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Comparison of Commercial Kits for Radioimmunoassay Ill


									dIN. CHEM.21/13, 1922-1926(1975)

Comparison of Commercial Kits for Radioimmunoassay:

Ill. Radioassay of Serum Folate

Norman P. Kubasik, Michael T. Volosin, and Harrison E. Sine

We evaluated six commercial radioassay kits in which                                    (3H) Folic Acid Assay Kit; Diagnostic             Products
tritium or iodine tracers are used to measure serum to-                              Corp., 9325 Venice Blvd., Culver City, Calif. 90230.
late. We assessed the adequacy of the literature sup-                                   1251-Folate Kit; RIA Products,     Inc., P.O. Box 914,
piled with the kits, within-run precision, reagent stability,                        Waltham,      Mass. 02154.
analytical recovery of added pteroylglutamic      acid and
                                                                                        “Total” 3H Folate Kit; RIA Products,        Inc.
N-methyltetrahydrofolic acid, results of analysis of pa-
                                                                                        Table 1 gives the salient features     (label, standard,
tients’ samples, and minimum detection limits. There
were serious discrepancies among manufacturers’  kits                                binder, etc.) of each kit. All manufacturers        employed
in each of these respects. Such discrepancies could af-                              pteroylglutamic     acid (PGA) as tracer and all but one
fect interpretation of results and of data on like samples                           used N-methyltetrahydrofolic      acid (MTHFA)          as the
derived from different laboratories that are using differ-                           standard. Four manufacturers    stated that beta-lacto-
ent kits. Laboratories are urged to assess carefully the                             globulin was the specific binder used and two stated
materials and method supplied by a manufacturer be-                                  that a binding protein     from milk was used. Three of
fore putting a kit to clinical use.                                                  the folate kits used a two-stage    assay system similar
                                                                                     to that described    by Rothenberg  et al. (2).
    Several          radioassay      procedures         have recently       been
                                                                                        A two-stage    assay consists of a sequential incuba-
described     for measurement       of serum folate (1-7). Ra-
                                                                                     tion system    in which the binder is first incubated
dioassay is an alternative       to the older microbiological
                                                                                     with   the unlabeled  standard or serum. The tempera-
assay, which is unavailable          to most hospitals and is
                                                                                     ture is lowered to 4 #{176}C, minimizes the dissocia-
subject   to certain    pitfalls   and problems.    The avail-
                                                                                     tion of unlabeled       folate from binder.           Tracer     is then
ability   of folate    radioassay      kits from commercial
                                                                                     added, which occupies            only the unoccupied            binding
manufacturers      now allows more laboratories       to deter-
                                                                                     sites.  The  two-stage      assay    system      is considered       non-
mine it routinely.      Kits are available     with either tn-
tiated        or iodinated          tracers.      In principle,       the latter          The one-stage     assay system          involves      competition
offer        distinct       advantages         over   the   former.    Gamma-
                                                                                     in the classical sense. Tracer, standard                or serum, and
labeling allows for higher specific activities                          and the
                                                                                     binder are incubated         together      in a competitive        radio-
elimination   of cumbersome       beta-counting                            tech-
                                                                                     logical inhibition    assay.
niques, resulting    in a savings of cost and time. The
                                                                                         The elimination       of serum blanks,           via assay tubes
problems    of sample quenching      are also eliminated.
                                                                                     that contain all the necessary            reactants     except specif-
     We have assessed the precision,     accuracy,   sensitivi-
                                                                                     ic binder, were available with two folate kits. A high
ty, and clinical usefulness     of six commercially       avail-
                                                                                     pH buffer and heating in a boiling water bath was
able folate radioassay    kits (three iodinated     and three
                                                                                     used to destroy       the capacity          of serum        proteins      to
tritiated),  and describe our findings here.
                                                                                     bind folate without an effect on the folate itself.
Methods and Materials                                                                    For particular     method       details,     one is referred          to
                                                                                     the inserts supplied      by each kit manufacturer.                  In all
   The following folate radioassay        kits were obtained
                                                                                     kits dextran-coated       charcoal        was used, followed             by
commercially    from November         27, 1974, to February
                                                                                     cold centrifugation         to separate           free folate         from
26, 1975, and evaluated     during the same period:
                                                                                     bound. In addition,      all incubation         steps were done in
   ‘251-Folate  Radioassay      Kit; Clinical   Assays,   Inc.,
                                                                                     the dark. All tnitium-labeled             folate supernates           were
237 Binney St., Cambridge,       Mass. 02142.
                                                                                     decanted    into 10 ml of “Aquasol”             liquid-scintillation
   3H-Folic Acid Radioassay       Kit; Clinical Assays, Inc.
                                                                                     solvent (New England          Nuclear,       Boston, Mass. 02118)
   ‘251-Folate;  Diagnostic     Biochemistry,     Inc., 10473
                                                                                     and counted      on a Beckman          LS-230 Liquid Scintilla-
Roselle St., San Diego, Calif. 92121.
                                                                                     tion System (Beckman           Instruments,          Fullerton,      Calif.
                                                                                     92634), with use of an automatic                external      standard-
   Clinical      Laboratories,  The Genesee    Hospital, 224 Alexander        St.,
Rochester,       N. Y. 14607.                                                        ization for quench corrections.             Iodine-labeled         folates
   Received       July 10, 1975; accepted  Sept. 22, 1975.                           were counted        on a Model            1185 Gamma              System

1922         CLINICAL CHEMISTRY,         Vol. 21, No. 13, 1975
                                 Table 1. Key Features of Six Commercial Kits for Folate Radioassaya
                                                                                                                                                               Use of            Normal
                                                                                                                                                               serum              range.
     Kit manufacturer                           Label1’        Standard                            Binder                             Assay type               blank             sg/Iiter
Clinical Assays                                  1251         MTHFAC                   Milk      binding      protein                Two-stage                 Nod                5-20
Clinical Assays                                  3H           MTHFA                    Milk      binding      protein                Two-stage                 Yes                4-16
Diagnostic       Biochemistry                    1251         PGA                      Beta-lacto          globulin                  One-stage                 Yes                2-14
Diagnostic       Products                        3H           MTHFA                    Beta-lacto          globulin                  One-stage                 No                    3-21
RIA Products                                     1251         MTHFA                    Beta-lacto globulin                           One-stage                 Yes                4-16
RIA Products                                     3H            MTHFA                   Beta-lacto          globulin                  Two-stage                 Yes                4-16
  a Dextran-coated     charcoal used in the separation in each case. A tabulation    of lot numbers, for those who are interested in them, can be
obtained from the authors or from the Editorial Office.
  hOn pteroylglutamic      acid.
  C N-Methyltetrahydrofol     ic acid.
  d Boiling is used. However, serum background        tubes are run with two patients’ sera. The average value of the two background tubes is then
used as the combined        nonspecific       binding   and background    correction    factor     for each serum sample being assayed.

      Table 2. Rating of the Literature Supplied                                                                 Table 3. Within-run Precision for
           with the Foiate Radioassay Kits                                                                        the Analysis of Serum Folate
                   Manufacturer                            Isotope        Rating0                                                                Mean                SD
                                                                                                       Kit                                    _____________
    Clinical Assays,Inc.                                     1251           8.5                  manufacturer                Isotope                      Mg/liter                    CV, %
    Clinical Assays,Inc.                                     3H             7.5               Clinical                         ‘‘l                4.13              0.07                  1.6
    Diagnostic        Biochemistry,           Inc.           1251           6.5               Assays                                             17.07              2.36                 13.8
     Diagnostic       Products        Corp.                  3H             8.5               Clinical                         3H                 1.24              0.28                 22.4
     RIA Products,          Inc.                             1251           2.5               Assays                                             11.37              0.29                  2.6
     RIA Products,          Inc.                             3H             3.0               Diagnostic                       ‘I                 4.33              0.32                  7.5
  a Perfect score, 11 .0.                                                                     Biochemistry                                         8.38             0.27                  3.2
                                                                                              Diagnostic                       3H                 2.98              0.66                 22.2
                                                                                              Products                                           16.97              0.54                  3.2
                                                                                                                               1251               3.30
                                                                                              RIA Products                                                          0.19                  5.7
(Seanle Analytic,    Des Plaines,        Ill. 60018). The radio-                                                                                 18.28              0.60                  3.3
activity of both tritium-        and iodine-labeled         samples                           RIA Products                     3H                  3.54             0.25                  7.1
was counted     for at least 5 mm, to reduce and negate                                                                                          15.79              0.31                  2.0
the effects      of counting        error     on assay       results.                           Different sample pools were used for the different                         kits. n   =   20 at
MTHFA       (barium    salt) was purchased            from Sigma                              each concentration.
Chemical    Co., St. Louis, Mo. 63178, and crystalline
PGA from Nutritional           Biochemicals          Corp., Cleve-
land, Ohio 44128. For pipetting,            we used MLA micro-
pipettes  (Medical     Laboratory        Automation,       Inc., Mt.                          logical samples      with the procedure          and reagents as
Vernon,   N. Y. 10550) equipped              with polypropylene                               supplied,    (b) precision    and accuracy      (including    speci-
tips. Manufacturer’s      directions      were rigidly followed,                              ficity) of the method        for biological    samples,     and (c)
and all assays were performed           in duplicate     by one op-                           comparison       data with other accepted           methods     (i.e.,
erator.                                                                                       microbiological)      and recovery experiments.          One man-
Results and Discussion                                                                        ufacturer    presented     a very uninformative          kit insert,
                                                                                              which gave no qualitative        or quantitative      information
Rating of Manufacturer’s                      Literature                                      on each reagent supplied.
    Table 2 shows our rating of the manufacturer’s                lit-
erature    included   with each kit. This evaluation             was                          Typical Standard Curves
based on an arbitrary         scoring procedure         similar     to                            Figure   1 shows typical     standard       curves obtained
that described      by Krynski      and Logan (8), in accor-                                  with the six kits. The range of the percent binding of
dance     with the American          Association       of Clinical                            the standards     encompasses      the effect of a number           of
Chemists     Committee     on Standards      and Controls       (9).                          variables    over a two-month       period of time and sum-
    Most manufacturers       were quite weak in presenting                                    marizes    five to six separate       standard      curves.    Vari-
statements      with supporting      data from original         work                          ables include reagent stability,        different    lots of mate-
and from citation      from the literature      to illustrate:    (a)                         rial, changes     in manufacturer         methodology        during
typical measured       values in the normal physiological                                     the course of this study,        and reagent         composition.
range and in the pathological         range obtained        for bio-                          Five of the six kits tested demonstrated             adequate     re-

                                                                                                                        CliNICAL CHEMISTRY.Vol. 21, No. 13, 1975                         1923
                                                  RIA PRODUCTS                                                                                DIAGNOSTIC   PRODUCTS




                                   6.25          12.5              25.0         50.0
                               folol..  og/.,I

                                                        CLINICAL   ASSAYS

                                                                                                                                  DIAGNOSTIC SIOCHSMI$TRY


                                                                                                                            f.Iot..   ng/mI

Fig. 1. Standard curves obtained for some commercial folate radloassay kits
The means are plottednd the bars Indicate the
                    a                            range for standards        during the two-month   time period tested

producibility    of standard  binding    over the period                                   h, 1 g of charcoal   per 20 ml of serum. The serum was
tested, as judged from standard     curve reproducibility                                  clarified   by repeated      centrifugation      and filtration.
(Figure 1).                                                                                The efficiency     of the adsorption          on charcoal      was
                                                                                           checked    by adding    [3H]-PGA        to the serum pool be-
Within-Run      Precision                                                                  fore treatment.    The radioactivity         of aliquots   of the
   Table 3 shows the within-run              precision      for the var-                   serum,    measured     before     and after     charcoal treat-
ious kits at two sample            concentrations         (normal      and                 ment, showed that the folate was removed with great-
folate-deficient).       All of the kits, except that of Clini-                            er than 90% efficiency.
cal Assays (1251), demonstrated              good precision in the                            MTHFA recovery was poor for two of the kits. For
normal range. Examination               of the data for the Clini-                         the other four, recoveries  ranged from 65.9% to
cal Assays (125!) kit showed that there was an appre-                                      129.9%.
ciable sample         blank     despite    the heating         step this                      The response of the kits with respect to the “fo-
method       incorporated      to reduce nonspecific             binding.                  late-free”   serum used as the base in these studies
In fact, the sample          blank was responsible             for about                   varied between     kits and between    runs with the same
80% of the counts observed                in a 17 ng/ml sample,                            kit.
hence the higher coefficient             of variation      for the nor-                        A range of 0 to 5.4 gig/liter was obtained      for this
mal range precision.         Kit within-run        precision      (CV) at                  serum, with only Clinical Assays 3H kit giving a con-
low folate concentrations           ranged from 1.6% to 22.4%.                             sistent “zero” value.
                                                                                               Because   of the poor MTHFA         recoveries  demon-
Analytical Recoveries                                                                      strated    by two commercial     folate kits (Table    4), it
 Table   4 shows the results of the recovery              of                               was of interest to determine if the kits were measur-
MTHFA     from “folate-free”      serum.    “Folate-free”                                  ing PGA in serum and not MTHFA. Table 4 also de-
serum  was prepared     by stirring    pooled    “normal”                                  scribes    the recovery        of added            PGA    from   “folate-free”
serum with Norit I neutralized    charcoal (Sigma) for 1                                   sera. With        both       of the kits in question                 one could

1924 CLINICALCHEMISTRY,Vol. 21, No. 13, 1975
                                                                                                   Twenty of each standard          were assayed, and Student’s
             Table 4. Analytical Recovery of Added                                                 t -test applied to determine        if there was a significant
              Compounds from “Foiate-free” Sera                                                    difference     between     count rates. Only two kits could
                                                Added          Recovered
            Kit                                                                   Recovered,       not differentiate      0.5 and 1.0 g of folate per liter from
  manufacturer            Isotope                        ag/liter                      %
                                                                                                   zero. A single kit (RIA Products’          3H kit) could distin-
N-Methyltetrahydrofolic                      acid addeda
                                                                                                   guish these from the “zero” standard,             but could not
Clinical                       1251              1.73                1.96            113.3
                                                                                                   distinguish     the 0.5 and 1.0 tg/1iter       standards   from
Assays                                           6.92                7.92            114.5
                                                17.3                15.08             87.2         each other.
Clinical                       3H                1.73                1.86            107.5         Comparative         Results for Patients’ Samples
Assays                                           6.92                8.70            125.7
                                                17.3                20.0             115.6            Twelve separate        pools of patients’     sera were divid-
                               1251              1.73                0.05                 2.9      ed into aliquots,      stored frozen, and assayed by use of
                                                 6.92                0.40                 5.8
                                                                                                   each kit. The results         (Table 6) indicate       that all kits
                                                17.3                 0.13                 0.8      gave their lowest values for samples               1-4. Classifica-
                                                                                                   tion of folate-deficient         samples     was based on the
Diagnostic                     3H                1.73                1.87            108.1
                                                                                                   manufacturer’s         suggested      normal      ranges.      Hence,
Products                                         6.92                8.99            129.9
                                                17.3                22.12            127.9         samples that are deficient         by one set of normals or kit
                               129               1.73                0.04                 2.3      may be borderline         or normal with another           set or kit.
RIA Products
                                                                                                   Inter-kit   variations      were considerable,        as evidenced
                                                 6.92                0.83                12.0
                                                17.3                 1.50                 8.7      by samples 5-12. In addition,            comparison       of individ-
                                                                                                   ual samples     and their means demonstrated                that kits
RIA Products                   3H                1.73                1.16                67.1
                                                                                                   with iodinated       tracers    gave distinctly      lower results
                                                 6.92                4.56                65.9
                                                17.3                18.1             104.6         than did kits with tritiated        tracers.

                                                        PGA added           PGA recovered
Pteroylg/utamic          acid added1’                                                                  From our data, it can be seen that results generat-
Clinical                               129                   2.5                  23.8             ed by current        “state of the art” radioassay                 methods
Assays                                                     10.0               >50                  for serum folate can vary considerably.                      One point in
                                                           25.0               >50                  particular     concerns the kits, both tritiated                and iodin-
Clinical                               3H                    2.5                  11.3
                                                                                                   ated, available       from Clinical Assays, Inc. In the liter-
Assays                                                     10.0               >50                  ature supplied         with the iodinated           kit, this manufac-
                                                           25.0               >50                  turer states that there is a strong correlation                    between
Diagnostic                             129                  2.5                 0.83               the results obtained          with their tritiated-folate             proce-
Biochemistry                                              10.0                >15                  dure and those obtained                with the iodinated-folate
                                                          25.0                >15                  procedure.      We, in fact, observed               that all iodinated
Diagnostic                             3H                    2.5                    3.2            kits in this study gave distinctly              (-‘50%) lower results
Products                                                   10.0                   13.0             than did all of the tritiated          kits (Table 6).
                                                          25.0                    31.3                 Variations     in assay particulars          and kit composition
RIA Products                           1251                  2.5                    3.2            among the manufacturers               were minor, the one excep-
                                                          10.0                    18.7             tion being Diagnostic             Biochemistry           (1251), in which
                                                          25.0                >50                  an assay pH of 9.3 was used, with PGA as the stan-
AlA Products                           3H                   2.5                     6.53           dard material.         Recently,     Givas and Gutcho               (10) re-
                                                          10.0                    49.0             ported the feasibility         of using the more stable PGA as
                                                          25.0                >50                  standard      material     at a pH of 9.3 in a tritiated            system.
  an   =    12 at each concentration.            The MTHFA           added was corrected           Our results with the Diagnostic                  Biochemistry         iodin-
for barium salt.                                                                                   ated system, however,             were unfavorable.           Additional-
   1’ n = 6 at each concentration.
                                                                                                   ly, it should be noted that Clinical Assays’ iodinated-
                                                                                                   folate system incorporates             a high pH (10.5) buffer in
                                                                                                   an initial heating step to denature                folate-binding        pro-
                                                                                                   teins. However,          a second       buffer,      with a lower pH
measure  PGA, as with all others compared      in this                                             (7.1), is added to adjust the pH of the reaction                        mix-
study. Most of the kits gave values greater than the                                               ture of 7.6 before the competition                      reaction.     In all
amount  of PGA added, and samples were not diluted                                                 other kits a similar (7.4-7.6) pH is used.
above the highest  standard  used in the commercial                                                    A number of recent reports have demonstrated                          dif-
kit.                                                                                               ferences      among       commercially          available       radioassay
                                                                                                   kits. Discrepancies          have been described               for digoxin
Minimum Detection                     Limits                                                       kits (11, 12), luteinizing           hormone         kits (13), vitamin
    Table 5 shows minimum                          detection          limits for each              B-12 kits (14), and human                  placental        lactogen      kits
kit. Folate  solutions were                       prepared           (0, 0.5, and 1.0              (15). This report indicates            that results with commer-
ag/liter)         from   the         standard          provided        with       each      kit.   cial folate radioassay           kits are, not surprisingly,             also

                                                                                                                     CLINICAL CHEMISTRY, Vol. 21, No. 13, 1975              1925
                                 Table 5. Minimum                     Detection          Limits for Six Commercial                                       Folate Kits
                                                                                                         0.5 pg/liter                                                              1.0 pg/liter
                                                              0 pg/liter                                                Different           from                                              Different     from
       Kit manufacturer                     Isotope        Mean          dpm          Mean          dpm                      0 pg/liter                         Mean         dpm                  0 pg/liter
Clinical Assays                              1251              7345                          6967                      Yes (P          =    .001)                         6196               Yes (P.001)
Clinical    Assays                           3H              10265                           9002                      Yes      (P=.001)                                  8061               Yes (P.001)
Diagnostic        Biochemistry               1251          66.8(%B/T)a               66.8(%B/T)                                     No                          67.0(%B/T)                           No
Diagnostic        Products                   3H                   5348                       5074                      Yes      (P.001)                                   4638               Yes (P=.001)
RIA Products                                 1251                 6460                       6594                                   No                                    6459                       No
RIA Products                                 3H                14401                       13819                       Yes (P=.001)                                  14021                   Yes(P=.001)
  n = 20 at each concentration.
  a Diagnostic Biochemistry     methodology               calls for counting      both free and bound for each sample and calculating                                       %B/T.

                                       Table 6. Analysis for Folate in Samples of Serum from Patients
                                                                                                                                                                                                  gested      No.
                                                                                           Serum no.                                                                                                          defi.
                                             2        3         4             5      6          7              8                9                 10           11           12        Mean         mals
   Manufacturer and
         isotope                                                                                    Folate, pg/liter                                                                                         sultsa
Clinical Assays 3H
Clinical   Assays 1251           5.6
                                 3.1        5.2
                                            2.8     3.9
                                                    2.6       4.2
                                                              2.9          9.4
                                                                           3.7      10.5
                                                                                     5.4       8.2
                                                                                               4.4             9.6
                                                                                                               4.8           16.0
                                                                                                                              6.2                19.5
                                                                                                                                                 10.2         22.7
                                                                                                                                                               5.9         14.3
                                                                                                                                                                           10.5       10.8
                                                                                                                                                                                       5.2        4-16
                                                                                                                                                                                                  5-20         1

Diagnostic              129      3.7        3.9     3.0        2.7         5.6       4.2       4.4             46            10.7                13.6          5.1          7.0         5.7       2-14         0
Diagnostic             3H        3.3        3.0     2.5        3.4         6.3       6.3       6.2             7.0           14.4                21.0         10.6         16.5         8.4       3-21         1

RIA    Products        1251      3.4        3.4     3.2        3.6         5.6       4.2       4.7             4.8           10.7                11.0          7.4         10.0         6.0       4-16         5
AlA    Products        3H        5.7        4.9     3.7       3.9         11.9      11.2       9.7          12.0             21.7                25.0         14.7         19.4       12.0        4-16         2
  a Based on the manufacturer’s             suggested normal         range.

                                                                                                     trahydrofolic              acid       for     a folate     binder.      J. Lab. GUn. Med. 83, 164
subject to variability     from manufacturer     to manufac-
turer. We caution clinical laboratories       to be aware of                                         (1974).

potential    problems   and pitfalls and to evaluate    a RIA                                        8. Krynski,   I. A., and Logan, J. E., Observations     on diagnostic    kits
                                                                                                     for the determination     of total cholesterol.   Clin. Biochem.      2, 105,
kit carefully    before using it as a service tool for aid in                                        (1968).
patient care.                                                                                        9. American  Association   of Clinical Chemists                                   Committee    on Stan-
                                                                                                     dards and Controls.    AACC policy regarding                                    reagent   sets and kits.
References                                                                                           Clin. Chem. 12,43 (1966).
1. Waxman,      S., Schreiber,     C., and Herbert,    V., Radioisotopic assay                       10. Givas, K. J., and Gutcho, S., pH dependence     of the binding of
for measurement       of serum    folate  levels. Blood 38,219 (1971).                               folates to milk binder in radioassay of folates. Gun. Chem. 21,427
2. Rothenberg,   S. P., daCosta, M., and Rosenberg, F., A radioassay                                 (1975).
for serum folate; use of a two-phase   sequential incubation,  ligand-                               11. Kubasik,       N.              P., Schauseil,     S., and Sine, H. E., Comparison           of
binding system. New Engi. J. Med. 286, 1335 (1972).                                                  commercial      kits              for radioimmunoassay:              I The radioimmunoassay
3. Miney, E. K., Wilcox,          E., and Morrison,     R. T., Estimation     of                     of serum digoxin                   using tritium tracer. Clin. Biochem.           7, 206 (1974).
serum and red cell folate         by a simple radiometric    technique.   Clin.                      12. Kubasik,      N.              P., Norkus,     N. S., and Sine, H. E., Comparison            of
Biochem. 6, 274 (1973).                                                                              commercial      kits              for radioimmunoassay:             II The radioimmunoassay
4. Tajuddin,  M., and Gardyna,              H. A., Radioassay          of serum folate,              of serum     digoxin                  using iodinated       tracer.     Clin. Biochem.    7, 307
with use of a serum blank and               nondialyzed    milk       as folate binder.              (1974).
Clin. Chem. 10, 195 (1973).                                                                          13. Wheeler, M. J., Woodrup,      P., and Watson,    D., Comparison     of
5. Waxman,     S., and Schreiber,   C., Measurement    of serum folate                               radioimmunoassay       methods for human luteinising     hormone.   Clin.
levels and serum folic acid-binding    protein by 3H-PGA radioassay.                                 Biochem.    8, 23 (1975).
Blood 42, 281 (1973).                                                                                14. Shaw,            W., and Bailey,    G., Evaluation                          of two vitamin   B-12
6. Dunn, R. T., and Foster,            L. B., Radioassay      of serum folate. Clin.                 assay kits           and L. leichmannii     bioassay.                         Clin. Biochem.   7, 320
Chem. 19, 1101 (1973).                                                                               (1974).
7. Kamen,    B. A., and Caston J. D. Direct radiochemical  assay for                                 15. Wheeler,               M. J., and              Watson,  D., Radioimmunoassay   kits for
serum folate: Competition     between 3H-folic acid and 5-methylte-                                 human           placental          lactogen.         Ann. Clin. Biochem.   11,49(1974).

1926       CLINICAL CHEMISTRY,             Vol. 21, No. 13, 1975

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