MACY,. E.; KEMENY,. M.; SAXON,. A. Enhanced. ELISA: How to measure less than 10 pico- grams of a specific protein (immunoglobulin) in less than 8 hours.
Enhanced
ELISA:
how to measure
less than
10 picograms of a specific protein (immunoglobulin) in less than 8 hours ERIC
MACY,
Diuision
MICHAEL
of Clinical
Cahfornia,
KEMENY,1
Immunology
Los Angeles,
AND
and Allergy,
CA 90024,
USA,
ANDREW
Department
SAXON*
of Medicine,
and TDepartment
UCLA
of Medicine,
School of Medicine,
Guy’s Hospital,
London,
University
mit the measurement of the antibody dividual B cells driven to high-rate
ABSTRACT
only
a relatively
short
culture
of
England
period,
produced Ig secretion as can
by inafter be done
In this paper we outline a flexible and rapid method to measure picogram quantities of isotype-specific immunoglobulin (Ig), including IgE. Only readily or
with hemolytic plaque or enzyme-linked immunosorbent assay (ELISA) spot assays (4-5). Radioimmunoassays can detect this level of Ig production, but for
commercially available reagents are required: isotypespecific, anti-human Ig murine monoclonal antibodies (Mab) to coat microtiterplates,polyclonalalkaline
reasons
phosphatase-coupled isotype-specific F(ab)’2 or Fab’ fragments as second antibodies, and an enhanced developing system that amplifies the signal-to-noiseratioof
the quantitatively bound second antibody. The procedure is detailed in the appendix to enable easy application, even if one has no previous experience with ELISAs. This system can be used to detect less than 10 picograms of Ig in cultures supernatants of cells that contain mixtures of various Igs and it can be used to
detect method
the product
of a single cell producing 1g. This to measurement of the
also will be applicable quantities of lymphokines
minute active molecules
and other
biologically
produced
in vitro and found in various fluids in vivo. MACY, E.; KEMENY, M.; SAXON, A. Enhanced ELISA: How to measure less than 10 picograms of a specific protein (immunoglobulin) in less than 8 hours. FASEB]. 2: 3003-3009; 1988. Key Words: IgE measurement immunoglobulin measurement enhanced ELISAs enzyme-linked immunosorbent assay immunoglobulin synthetic rate
THE
ABILITY
TO REPRODUCIBLY
MEASURE
in
an
isotype-
specific fashion small quantities of immunoglobulin (Ig)1 produced in vitro is important for studies of human humoral immunity (1). Naturally occurring plasma cells can produce 2000 molecules of Ig/cell per second (2). Spontaneous Ig-producing peripheral blood lymphoblastoid cells or B lymphocytes driven to high-rate antibody production produce approximately an order of
magnitude the ability
less, about to measure
092-6638181002-30010.50.
1 pg/cell per day (3). Therefore, less than 10 pg of Ig would per-
© FASEB
of speed, ease of assay, and
safety, we
have
adapted ideas and methods from our own and other laboratories with regard to ELISA technology to provide for a rapid and sensitive ELISA system to measure all Ig classes
with
reagents
that
are available
to all inves-
This methodology could readily be adapted for other proteins found in picogram concentrations. tigators.
A sensitive and outlined.
recently
specific Using
enhanced ELISA a combination
for IgE of those
was re-
other isotype-specific murine monoclonal (Mab) anti-Ig’s as coating antibodies, and Fab’ or F(ab)’2 alkaline phosphatase (Alk-phos)-coupled developing antibodies in enhanced sandwich ELISA assays, we have confirmed those findings for IgE and extended the methodology, so that we are now able to assay for IgG, IgA, 1gM, or IgE with sensitivity and specificity at less than 10 pg and detect the product of a single Igproducing cell. agents,
MATERIALS Regular
and
AND enhanced
METHODS ELISAs
Detailed methods and reagent preparation are described in the appendix. Briefly, microELISA plates are coated with monoclonal antibodies or antigen for 2 h. The plates are washed three times and then blocked with bovine serum albumin (BSA) or ovalbumin (OA) for 1/2 h. Serum dilutions, standards, or culture supernatants to be assayed are added, diluted if needed in
‘Abbreviations: ELISA, enzyme-linked immunosorbent assay; Alk-phos, alkaline phosphatase; INT, iodonitrotetrazolium blue; BSA, bovine serum albumin; OA, ovalbumin; OD, optical density; p-NPP, para nitrophenyl phosphate; Mab, murine monoclonal antibody; Ig, immunoglobulin; BBS, borate-buffered saline; PBS, phosphate-buffered saline.
3003
BSA or OA, and incubated for 2 h. The plates are then washed three more times. The second antibody, Alkphos coupled, is added for 2 h, and then the plates are washed three times each with two different wash solutions. The developing reagents are then sequentially added. The complete assay can be accomplished in 8 h. Data
1.0
O.D.IgE-PS100pI
o
o
Cells from a patient with atopic disease and high serum IgE (>10,000 IU/ml) were obtained by buoyant density centrifugation, separated into the B and T cell fractions, and cryopreservcd in a standard fashion (see ref 7 for description). The cryopreserved B cell fractions, known to spontaneously produce IgE as well as IgG and 1gM, were thawed, washed, and cultured at four different cell concentrations (5000, 10,000 50,000, and 100,000/well) in 60 replicate microculture wells in 200 l final volume in complete RPMI (8). The supernatants were harvested on day 5 and Ig isotype deter(IgE,
ELISA samples.
on
IgG,
each
and
well’s
1gM)
were
supernatant
done
by
by enhanced
using
60-!il
RESULTS The most
Ig for which difficult
it has been
to accurately
most important as well as measure
in the
picogram
range is IgE, so we focused on the ability of this assay system to measure IgE. Figure 1 is an example of an enhanced ELISA IgE standard curve that shows a comparison of the standard IgE preparation from the World Health Organization (WHO) with the IgE paraprotein PS, a IgE myeloma protein used as a standard in our own and in many other laboratories. All data shown in Fig. 1 were obtained from a single 96 wellenhanced ELISA plate by using a 30-mm substrate and a 30-mm developer incubation. Although the two standards showed somewhat different curves, both were linear from 3.9 to more than 250 pg/well (r values of .99). Two different but parallel PS assays are also shown in Fig. 1, where the specified number of picograms of IgE were pipetted into the wells as either 20 or 100 tl. We were concerned that adding 20-gil samples might introduce a greater chance of error. With 2O-jl samples the wells were filled with 80 /Ll of 0.5% BSA to a total volume of 100 ed/well, and the curves were virtually identical to those obtained with 100-tl samples. When standards from the United Kingdom (UK), National Institutes of Health (NIH), and WHO were compared with PS, PS produced a twofold higher opticaldensity
3004
Vol. 2
Nov.
0 0.4
y -0.01 74 + 0.0015x WHO: y-0.0485+O.OO29 PS 100il: PS 20tl: y=0.0302+0.003x
0.2
0
100
200
-
-
0.99 0.99 0.99
400
300
pg IgEweII Figure 1.Enhanced
cell culture
minations
0.6
0
analysis
and
O.D. IgE . WHO
ID O.D.IgE.PS20sI
ELISA plates were read on Bio-Tek instruments model EL31O hard wired to a Macintosh II computer. The data were processed with the ELISA Master program,2 and graphed using Cricket Graph. Limiting dilution curve fit, mean number of precursors, and 95% confidence intervals were done with the maximum likelihood method (6). Cells
Io
08
1988
IgE ELISA, WHO standard vs. PS myeloma. Increasing amounts of WHO and PS 1gB were assayed. After the second antibody (Alk-Phos Fab’ anti-IgE) was washed out, substrate was reacted for 30 mm, followed by developer incubation for 30 mm. The PS IgE was added in either 100 or 20 jil (El) and then topped up to 100 &l volume with BSS 1% BSA. The WHO IgE
(#{149})
(0)
was added in 100 ol.
(OD) than
than the NIH standard and a twofold lower OD the UK and WHO standards with equivalent amounts added in our assay (results not shown). Thus all four standard IgE preparations assayed within a fourfold range and PS were in the middle of the range. Figure 2 shows the plate-to-plate variation between four IgE-enhanced ELISA platesdone on the same day with two different substrate incubation times. There is no significant plate-to-plate variation in signal from standard curves with identical incubation times. With the longer substrate incubation, there was a marked increase in signal: 170-243% over the range of dilutions used. Figure 3 shows typical saturation between 1000 and 2000 pg of IgE/well in our IgE-enhanced ELISA when developed with 45-mm incubations for both substrate and developer. IgG, IgA, or 1gM added at up to 28,800 pg/well did not produce a measurable signal in the IgEenhanced ELISA nor did all possible reciprocal combinations with the other isotype-specific enhanced ELISAs (data not shown). The useful linear portion of typical enhanced ELISA curves has been between 0 and 500 pg Ig/well. Equivalent sensitivity and specificity were obtained with enhanced ELISAs measuring IgG, IgA, and 1gM by using the specified coating and developing antibodies (see appendix). Data for IgA, 1gM, and IgE assays are shown in Fig. 4, which demonstrates the increased sensitivity of the enhanced development compared with regular p-nitrophenyl phosphate (p-NPP) development. Identical primary and secondary antibodies for each isotype were used on each plate. One-half of each plate
2Giftgenerouslyprovidedby Dr. Richard Deem, UCLA ment of Microbiology and Immunology, Los Angeles, USA.
The FASEB Journal
Depart-
CA 90024,
MACY
ET AL.
B
A 1.8 1.5
P1.1.83
#{149} P111.11
#{149} Plawes
#{149} P1.1.12
45 mInutes withsubstrate
25 mInutes with substrate
1.2 0.9 0.6 0.3
0
7.75
15.5
31.25
62.5
125
250
500
1000
pglgE/w.II 0
7.75
15.5
31.25 pg
Figure 2.
Reproducibility
cubated
substrate
over
the linear
portions
of the curves.
250
is a 170-243%
There
500
1000
IgE/well
standards. Plates 1 and between the pairs of plates increase in signal with 45-mm
2 were inincubated compared
incubation.
was developed with the regular reagents (p-NPP) and the other half was developed with the enhanced reagents to provide a direct comparison between the two development systems. The enhanced ELISAs provided much better detection of low levels of all Igs and could readily distinguish less than 10 pg Ig from background. The enhanced ELISA, compared with the regular development, produced ODs from 5.6 to 13.5 times higher for 10-pg Ig determinations, with no significant increase in the background OD of the blank wells. To demonstrate the ability of the enhanced ELISA to identifythe Ig produced by individual cellsaftera relatively short culture, B cells known to spontaneously secrete IgE as well as IgG and 1gM from an individual with atopy, and a serum IgE greater than 10,000 IU/ml, were cultured at limiting dilution (6). Sixty replicate wells were set up each with 5000, 10,000, 50,000, or 100,000 cryopreserved B fraction cells in a final culture volume of 200 tl.After 5 days, supernatants from each well were divided into 60-1d aliquots and assayed for IgE, IgG, and 1gM. Individual wells that were positive for IgE or IgG with a single precursor produced about 40 pg Ig/cellper 5-day culture,whereas 1gM-positive wells produced approximately 10 pg Ig/cell per 5-day culture. Figure 5 represents limiting dilution curves for spontaneous IgG-, IgE-, and 1gM-producing cells. The number of B fraction cells added that produce 37% negative wells corresponds to a mean of one precursor per well. Based on B fraction cells, one cell of 3500 (95% confidence interval, 2700-4400) was an IgGproducing cell, one cell of 5100 (95% confidence interval, 4100-6500) made IgE, and one cell of 30,800 (95% confidence interval, 23,700-40,100) spontaneously synthesized 1gM. Figure 6, which was generated from the same limiting dilution data depicted in Fig. 5, shows that the mean IgG, 1gM, and IgE produced per well was linear from 5000 to 100,000 B fraction cellscultured. This demonstrates the absence of a cell density effect and ENHANCED ELISA
125
the enhancedELISA. Four plateswere used to assay identical PS IgE for 25 mm (A) and plates 3 and 4 were incubated for 45 mm (B). The variation of
with substrate at the same times is not significant to 25-mm
62.5
validates
that we were measuring the Ig from individual The consistency of the data shown by measuring the total amount of IgG and IgE produced in wells containing the largest number of cells is within a factor of 2.5 of that calculated by multiplying the predicted number of precursors in the well by 40 pg precursors.
Ig/precursor
produced
in 5 days.
If every positive precursor (i.e., spontaneous Igproducing lymphoblastoid cell) was able to divide every 24 h, after 5 days each culture with a single initial precursor would contain a maximum of 31 cells/day of Ig production (16 cells + 8 cells + 4 cells + 2 cells + 1 precursor), providing an estimate of approximately 1 pg Ig/cell per day. If the cells proliferated less, the estimate of the corresponding amount of Ig/cell would increase.
S
S
2.0
1.6
1.2 0
0
.DE-PS1
0.8 0.4
0
400
800
1200
1600
2000
pg IgE/weII Figure
3. IgE-Enhanced
ELISA
saturation
curve.
An IgE en-
hanced ELISA with PS IgE
was performed with a 45-mm incubation period for both substrate and developer. The linear working part of the curve was as much as approximately 500 pg/well, with saturation occurring around 2 ng/well.
3005
A
B
0.6
IgE (E) 60:
y
-
0.0014
+
S
r -0.98
0.0066x
IgA(E)
10:
y
0.0261
-
+
0.0168x
r -0.98
0.5
0.20
0
0.4
IgE(R)120
0
0.li
0
#{149} IgE(E)60
In
S
h
IgA(R)50
A
IgA(E)10
0.3
0
0.1(
0
0.2
IgA(R)50:
y-0.0044+0.003x
r-0.99
0.1 r-0.90 0
5
10
15
20
25
30
35
pg IgA/well pg IgE/well
C IgM(E)11:
yO.0432+O.029lx
r-0.99
U
08
Figure 4. Comparison of the enhanced vs.regularELISA method for detection of low levels of IgE (A), IgA (B) and 1gM (C). Plates o
0.8
IgM(R)50
were incubated with Mab coating reagents (Ig standards applied) and then the lefthalfof each plate was developed by using the enhanced ELISA method (E); the right half was developed with the unenhanced regular (R) technique. Each isotype was compared on its own plate. The enhanced ELISA was done with a 45-mm substrate incubation and then scanned at 490 O.D. after a 60-mm
#{149} IgM(E)11 0.4
developer incubation. The regular ELISA was incubated in the dark for 120 mm with its substrate and then read at 405 O.D.. Curve fitwas calculatedusing CricketGraph on a Macintosh II
r-0.99
02
computer. 0
5
10
15
20
25
30
35
pg 1gM/well
DISCUSSION The enhanced ELISA was originally described by Stanley and co-workers (9) in 1985, and the enhancing reagents are now available from Gibco/BRL (Bethesda Research Labs., Gaithersburg, Md.) for use with any Alk-Phosdeveloped ELISA. The reagents may also be produced from the constituent chemicals and enzymes (see appendix). Previously, the rate of signal development restricted the lower limits of detection in ELISAs because background development would rise to obscure low signals. Steps such as developing in the dark for a period of several hours to overnight have been used to boost the signal but this did not yield a major change in the signal-to-noise ratio. The enhanced ELISA uses alkaline phosphatase coupled to the developing antibody to generate NAD from NADP. The NAD is cycled in the development step between alcohol dehydrogenase and diaphorase to reduce iodonitrotetrazolium blue (INT) to a colored form and thereby rapidly generate a strong signal before the usual nonspecific rise in background signal noise occurs. The limiting factor in the enhanced ELISA is the behavior of the antibodies used. We use a variety of read+
3006
Vol. 2
Nov. 1988
ily available high-affinity mouse, anti-human heavy chain-specific monoclonal antibodies for the coating step and Alk-Phos-coupled Fab’ [anti-human IgE produced by IQ(Bio) Limited, Cambridge, UK, using a rabbit anti-IgE antiserum from Dr. M. Kemeny] or F(ab)’2 [goat anti-human IgA and 1gM from Tago, Burlingame, Calif. or anti-human IgG (A-0287) from Sigma Biochemicals, St. Louis, Mo.] as second antibodies. The use of Fab’ or F(ab)’2-labeled reagents as second antibodies reduces nonspecific Fc region interactions and lowers background color development in the assays. Aliquots of an alkaline phosphate-coupled Fab’ antiIgE reagent and the Mab anti-human IgE antibodies (CIA-E-7.12 and CIA-E-4.15) will be available to research investigators from the National Institute of Allergy and Infectious Disease/NIH Serum Bank, Bethesda, Md., in care of BioTek Research Labs, 1600 East Gude Drive, Rockville, Md. 20850, USA. The other Alk-Phos-labeled developing antibodies are commercially available as noted previously. Mab antihuman IgG, Hb 60; anti-IgM, Hb 57, and anti-IgA, CHE 86, are available from the American Type Culture Collection (ATCC), Rockville, MD. Other high-
The FASEB Journal
MACV El AL.
affinity, heavy chain-specific monoclonal antibodies could be substituted. Difficulties in the measurement of low levels of IgE found in vivo and particularly produced in in vitro systems emphasize the need for sensitivity and specificity of the enhanced ELISA. IgE was initially reported to be produced by fetal cells as early as in the 11th week of fetal gestation, but it has been shown that it is likely to be caused by detection of private light-chain determinants by the early polyclonal anti-IgE antisera (10). Similarly, the interpretation of data regarding the in vitro production of IgE from mononuclear cells driven by a variety of stimuli has been difficult because of problems in accurately measuring the small amounts present, even when the issue of polyclonal antisera binding to idiotypic or similar nonisotype restricted elements in known (1). Thus, laboratories report as much as a 100-fold difference in the IgE obtained with the same culture system, and measurement of levels of IgE below 5 ng/ml was highly variable in an interlaboratory evaluation, although, as has been noted, the samples provided in that assay may have contributed to some of the variation (1). The ready availability of the enhanced ELISA reagents and antisera for all of the Ig isotypes as provided for later in this paper should obviate these problems for investigations regarding human Ig. The enhanced ELISA is sensitive enough to detect Ig produced by individual, spontaneous, high-rate Igsecreting cells from a culture of only several days. Thus the enhanced ELISA system is readily adapted to measuring Ig produced by cells brought together in limiting dilutions, and has the intrinsic ability to study interactions between individual cells. Products other than Ig, if produced in similar quantities, could also be assayed.
3000
2000’ 0 0 C
1000
0
20000
40000 B
60000
80000
100000
120000
fractioncells/well
Figure 6. The mean amounts of IgG, IgE, and 1gM produced in each well in the limiting dilution data graphed in Fig. 5. The total
amount of IgG, dilution
IgE,
was divided
number ofB
and
1gM produced
by the number
by
all the wells at each
of wells and then plotted
vs. the
fraction cellsadded.
In conclusion, the enhanced ELISA is sensitive below the range of 10 pg of IgG, IgA, 1gM, and IgE in a 100-tl sample. The assay can be completed within 8 h and isreadilyreproducible. With the use of other coating proteins or antibodies, the assay is easily adaptable for the assay of antigen-specific antibody of any isotype or, for that matter, any protein for which high affinityspecific antibodies are available. The production of Fab’ or F(ab’)2 alk-phos-coupled reagents may be viewed as a limiting factor in achieving the same sensitivity for the assay of any protein, but such antibodies can be prepared by commercial concerns from an investigator’s unlabeled reagent if they are not already available. We have used this system to measure antitetanus toxoid and antidiphtheria toxoid IgG and 1gM produced in vitro by a single cell as well as serum IgE against fetal calf sera with equivalent results. This enhanced ELISA methodology should be useful in providing sensitive assays to measure minute quantities of biologically active molecules produced in vitro, such as interleukins, as well as for measuring levels of such proteins in body fluids.
E!I
This project was supported by U.S. Public Health Service grants AI-07126, AI-15251, AI-15332, CA-12800, CA-23l75, and by American Cancer Society grant IM-482.
APPENDIX
20000
30000
60000
ELISA
procedure:
B fractIoncells/ well
1) Polystyrene microtiter plates, Immulon 2-MicroELISA plates, (Dynatech Labs, Alexandria, VA) are coated with various Mab anti-human Ig at 2 g/ml in borate-buffered saline (BBS) (100 tl/well) at room tem1gM production. 60 replicate wells were set up at each of the four perature in a humidified box for from 2 h to overnight. indicated numbers of cells per well (5,000, 10,000, 50,000, and To measure IgE, a combination of Mab CIA-E-7.12 100,000). Each 220-tl well was split into 60-tlaliquots and assayed (1 Lg/ml of each) is used (12). To assay forlgG, IgE, and 1gM. Curve fitwas done by the maximum likeli- and CIA-E-4.15 hood method. for IgG, Mab Hb 60 at 2 tg/ml is used. To assay for Figure
5. Limiting
dilution of peripheral blood, spontaneous Igproducing lymphoblastoid B cells. B fraction cells were set up in 5-day culturesin cRPMI and then assayed for total IgG, IgE, and
ENHANCED ELISA
3007
IgA, Mab CHE B6 at 2 sg/ml is used and for 1gM, Hb 57 at 2 ig/ml is used. Mab Hb 60 and Hb 57 can be obtained from ATCC. CHE B6 was obtained from Dr. F. Kearney (University of Alabama, Birmingham) through the ATCC. The Mab’s are grown as ascites in BALB C mice and enriched by 50% ammonium sulfate precipitation. The coating antibodies are kept in BBS at a concentration of 2 mg/ml and diluted 1:1000 before use. Protein antigens, such as tetanus toxoid or diphtheria toxoid, are used at 2 tg/ml in BBS. The coating mixture is used only once and then discarded. 2) After the coating material is discarded, the wells should be washed three times (300 tl/well) with phosphate-buffered saline (PBS)/Tween and then blocked with 0.5% BSA in PBS/Tween (150 1il/well) for 1/ h at room temperature. Use OA to block the plates and as a sample diluent if the antibody to be measured is directed against BSA or haptenated BSA. 3) Discard the blocking solution and then add either
J.
the
culture
supernatants
or
material
to
be
assayed
(diluted tl/well.
with 0.5% BSA) to a final volume of 100 If less than 100 tl/well of culture supernatant is added, add 0.5% BSA to a total volume of 100 tl/well. Allow
the
plate
to incubate
in a humidified
box
least 2 h at room temperature. 4) Discard the contents of the wells. Wash with PBS/Tween
(300 1il/well).Add
for
at
four times
100 tl/well of the
alkaline phosphatase (Alk-Phos)-coupled developing antibody diluted in 0.5% BSA. Rabbit Fab Alk-Phosaffinity-purified anti-IgE from Dr. M. Kemeny (Guy’s Hospital, London) was used at a 1:3000 dilution. The goat Fab’2 Alk-Phos anti-IgA and anti-IgM (Tago), and anti-IgG (Jackson Immunoresearch, Avondale, Pa.) are used at one to two times the working dilution provided on the specification sheet from the company. This usually works out to a 1:3000 dilution. For the data shown, all of the antisera are diluted 1:3000. The second antiserum is incubated at room temperature in a humidified box for at least 2 h to overnight. 5) Discard the second antibody. Wash three times with PBS/Tween using 300 tl/well. Wash three times with Tris/NaCl using 300 tl/well. At this point there are two options. If the regular (unenhanced) development system is to be used, add the regular developing (p-NPP) solution (100 id/well). Develop in the dark until the absorbance at 405 nm of the highest concentrations on the standard curve reaches 1 to 2. For the enhanced development system, continue with step 6. 6) Add
50 id/well
of substrate
solution
(see
later
If you make equal
parts
watching to pink 3008
Any nonspecifically generated NAD4
your own NADP of substrate
and
bound will cause
solution, amplifier
Alk-Phos or signal noise.
test it by adding in a test
tube
and
for 15 mm for any sign of color development or purple. NADP degrades easily, and preVol. 2
Nov. 1988
in a plate
reader
until
the
absorbance
at 490
nm
in the
highest
concentration wells is near 2. The wells are typically saturated in the IgE assay with about 1 ng of IgE. Complete color development generally occurs within 30 mm at 20#{176}C or in an equivalent period of time to the incubation casionally it will take
allowed
with
the
substrate.
Oc-
slightly longer. If you make your own amplifier, it may change to a light pink on its own within 1 day. This will cause a decrease in the signal-tonoise ratio because of the increase in background of the assay; therefore, amplifier solution should be made fresh daily. The BRL amplifier solution is generally more stable and may be stored reconstituted according to the
manufacturer’s
instructions.
Work
quickly
when
adding reagents in steps 6 and 7. Keep the plates in the same order, so that all the wells have exactly the same amount of time with all of the reagents added. Multichannel pipettes are required to rapidly add the substrate and developer. 8) Stop the assay, if desired, with 20 1d/well of 0.75 M H2S04. We have often found it advantageous to serially read the plate until the absorbance of a predetermined well on the standard curve reaches a desired point. We used a BIO-TEK EIA Autoreader model EL31O, which has this capability, but other ELISA readers also have this feature. Plate color, once stopped, has been noted to be stable for at least 48 h if the plate is kept from drying out. The total time needed is 2 h to coat the plate, 1/ h to block, 2 h with the sample, 2 h with the second antibody, and about 1 h to develop. The total time needed (exclusive of washing) is about 8 h. The assay is usually done by precoating the plates overnight, which allows the assay to be easily completed in 1 day. It is theoretically possible to store coated plates frozen, but it has been easier to store the coating proteins as concentrated stock solutions and coat plates as needed.
in-
structions) and allow to incubate at room temperature for 35-45 mm. The longer the incubation, the more NAD will be generated and the greater will be the signal produced by the amplification step. For this step the wells should be uncovered. This is the most critical step in the assay. spontaneously
weighed, unopened vials should be used fresh for every assay. If your test of the substrate and developer produces spontaneous color change, it is possible to discard the substrate in the wells and rewash four times with Tris/NaC1 and repeat step 6. We have had no problems with the BRL enhancing kit reagents. 7) Add 50 id/well of the amplifier solution. For the remainder of this step, the assay should be carefully watched. If a large amount of Ig is present in some of the wells, color development will be noted quickly. Observe the plate for dark purple in the highest concentrations in the standard curve, or scan every few minutes
Reagents
and
buffers:
The following reagents-buffers are needed. Constituent chemicals, where not specified, were obtained from Sigma or an equivalent supplier. A) Coating buffer to dilute Mab anti-Igs or other proteins, BBS consisted of H3BO4 (mol wt 61.84), 10.3 g/liter; NaCl (mol wt 58.44), 7.3 g/liter:pH to 8.5. B) Substrate solutionuse as directed in the BRL kit or make as follows. Tris-
The FASEB Journal
MACV El AL.
HC1 (mol wt 157.6), 11.82 g/liter; MgCl2 (4.9 M stock), 306 id/liter; NaN3 (mol wt 65.0), 0.2 g/liter: pH to 8.8 at 20#{176}C. Add 10 mg NADP to each 40 ml just before use; may use Sigma preweighed vials. This makes enough for eight 96-well plates by using 50 id/well. C) Amplifier solution - use as directed in the BRL kit or make as follows. Dissolve 1.2 ml ethyl alcohol (200 proof) and 100 mg iodonitrotetrazolium violet (INT) (mol wt 505.7), Sigma grade 1, using a stirring bar at 20#{176}C for 10-15 mm in a total volume of 40 ml 0.025 M
NaPO4
buffer,
pH
7.0,
with
0.02%
NaN3.
After
the
material is completely dissolved, add 1.2 mg diaphorase (EC 1.6.4.3) (from microorganisms), Boehringer (Indianapolis, md.) and 7.5 mg alcohol dehydrogenase (EC 1.1.1.1), Boehringer. Swirl gently to dissolve and use within several hours. This is enough for eight 96well plates by using 50 1d/well. D) Blocking solution and standard diluent: BSA (fraction V) or OA, 5 g/liter, in PBS/Tween. E) Stop solution: 0.75 M H2S04. F) PBS/Tween: Tween 20, 0.5 mi/liter, in PBS. G) Final wash -Tris/NaC1: Tris base (mol wt 121.14), 6.04 g/liter; NaCl (mol wt 58.44), 8.76 g/liter; NaN3 (mol wt 65.0), 0.5 g/liter: pH to 7.5. H) 10 X PBS stock with azide (PBS) - NaC1 (mol wt 58.44), 30.4 g/liter; NaH2PO4. H20 (mol wt 137.99), 15.4 g/liter; Na2HPO4.7H20 (mol wt 268.07), 77.3 g/liter; NaN3 (mol wt 65.0), 2.0 g/liter: pH to 7.0. Dilute 10-fold before use as PBS. I) Carbonate buffer-NaHCO3 (mol wt 84.01), 8.4 g/liter; MgCl2 (4.9 M stock), 674 1d/liter;NaN3 (mol wt 65.0), 0.1 g/liter: pH to 10.3. J) Regular developing solution - Dissolve 0.05 g of p-Nitrophenyl phosphate (mol wt 371) in 30 ml of carbonate buffer just before use. This is enough for three plates with a 100-sal/well.
ENHANCED
ELISA
REFERENCES
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Receivedfor publication May 16, 1988. Accepted for publication June 20, 1988.
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