.0 RISK ANALYSIS patients with non-neoplastic conditions (including but not limited to AFP 10000 ng/ml ND*
first trimester pregnancy, menstruation, endometriosis uterine CEA 10000 ng/ml ND*
The MSDS and Risk Analysis Form for this product are available fibrosis, acute salphingitis, hepatic diseases and inflammation of HCG 10000 mIU/ml ND*
on request from Monobind Inc. peritoneum or pericardium). RF 1000 kIU/ml ND*

12.1 Assay Performance TABLE I 15.0 REFERENCES

1. It is important that the time of reaction in each well is held Expected Values for CA 19-9 AccuBind® ELISA Test System
constant to achieve reproducible results. Healthy and non-pregnant subjects < 40 U/ml 1. Zamcheck, N, Adv Intern Med, 19, 413 (1974).
2. Pipetting of samples should not extend beyond ten (10) 2. Rayncao G, Chu TM, JAMA, 220, 381 (1972).
minutes to avoid assay drift. It is important to keep in mind that establishment of a range of 3. Harrison, Principles of Internal Medicine, McGraw Hill Book
3. Highly lipemic, hemolyzed or grossly contaminated values, which can be expected to be found by a given method for Company, New York, 12th Ed.
specimen(s) should not be used. a population of "normal" persons, is dependent upon a multiplicity 4. Wild D, The Immunoassay Handbook, Stockton Press, 444
4. If more than one (1) plate is used, it is recommended to repeat of factors: the specificity of the method, the population tested and (1994).
the dose response curve. the precision of the method in the hands of the analyst. For these 5. Hasholzner U, Steiber P, Baumgartner L, Pahl H, Meier W,
5. The addition of substrate solution initiates a kinetic reaction, reasons, each laboratory should depend upon the range of Fateh-Moghadam A, “Methodological and clinical evaluation of
which is terminated by the addition of the stop solution. expected values established by the Manufacturer only until an three automated CA 19-9 assays compared with CA 19-9 II
Therefore, the substrate and stop solution should be added in in-house range can be determined by the analysts using the RIA (Centocor)”, Tumor Diagnosis & Ther, 15,114-117(1994).
the same sequence to eliminate any time-deviation during method with a population indigenous to the area in which the 6. Hasholzner U, Steiber P, Baumgartner L, Pahl H, Meier W,
reaction. laboratory is located. Fateh-Moghadam A, ”Clinical significance of the tumor
6. Plate readers measure vertically. Do not touch the bottom of markers CA 19-9 II and CA 72-4 in ovarian carcinoma”, Int J
the wells. 14.0 PERFORMANCE CHARACTERISTICS Cancer , 69, 329-34 (1996).
7. Failure to remove adhering solution adequately in the 7. Ovarian Cancer – NIH Consensus Conference, JAMA, 273,
aspiration or decantation wash step(s) may result in poor 14.1 Precision 491-497 (1995).
replication and spurious results. The within and between assay precision of the CA 19-9 8. Daoud E, Bodor G, Weaver C, Landenson JH and Scott MG,
8. Use components from the same lot. No intermixing of reagents AccuBind® ELISA test system were determined by analyses on “CA 19-9 concentrations in malignant and non-malignant
from different batches. three different levels of control sera. The number, mean value, disease”, Washington University Case Conference, Clin
9. Patient specimens with CA 19-9 concentrations above standard deviation (σ) and coefficient of variation for each of these Chem, 37, 1968-74 (1991).
500U/ml may be diluted (for example 1/10 or higher) with control sera are presented in Table 2 and Table 3. 9. De Bruijn HWA, Van Der Zee AGJ & Alders JG, “The value of
CA19-9 zero calibrator and re-assayed. The sample’s Cancer Antigen 125 (CA 19-9) during treatment and follow up