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					                                                                    specimen's activity can be correlated with free - Chorionic                               Note: The calibrators, human serum based, were
                                                                    Gonadotropin concentration.                                                                calibrated using a reference preparation, which was          REAGENT PREPARATION:
  Diagnostic Automation, Inc.                                                                                                                                  assayed against the WHO 1st IRP (75/551).
                                                                                                                                                                                                                            1. Wash Buffer
         23961 Craftsman Rd, Suite E/F                              PRINCIPLE                                                                          B.      Enzyme-Conjugate       and   Biotinylated    Monoclonal
                                                                                                                                                                                                                                  Dilute contents of Wash Concentrate to 1000ml with distilled
             Calabasas, CA 91302                                                                                                                               antibody —13ml/vial - Icon E                                       or deionized water in a suitable storage container. Store at
                                                                    Immunoenzymometric sequential assay:
   Tel:( 818)-591-3030,Fax:( 818)-591-8383                                                                                                                     One (1) vial containing enzyme labeled antibody,                   room temperature 20-27C for up to 60 days.
                                                                    The essential reagents required for an immunoenzymometric                                  biotinylated monoclonal mouse IgG in buffer, dye, and        3. Working Substrate Solution
          Email:onestep@rapidtest.com                               assay include high affinity and specificity antibodies (enzyme                             preservative. Store at 2-8C.                                   Mix equal volumes of Solution „A‟ and Solution „B‟ in a clean
           Website: www.rapidtest.com                               and immobilized), with different and distinct epitope recognition,                                                                                         container. Use within 60 minutes. For example, add 1ml of A
                                                                    in excess, and native antigen. In this procedure, the                              C.      Streptavidin Coated Microplate -- 96 wells - Icon              and 1ml of B for two (2) eight well strips (A slight excess of
                                                                    immobilization takes place during the assay at the surface of a                            One 96-well microplate coated with streptavidin and             solution is made. Discard the unused portion).
                                                                    microplate well through the interaction of streptavidin coated on                          packaged in an aluminum bag with a drying agent. Store
                                                                    the well and exogenously added biotinylated monoclonal anti-                               at 2-8C.
                                                                    fß-hCG antibody.                                                                                                                                        TEST PROCEDURE
       Free -HCG (f-hCG)                                          Upon mixing monoclonal biotinylated antibody, and a serum
                                                                                                                                                       D.      Wash Solution Concentrate – 20 ml - Icon
                                                                                                                                                               One (1) vial containing a surfactant in buffered saline. A   Before proceeding with the assay, bring all reagents, serum
 Chemiluminescence Immunoassay                                      containing the native antigen, reaction results between the
                                                                    native antigen and the antibody, forming an Antibody-Antigen
                                                                                                                                                               preservative has been added. Store at 2-30C.                references and controls to room temperature (20 - 27 C).
                   Cat. No. 9043-C                                  complex. Simultaneously the biotin attached to the antibody                        E.      Substrate A --7ml/vial - Icon SA                             1.     Format the microplates‟ wells for each serum reference,
                                                                    binds to the streptavidin coated on the microwells resulting in a                                                                                              control and patient specimen to be assayed in duplicate.
                                                                                                                                                               One (1) bottle containing Luminol in buffer.     Store at
Intended Use: The Quantitative Determination of Free Beta           complete immobilization of the complex. The interaction is                                                                                                     Replace any unused microwell strips back into the
                                                                                                                                                               2-8C.
(ß) Chorionic Gonadotropin Subunit Concentration in                 illustrated by the following equation:                                                                                                                         aluminum bag, seal and store at 2-8C.
Human Serum by a Microplate Immunoenzymometric assay                                                                                                                                           B
                                                                                                                                                       F.      Substrate B -- 7ml/vial - Icon S                             2.     Pipette 0.025 ml (25µl) of the appropriate serum reference,
                                                                                                                         ka
                                                                                                                                                               One (1) bottle containing hydrogen peroxide (H 2O2) in              control or specimen into the assigned well.
                                                                                                  Btn
SUMMARY AND EXPLANATION OF THE TEST                                              Ag(fhCG) +             Ab(m)                 Ag(fhCG) -BtnAb(m)             buffer. Store at 2-8C.
                                                                                                                                                                                                                            3.     Add 0.100 ml (100µl) of the biotinylated antibody to each
                                                                                                                         k-a
                                                                                                                                                       H.      Product Insert.                                                     well. It is very important to dispense all reagents close
Human chorionic gonadotropin (hCG) concentration increases          Btn                                                                                                                                                            to the bottom of the coated well.
                                                                         Ab(m) = Biotinylated Monoclonal Antibody (Excess Quantity)
dramatically in blood and urine during normal pregnancy. hCG
is secreted by placental tissue, beginning with the primitive       AgI(fhCG) = Native Antigen (Variable Quantity)                                                                                                         4.     Swirl the microplate gently for 20-30 seconds to mix and
                                                                                                                                                       Note 1: Do not use reagents beyond the kit expiration date.
trophoblast, almost from the time of implantation, and serves to                                                                                                                                                                   cover.
                                                                    Ag(fhCG) -BtnAb(m)= Antigen-Antibody complex (Variable Quan.))                    Note 2: Opened reagents are stable for sixty (60) days when
support the corpus luteum during the early weeks of pregnancy.                                                                                                 stored at 2-8C.                                             5.     Incubate 60 minutes at room temperature.
hCG or hCG similar glycoproteins can also be produced by a          ka     = Rate Constant of Association                                              Note 3: Above reagents are for a single 96-well microplate.          6.     Discard the contents of the microplate by decantation or
wide variety of trophoblastic and nontrophoblastic tumors. The      k-a = Rate Constant of Disassociation
                                                                                                                                                       Required But Not Provided:                                                  aspiration. If decanting, tap and blot the plate dry with
measurement of hCG, by assay systems with suitable sensitivity
                                                                    Simultaneously, the complex is deposited to the well through                                                                                                   absorbent paper.
and specificity has proven great value in the detection of                                                                                             1. Pipette capable of delivering 50µl volumes with a precision
pregnancy and the potential diagnosis of early pregnancy            the high affinity reaction of streptavidin and biotinylated                                                                                             7.     Add 300µl of wash buffer (see Reagent Preparation
                                                                    antigen. This interaction is illustrated below:                                       of better than 1.5%.
disorders. Free ß-hCG subunit testing has improved the                                                                                                                                                                             Section), decant (tap and blot) or aspirate. Repeat two (2)
                                                                                                                                                       2. Dispenser(s) for repetitive deliveries of 0.100ml and 0.300ml
                                                                    Ag(fhCG) - Ab(m) + StreptavidinC.W.  immobilized complex (IC)
                                                                                Btn                                                                                                                                                additional times for a total of three (3) washes. An
diagnostic probability of abnormal pregnancy/disease states                                                                                               volumes with a precision of better than 1.5%.
(1).                                                                                                                                                   3. Microplate washers or a squeeze bottle (optional).                       automatic or manual plate washer can be used. Follow
                                                                    StreptavidinC.W. = Streptavidin immobolized on well
                                                                                                                                                       4. Microplate Luminometer.                                                  the manufacturer’s instruction for proper usage. If a
Patients with trophoblastic diseases produce ordinary and
                                                                    Immobilized complex (IC) = Ag-Ab bound to the well                                                                                                             squeeze bottle is employed, fill each well by
irregular forms of hCG; e.g. nicked hCG, hCG missing the the                                                                                           5. Absorbent Paper for blotting the microplate wells.
ß-subunit C-terminal segment, hyperglycosylated hCG and free        After a suitable incubation period, the antibody-antigen bound                     6. Plastic wrap or microplate cover for incubation steps.                   depressing the container (avoiding air bubbles) to
ß subunit. On the other hand common epithelial tumors of the        fraction is separated from unbound antigen by decantation or                       7. Vacuum aspirator (optional) for wash steps.                              dispense the wash. Decant the wash and repeat two
urogenital tract frequently express the free ß-Subunit of hCG       aspiration. Another antibody (directed at a different epitope)                     8. Timer.                                                                   (2) additional times.
with no concomiotant expression of its heterodimer partner, the     labeled with an enzyme is added. Another interaction occurs to                     9. Quality control materials.                                        8.     Add 0.100 ml (100µl) of the Anti HCG-enzyme labeled
common -subunit of the glycoprotein hormone. (5) While most        form an Enzyme labeled Antibody-Antigen-Biotinylated-                              .                                                                           antibody to each well.
hCG assays do a very good job of monitoring the normal              Antibody complex on the surface of the wells. Excess enzyme
                                                                    is washed off via a wash step. A suitable substrate is added to
                                                                                                                                                       PRECAUTIONS                                                          9.     Swirl the microplate gently for 20-30 seconds to mix and
pregnancies, still there needs to be a system of differential
                                                                    produce color measurable with the use of a microplate                                                 For In Vitro Diagnostic Use                              cover.
diagnosis of ovarian tumors, epithelial tumors and trophoblastic
malfunctions. That is where determination of free -subunit,        spectrophotometer. The enzyme activity on the well is directly                          Not for Internal or External Use in Humans or Animals           10. Incubate 60 minutes at room temperature.
free -subunit, nicked hCG and nonnicked hCG etc are of             proportional to the native free antigen concentration. By
                                                                                                                                                       All products that contain human serum have been found to be          11. Discard the contents of the microplate by decantation or
individual value.                                                   utilizing several different serum references of known antigen
                                                                                                                                                       non-reactive for Hepatitis B Surface Antigen, HIV 1&2 and HCV            aspiration. If decanting, blot the plate dry with absorbent
                                                                    concentration, a dose response curve can be generated from
Although fß-hCG normally constitutes less than 1% of the total                                                                                         Antibodies by FDA licensed reagents. Since no known test can             paper.
                                                                    which the antigen concentration of an unknown can be
hCG concentrations in normal pregnancy, it constitutes a                                                                                               offer complete assurance that infectious agents are absent, all
                                                                    ascertained.                                                                                                                                            12. Add 300µl of wash buffer (see Reagent Preparation
significant part (as much as 26% of hCG) in trophoblast disease                                                                                        human serum products should be handled as potentially
                                                                                                                    k
                                                                                                                                                                                                                                Section), decant (tap and blot) or aspirate. Repeat two (2)
(2,3). There is also increasing evidence that free beta subunit                                                      b                                 hazardous and capable of transmitting disease.             Good
                                                                                                                                                                                                                                additional times for a total of three (3) washes. An
may be better than total hCG measurement in assessing                                           Enz                           Enz                      laboratory procedures for handling blood products can be found
                                                                                       (IC) +         Ab(x-fhCG)                   Ab(x-fhCG) - IC                                                                            automatic or manual plate washer can be used. Follow
Down‟s Syndrome (4).                                                                                                                                   in the Center for Disease Control / National Institute of Health,
                                                                                                                                                                                                                                the manufacturer’s instruction for proper usage. If a
                                                                                                                    k
                                                                                                                     -b                                "Biosafety in Microbiological and Biomedical Laboratories," 2nd
In this method, fß-hCG calibrator, patient specimen or control is   Enz                                                                                                                                                         squeeze bottle is employed, fill each well by
                                                                       Ab(x-fb-hCG)   = Enzyme labeled Antibody (Excess Quantity)                      Edition, 1988, HHS Publication No. (CDC) 88-8395.
first added to a streptavidin coated well. Biotinylated                                                                                                                                                                         depressing the container (avoiding air bubbles) to
                                                                    Enz
monoclonal antibody (specific for fß-hCG) is added and the                Ab(x-fhCG) – IC = Antigen-Antibodies Complex                                                                                                         dispense the wash. Decant the wash and repeat two
reactants mixed. Reaction between the fß-hCG antibody and           kb = Rate Constant of Association                                                  SPECIMEN COLLECTION AND PREPARATION                                      (2) additional times.
native fß-hCG forms complex that binds with the streptavidin
                                                                    k-b = Rate Constant of Dissociation                                                The specimens shall be blood; serum in type and the usual            13. Add 0.100 ml (100µl) of working substrate solution to all
coated to the well. The excess serum proteins are washed away
                                                                                                                                                       precautions in the collection of venipuncture samples should be          wells (see Reagent Preparation Section). Always add
via a wash step. Another enzyme labeled monoclonal antibody
                                                                                                                                                       observed. For accurate comparison to established normal                  reagents in the same order to minimize reaction time
specific to f-hCG is added to the wells. The enzyme labeled                                                                                           values, a fasting morning serum sample should be obtained.               differences between wells.
antibody binds to the f-hCG already immobilized on the well        REAGENTS                                                                           The blood should be collected in a plain redtop venipuncture
through its binding with the biotinylated monoclonal antibody.                                                                                         tube without additives or anti-coagulants. Allow the blood to clot   14.     Incubate at room temperature for five (5) minutes.
                                                                    Provided:
Excess enzyme is washed off via a wash step. A color is                                                                                                for samples. Centrifuge the specimen to separate the serum           15. Read the relative light units in each well for 0.2-1.0 seconds.
generated by the addition of a substrate. The intensity of the      A.       f ß -hCG Calibrators --1ml/vial - Icons A-F
                                                                                                                                                       from the cells.
                                                                                                                                                                                                                             The results should be read within thirty (30) minutes of
color generation is directly proportional to the concentration of            Six (6) vials of references fß--hCG Antigen at levels of                  Samples may be refrigerated at 2-8oC for a maximum period of
the f-hCG in the sample.                                                                                                                                                                                                   adding the substrate solution.
                                                                             0(A), 5(B), 10(C), 25(D), 50(E) and 100(F) mIU/ml. Store                  five (5) days. If the specimen(s) cannot be assayed within this
The employment of several serum references of known free -                  at 2-8C. A preservative has been added.                                  time, the sample(s) may be stored at temperatures of -20oC for
Chorionic Gonadotropin levels permits the construction of a                                                                                            up to 30 days. void repetitive freezing and thawing. When
dose response curve of activity and concentration. From                                                                                                assayed in duplicate, 0.050ml of the specimen is required.
comparison to the dose response curve, an unknown
QUALITY CONTROL                                                          EXAMPLE 1                                                                            concentration is obtained by multiplying the result by the       REFERENCES
                                                                                                                                                              dilution factor (100).
Each laboratory should assay controls at levels in the low,                                                                                                                                                                    1. Henry JB, Clinical Diagnosis and Management by
medium and high ranges of the dose response curve for                                                                                                    7. Each component in one assay should be of the same lot




                                                                                                                                         Concentration
                                                                                                                                                                                                                                  Laboratory Methods, W.B. Saunders Company (1996) p




                                                                                              Well Position
monitoring assay performance. These controls should be                                                                                                      number and stored under identical conditions.                         486-491.




                                                                                                                       RLU (B)
                                                                                     Sample




                                                                                                                                 Mean
treated as unknowns and values determined in every test




                                                                                                                RLU
                                                                              I.D.
procedure performed. Quality control charts should be                                                                                                    B. Interpretation                                                     2. Osturk M., et al., Endocrinology, 120, 549-58 (1987)
maintained to follow the performance of the supplied reagents.
Pertinent statistical methods should be employed to ascertain                                                                                            If computer controlled data reduction is used to interpret the        3. Cole La, et al., Serono Symposia Publ., 65, 59-78 (1989)
trends. Significant deviation from established performance can                                                                                           results of the test, it is imperative that the predicted values for
indicate unnoticed change in experimental conditions or                                                                                                  the calibrators fall within 10% of the assigned concentrations.       4. Macri JN., and Spencer K., Am J Obstet Gynecol, 174, 1668-
                                                                                              A1               406
degradation of kit reagents. Fresh reagents should be used to                 Cal A                                      413             0                                                                                        69 (1996).
determine the reason for the variations.                                                      B1               421
                                                                                              C1               2744                                      PERFORMANCE CHARACTERISTICS                                           5. Cole LA. “Immunoassay of human chorionic gonadotropin
RESULTS                                                                       Cal B                                     2832             5                                                                                        and its free subunits, and metabolites.” Clin.Chem
                                                                                              D1               2921                                      A.    Precision                                                          43;12:2233-2243. 1997.
A dose response curve is used to ascertain the concentration of                               E1              12931                                                                                                            6. Cole LA, Isozaki T. Palomaki G, Canick J, Kellner L, Saller
fß-Chorionic Gonadotropin in unknown specimens.                              Cal C                                     12664            10               The within and between assay precision of the fß-hCG CIA
                                                                                              F1              12397                                                                                                               D, Cuckie H. “Detection of -Core fragment in second
                                                                                                                                                         Microplate Procedure were determined by analyses on three
                                                                                                                                                                                                                                  trimester Down‟s syndrome pregnancies.” Early Human
1.   Record the RLU‟s obtained from the printout of the                                       G1              25944                                      different levels of control sera. The number, mean value,
                                                                             Cal D                                     26384            25                                                                                        Development 1996;47:S47-49.
     microplate reader as outlined in Example 1.                                                                                                         standard deviation and coefficient of variation for each of these
                                                                                              H1              26824                                                                                                            7. Cole LA, “ core fragment.( -core, UGP or UGF)” Tumor
                                                                                                                                                         control sera are presented in Table 3 and Table 4.
2.   Plot the RLU‟s for each duplicate serum reference versus                                                                                                                                                                     marker Update 1994;6:69-75.
                                                                                              A2              53935
     the corresponding hCG concentration in mIU/ml on linear                  Cal E                                    57086            50                                         TABLE 2                                     8. Yamanaka N, Kawabata G, Morisue K, Hazama M,
     graph paper.                                                                             B2              60238
                                                                                                                                                                    Within Assay Precision (Values in mIU/ml)                     Nishimura R.”Urinary hCG -core fragment as atumor
3.   Draw the best-fit curve through the plotted points.
                                                                              Cal F
                                                                                              C2              106907
                                                                                                                       108920           100              Sample            N        X                        C.V.                marker for bladder cancer.” Nippon Hinyokika Gakkai
4.   To determine the concentration of uCG for an unknown,                                                                                                                                                                        Zasshi 1993;84:700-706.
                                                                                              D2              110934                                     Level 1          20       3.5        0.15            4.3%
     locate the average absorbance of the duplicates for each
                                                                                                                                                         Level 2          20      10.5        0.55            5.2%             9. Kinugasa M, Nishimura R, Hasegawa K, okamura M, Kimura
     unknown on the vertical axis of the graph, find the                                      E2               3194
                                                                           Ctrl 1                                       3269            2.2              Level 3          20      49.6        1.50            3.0%                A, Ohtsu F.”Assessment of urinary -core fragment of hCG
     intersecting point on the curve, and read the concentration
                                                                                              F2               3344                                                                                                               as a tumor marker of cervical cancer.” Acta Obstet Gynecol.
     (in mIU/ml) from the horizontal axis of the graph (the
                                                                                                                                                                                  TABLE 3                                         Jpn 1992;11:188-94.
     duplicates of the unknown may be averaged as indicated).                                 G2              30957
                                                                           Patient 1                                   32255            31.3                         Between Assay Precision* (Values in mIU/ml)
                                                                                              H2              33554                                                                                                            10. Cole LA.”stability of hCG free -subunit in urine.” Prenat.
Note: Computer data reduction software designed for IEMA                                                                                                                                                                           Diagnosis 1996;17:185-89.
assays may also be used for the data reduction.                                               A3              11415                                      Sample            N        X                        C.V.
                                                                            Patient 2                                  11534            9.1              Level 1          10       3.1        0.17            5.5%             11. Sancken U, Bahner D.”The effect of thermal instability of
                                                                                              B3              11653                                                                                                                intact human chorionic gonadotropin (ihCG) on the
                                                                                                                                                         Level 2          10      11.2        0.71            6.3%
                             FIGURE 1                                                                                                                    Level 3          10      48.5        1.75            3.6%                 application of its free -subunit as a serum marker of
                                                                                                                                                                                                                                   Down‟s syndrome screening.” Prenat. Diagnosis
                                                                         *The data presented in Example 1 and Figure 1 are for                           *As measured in ten experiments in duplicate.                             1995;15:731-738.
           120000
                                                                         illustration only and should not be used in lieu of a dose                                                                                            12. Javadpour N.”Current status of tumor markers in testicular
           100000
                                                                         response curve prepared with each assay.                                        B. Sensitivity                                                            cancer.” A Practical Review. Eur Urol 1992;21:34-36.
                                                                                                                                                                                                                                   (Suupl).
                                                                         Q.C. PARAMETERS                                                                 The chorionic gonadotropin procedure has a sensitivity of 0.025
            80000

                                                                                                                                                         mIU. This is equivalent to a sample containing 1 mIU/ml fß-hCG        13. Canick JA, Kellner DN Jr, Palomaki GE, Walker RP,
                                                                                                                                                                                                                                   Osathanondh R.”Second trimester levels of maternal urinary
                                                                         Maximum Absorbance (Calibrator „F‟) = >1.2                                      concentration.
     RLU




            60000
                                                                                                                                                                                                                                   gonadotropin peptide in Down syndrome pregnancy.”
                                                                         Maximum Absorbance (Calibrator „A‟) = < 0.1
                                                                                                                                                         C. Specificity                                                            Prenat. Diagnosis 1995;15:752-759.
            40000


                                                                         LIMITATIONS OF PROCEDURE
            20000                                                                                                                                        The cross-reactivity of the fß-Chorionic Gonadotropin CIA
                                                                         A. Assay Performance                                                            method to selected substances was evaluated by adding the
                0                                                                                                                                        interfering substance to a serum matrix at various
                    0   20     40       60      80      100        120

                                    mIU/ml                               1. It is important that the time of reaction in each well is held               concentrations, The cross-reactivity was calculated by deriving
                                                                            constant for reproducible results. Pipetting of samples                      a ratio between dose of interfering substance to dose of
                                                                            should not extend beyond ten (10) minutes to avoid assay                     Chorionic Gonadotropin needed to produce the same
                                                                            drift. If more than one (1) plate is used, it is recommended to              absorbance.
                                                                            repeat the dose response curve.                                              Substance                          Cross Reactivity
                                                                                                                                                         fß-hCG subunit                            1.0000
                                                                         2. Addition of the substrate solution initiates a kinetic reaction,             Intact Chorionic Gonadotropin (hCG)     < 0.0001
                                                                            which is terminated by the addition of the stop solution.                    Follicle Stimulating Hormone (FSH)      < 0.0001
                                                                            Therefore, the addition of the substrate and the stopping                    Luteinizing Hormone (LH)                < 0.0001
                                                                            solution should be added in the same sequence to eliminate                   Thyroid Stimulating Hormone (TSH)       < 0.0001
                                                                            any time deviation during reaction.

                                                                         3. Plate readers measure vertically. Do not touch the bottom of
                                                                            the wells.

                                                                         4. Failure to remove adhering solution adequately in the                                                                                                                    Revision:2 Date: 072302
                                                                            aspiration or decantation wash step(s) may result in poor                                                                                                           F/Insert/List D/Free B HCG_9043-C
                                                                            replication and spurious results.

                                                                         5. Sample(s), which are contaminated microbiologically, should
                                                                            not be used in the assay. Highly lipemeic or hemolysed
                                                                            specimen(s) should similarly not be used.

                                                                         6. Patient specimens with fß-hCG concentrations above 100
                                                                            mIU/ml may be diluted (for example 1/100) with normal male
                                                                            serum (fß-hCG < 1 mIU/ml) and re-assayed. The sample‟s

				
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