A flow microsphere inhibition immunoassay (FMII) for detecting paratope binding anti-idiotypic antibodies.

IMMUNOLOGICAL INVESTIGATIONS, 21(6), 565-580(1992)

Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/25/14 For personal use only.

A FLOW MICROSPHERE INHIBITION IMMUNOASSAY (FMII) FOR DE'IECIING PARATOPE BINDING ANTI-IDIOTYPICANTIBODIES

J. E. Brown and A. J. Ainsworth College of Veterinary Medicine, Mississippi State University Mississippi State, MS 39762

ABSTRACT A simple, rapid and reproducible flow microsphere inhibition immunoassay (FMII)has been developed to detect the ability of paratope specific anti-idiotypic antibody (anti-Id or AB2) to inhibit antigen binding to the corresponding paratope of the Id (AB1). To evaluate the FMII as a measurement of paratope binding anti-Id, an avian model was used to produce Id and anti-Id antibodies for the study. Both antibody to bovine serum albumin (BSA Id) and anti-BSA Id were produced in white leghorn chickens and affinity isolated from egg yolks. The anti-BSA Id samples were incubated with BSA Id coated microspheres, then without rinsing, fluoresceinated BSA (BSA-FITC) was added for a short incubation period and the resulting decrease in fluorescent intensity was used to calculate the extent of inhibition. For validation, statistical comparisons of the line equations generated by BSA dilution curves and antiBSA Id dilution curves were performed. Replications within each ligand were not significantly different which indicated the assay was reproducible for determining the presence of paratope reactive anti-BSA Id used in this model. INTRODUCTION Numerous network theories accounting for immune regulation in mammals have been postulated. One theory states that antibodies can become targets of other antibodies in the immune system (1). These interactions are known as idiotype-anti-idiotype reactions and can be exploited to manipulate the immune system (2). Research has demonstrated that an antibody, (Id or ABI), from one animal injected into another animal of the same or different species can induce a second set of antibodies ,the so-called anti-idiotypes (anti-Id or AB2). Anti-Id antibodies induced to the paratope of the Id are capable of mimicking antigen and thereby inducing protective immune responses in the 565 Copyright 0 1992 by Marcel Dekker, Inc.


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BROWN AND AINSWORTH

absence of the original antigen (3). The potential for alternative vaccine production has been a primary reason for much of the excitement centered around the idiotype network and anti-Id. Numerous methods are described in the literature for detecting anti-Id antibodies and are based on several methods such as, hemagglutination (4, 51, radioimmunoassay (6, 71, in situ staining of cells in tissues (71, agar gel diffusion (8) and ELISA (9, 10) to name a few. Each of these methods has its merits and drawbacks. Hemagglutination methods are sensitive; however, the test is susceptible to erroneous results due to serum factors which can cause lysis of the indicator red blood corpuscles in addition to the fact that fresh cells must be constantly produced. The dangers of radioimmunoassays are apparent and cause additional hazardous waste disposal problems. In situ tissue staining and agar gel diffusion methods are useful for detection of anti-Id producing cells or anti-Id antibody, respectively, but do not quantify the results numerically, or distinguish between framework or paratope binding anti-Id. The substrates for the ELISA are suspect carcinogens and the coating process takes an extended period of time. Assays for monoclonal or polyclonal anti-Id for potential use in vaccination trials must not only detect the anti-Id but also determine if the anti-Id binds to an Id within the paratope. One of the methods of ensuring the presence of a paratope binding anti-Id is to determine the amount of inhibition of antigen binding to the Id that the anti-Id demonstrates in a blocking assay. Many of the assays designed for determining inhibition of antigen binding to the Id are extremely specialized or possess some of the drawbacks noted above. The objective of the present work was to develop a rapid, reproducible assay that was highly sensitive and specificbut that could also be easily adapted to other idiotypic antibody studies where paratope reacting anti-Id are under study. It was also considered necessary to eliminate the use of hazardous radioisotopes, cumbersome washing procedures, and long waiting period for results. For these reasons the development of a flow microsphere based assay was attempted. S e r u m derived proteins can be attached to polystyrene microspheres both in a covalent and non-covalent manner (1 1-15). Protein coated microspheres have been employed as supports for immunoassays since the 1960's; however, until recently there has been little use of microsphere based flow cytometry. The concept was documented in the scientific literature as early as 1977 when Horan and Wheeless first described a method for the development of a flow cytometer assay based on a concept by Fulwyler in which microspheres were used as the supporting matrix for one of the reactants along with a fluorochrome labeled reactant which the flow cytometer counted and analyzed (16,17). The Flow Microsphere Immunoassay (Fh4IA) previously mentioned employs use of a capture reagent coated onto the surface of homogeneous monodispersed

PARATOPE BINDING ANTI-IDIOTYPIC ANTIBODIES

567


microspheres that act as an accumulator thus removing analyte from a sample for use in the detection procedure specific for the analyte. The objective of the present work was to develop a rapid, reproducible assay that was highly sensitive and specific but that could also be easily adapted to other idiotypic antibody studies where paratope reacting anti-Id are under study. This paper reports the development of the Flow Microsphere Inhibition Immunoassay (FMII) a micrcrosphere based assay discrete from the FMIA to detect paratope binding anti-Id by measuring inhibition of fluorescent analyte binding. MATERIALS AND METHODS ANIMALS AND INOCULATIONS

White leghorn chickens were purchased from disease controlled stock (SPAFAS, Inc., Roanoke, IL) as either active laying hens or as eggs which were hatched and matured to egg laying age. Chickens were housed individually in cages so that eggs from each hen could be kept separate and identified. Chickens were divided into three experimental groups (4 - 6 birds per group) according to the type of antigen used to immunize them and were designated as nonimmune immunoglobulin (IgG), bovine serum albumin (BSA), Id , anti-BSA Id. The non-immune IgG group also served as the negative control for BSA Id (antiBSA) responses. Booster inoculations were given intraperitoneally every 21 to 28 days. The BSA Id group was inoculated with 6.25 mg of BSA (Sigma Chemical St Louis, A-7030, Fraction V) as a 50:SO mixture in Freund's incomplete adjuvant (IFA). The anti-BSA Id group was inoculated with 500 pg of affinity purified BSA Id (isolation described later) as a 50:50 mixture in IFA. Care and use of animals followed the guidelines established for biomedical research by the United States of America National Institutes of Health - Public Health Service. ISOLATION OF IgG - FROM YOLK Isolation of IgG from yolk was performed using a modification of a described method (18). Briefly, egg yolks were separated from the whites, the yolk volume measured and two volumes of 0.01 M phosphate buffered saline, pH 7.4 (PBS) added. Upon mixing thoroughly, 3.5% (w/v) of polyethylene glycol (PEG, P-2139, m.w. 8000, Sigma Chemical Co., St; Louis, MO.) was added to the yolk solution and dissolved. Mixing was stopped once the PEG was in solution and the mixture was allowed to coagulate. The slurry was centrifuged at 14,OOOxg for 10 minutes and the supernatant decanted through a pre-wetted pad of layered cotton and gauze. The filtrate was then adjusted up to a 12%PEG concentration, the PEG dissolved, centrifuged at 14,000 x g for 10 minutes and the pellets retained. The pellet was dissolved in PBS to the original yolk volume and PEG added to 12% (w/v) and dissolved. The mixture was immediately centrifuged at

BROWN AND AINSWORTH

568

14,000 x g for 10 minutes, the supernatant discarded and the pellet centrifuged twice to extrude residual PEG. The final pellet was redissolved with PBS to onetenth the original yolk volume. Purity of yolk IgG was confirmed by agarose gel electrophoresis (Paragon, Beckman Instrument, Norcross, GA) and protein concentration by a bicinchoninic acid method (Pierce Chemicals, Rockford, IL).


AFFINITY CHROMATOGRAPHY

Affinity supports (19) for BSA Id and anti-BSA Id isolation were made by coupling BSA to Sepharose 4B (Sigma Chemical Co.) using the cyanogen bromide method (20) or coupling BSA Id to an adipic acid dihydrazide modified gel (21). One hundred fifty mg of BSA or 100 mg of BSA Id was conjugated to 50 ml of the appropriate gel. Batch isolations were performed by combining 50 ml of isolated yolk IgG plus an equal volume of PBS with 50 ml of affinity gel support and mixing for 60 minutes at 22% in a roller bottle. The mixture was washed with PBS until the optical density (O.D.) at 280 nm was baseline (essentially zero) and loaded into a 1.6 x 35 cm column. The gel was washed with 1.5 M sodium chloride, 0.01 M phosphate buffer, pH 7.5, to remove any nonspecific or loosely bound protein. Once this protein was removed, as determined by a return of the O.D. to baseline, 0.5 M glycine, 0.2 M sodium chloride, pH 2.0 was used to elute specifically bound IgG. The pH of the isolated IgG was adjusted to 7.5 with 6 N NaOH, dialyzed once against PBS and concentrated using an ultrafiltration cell with a 43 mm diameter IM ' 30 membrane ( Amicon Division W. R. Grace & Co, Beverly, MA ). Samples were stored at -2O'C until needed. Upon removing samples for use, sodium a i d e was added to a final concentration of 0.02%. DETECTION OF BSA ID

The BSA Id antibodies were detected using a modification of a previously described enzyme linked immunosorbent assay (ELISA) (22). Briefly, 96 well ELISA microtitration plates (Titertek, McLean, VA) were coated with a concentration of 5 mg BSA/ml (0.1 ml per well) in 0.01M PBS for three hours at 37°C. After coating, the plates were washed three times in PBS. Crude BSA Id and affinity purified BSA Id antibody titers were determined using the following procedure. Between each step, plates were washed three times by dispensing 0.3 ml of 0.01M PBS into all wells and the fluid discarded. Samples of BSA Id to be tested were diluted two-fold beginning at 1:500 in 0.01M tris base, 0.001M ethylenediaminetetraacetic acid, 0.5M sodium chloride, and 0.003M sodium azide, pH 7.4 (TEN buffer) and 0.1 ml of diluted sample added to the appropriate wells. The test plate was covered and incubated for 30 minutes at 22'C. After washing, 0.1 ml of rabbit anti-chicken alkaline phosphate conjugate (ICN ImmunoBioIogicals, Lisle, IL)diluted 1:2000 in TEN buffer containing 1% heatinactivated normal rabbit serum was dispensed into the wells and incubated for


569

30 minutes at 22°C. Upon washing the wells, 0.1 ml of p-nitrophenyl phosphate substrate (1 mg/ml in 0.9M diethanolamine buffer, pH 9.8, Substrate tablet 104, Sigma Chemical Co., St Louis, MO.) was added to all wells, the wells incubated for 30 minutes to allow for color development and the optical density at 405 nm determined for each well using a spectrophotometric plate reader. Appropriate negative (non-immune IgG) and positive (polyclonal anti-BSA) controls were included in all assays. Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/25/14 For personal use only.

FLOW MICROSPHERE INHIBITION IMMUNOASSAY (FMII)

BSA Id couplinn - to carboxvlated microspheres. BSA Id antibodies were covalently coupled to 3 pm carboxylated polystyrene microspheres (Epicon, Pandex Co., Mundelein Ill) using a carbodiimide procedure available as a kit (Polysciences,Warrington, PA). Two hundred fifty pl of a 5 % by weight suspension of microspheres (approximately 2 x lo9 microspheres) were washed 3 times by centrifugation at 14000 X g for 6 min. using the following sequence: once with carbonate buffer, pH 9.5 and twice with phosphate buffer pH 7.5. The microspheres were resuspended in 0.6 ml phosphate buffer containing 2% carbodiimide and incubated 3.5 - 4 hr at 22'C with rotational mixing. The microspheres were washed three times by centrifugation in borate buffer, pH 8.2 . After the final wash, the microspheres were resuspended in borate buffer and 200 or 400 pg of BSA Id added to the microspheres. The mixture was incubated overnight with gentle rotational mixing at room temperature. The supernate was removed and the protein content determined to calculate the protein bound to the microspheres. To block the remaining binding sites, the resulting pellet was resuspended in 1 ml of 0.1M ethanolamine, incubated for 30 min, then centrifuged again with 1 ml glycine, pH 7.4,(10 mg/ml) added to the pellet and incubated for an additional 30 min. The microspheres were pelleted, resuspended and stored in 0.1M PBS, pH 7.5,containing 10 mg/ml of glycine and 0.02% sodium azide at 4'C until used. The number of microspheres per ml was determined using a standard hemocytometer method on 1:lOO dilution of the suspension. Fluoresceination of BSA. Conjugation of BSA with fluorescein isothiocyanate (FITC) was performed based on the method of Woods, et al., (23). Briefly, powdered FITC was added at a concentration of 12 -20 pg/mg of BSA in carbonate buffer, pH 9.6 . Five ml of the mixture was passed through a 2.5 x 40 cm Sephadex G-25 column equilibrated with O.OlM PBS to separate BSA bound FITC (BSA-FITC) from unbound FITC. Fractions containing protein were determined spectrophotometrically (280 nm). The BSA-FITC fractions were pooled, concentrated an ultrafiltration cell with a 43 mm diameter PM 30 membrane ( Amicon Division W. R. Grace & Co, Beverly, MA ) and the FITC to protein ratio


570

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determined (FP ratio = 1.4). The conjugate was stored at 4% in PBS containing 0.02% sodium azide. DescriDtion of the FMII method. The FMII was based on the ability of paratope binding anti-Id antibodies to block the binding of the antigen to its complementary paratope of the Id. The concentration of Id coated microspheres was adjusted so that 50 pl would contain 500,000 microspheres. For determination of blocking ability, 50 pl of microspheres were incubated 30 min with 50 pl of anti-BSA Id. Fifty p1 of BSA-FITC was then added to the mixture and incubated for 30 min. All incubations were performed at 22°C. The mean fluorescent intensity (ELI) of each sample was determined by measuring the sample fluorescence using a fluorescent activated cell sorter (FACS) (FACStar, Becton-Dickinson, Mountain View, CA) to evaluate 5,000 events from each sample. Fluorescent data were collected in the log channel. The mean fluorescent intensity (El)of individual samples was determined by placing left and right boundary markers (gating) at the base of the peak of the computer generated display (histogram) of each sample. The computer program would then calculate the mean fluorescent intensity of the events between the markers. Once an individual becomes familiar with gating the results so generated, data can be rapidly analyzed. Included in assays as controls were: microspheres + BSA-FITC (maximum FL1); microspheres + chicken non-immune IgG + BSA-FITC (negative control); pre-incubation of the test sample + chicken non-immune I& prior to running the assay (allotype control); and preincubation of the test sample + BSA Id prior to running the assay. Percent inhibition was calculated using the following equation:

-

(Maximum FL1-neg FL1) (unknown FL1-neg FL1) x

100

(1)

(Maximum FLl- neg FLl)

FMII validation. For validation, BSA and anti-BSA Id were diluted in series of four dilutions with five replicates of the dilution series. Serial dilutions were performed for BSA concentrations ranging from 100 to 4 ng/ test and for antiBSA Id with concentrations ranging from 68.35 pg/test to 240 ng/test. Testing for homogeneity of equations for the results of the replications was chosen to determine if the FMII produced a linear and reproducible response. STATISTICAL ANALYSIS

Validation of the FMII was accomplished by a regression analysis testing the replications within each ligand for equality of slopes and intercept as well as homogeneity of the slopes of lines with percent inhibition versus loglo


57 1


(concentration) as test variables (24). The regression analysis tested the data of each of the replicates within a given ligand for equality of slope and intercept. Dependant upon the finding that the replicates within each ligand showed no significant differences (p 1.05) an equation describing a line for each ligand was determined. The standard error of the means for each dilution within each ligand were used to compute a confidence interval (CI) based on two sided t0.95 values of 2.776 for 4 degrees of freedom. RESULTS DETERMINATION OF PERCENT INHIBITION USING THE FMII

The mean channel fluorescence (FLU was determined for each sample through analysis of the distribution of measured fluorescence associated with each of 5000 individual microspheres by the software associated with the instrument. The program also allows for setting of gates to limit the inclusion of signals which were not within the normal distribution. The means so determined for each sample were used to calculate the percent inhibition using eq. 1. DETERMINATION OF ID CONCENTRATION TO COAT ON MICROSPHERES AND OPTIMIZATION OF BSA-FITC

Preliminary results of protein determinations on coupling supernates showed that 194 pg and 122 pg were bound to the total number of microspheres for coupling in the presence of 400 pg and 200 pg of BSA Id, respectively. Based on these results the microspheres were coupled in the presence of 400 pg BSA Id to keep the BSA Id at the higher coupling rate. Comparisons of amounts of protein coupled using the carbodiimide procedure to that of non-covalent binding on non-carboxylated microspheres also showed the carboxylated microspheres binding more protein on a microsphere to microsphere basis (data not shown). The results of comparison between dilution curves for BSA as the blocking ligand with and without washing between reagent additions are shown in Figure la. There was no significant difference between results of the FMII with the microspheres washed free of reactants between incubations and with the wash steps omitted. The optimum working dilution for the BSA-FITC proved to be 1:64000, giving the highest fluorescent intensity at which the sensitivity of the test appeared to be greatest. Preliminary results indicated that although using the conjugate at higher concentrations gave increased FL1, the test was less sensitive and the inhibition curve slope was much steeper (data not shown). The low end sensitivity of the F M I I with BSA as the blocking analyte was increased from 15 ng to 5 ng when 1%NRS was added to the TEN buffer used to

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572

100

80 .r( .r( Y

P60

#-


8

&

40

20

0 1 0 - 1 100

101

102

103

lo4

105

ng BSA

ng BSA

FIGURE 1 FMII results for effects of two treatments on the blocking of BSA-FITC to Id coated microspheres by BSA. Figure la) shows similarities in percent inhibition of binding of BSA-FITC with and without washing by replacement of fresh diluent between each reagent incubation to remove the unreacted ligands : washed non- washed . Figure l b ) shows changes in the

-

-

-

I

percent inhibition of binding of BSA-FITC usingTEN buffer as the diluent for BSA-FITC with and without 1%normal rabbit serum (NRS) added: TEN alone

-

TEN+lWNRS

.


573


a).

FIGURE 2 FMII histogram showing the effects of blocking of BSA-FITC to Id coated microspheres. Units on the abscissa in log scale are mean FL 1 intensity and the units on the ordinate are numbers of events. Figure 2a) demonstrates the maximum green channel fluorescence (FLI) with unblocked BSA-FlTC reacted Id coated microspheres ( ) compared to microspheres blocked ( -) by incubation with anti-BSA Id (AB2) then reacted with BSA-FITC. Figure 2b) shows same reaction of 2a) with no significant change in FLl when the AB2 was ) and without ( -) non-immune IgG. Histogram preincubated with ( printout image was duplicated with an image scanner and bit processed with a graphics program for clarity without changing the original curve.

---

---

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TABLE I


Representative experiment showing the specificity of inhibition of antigen binding by BSA anti-Id as determined by the flow microsphere inhibition immunoassay (FMII). Sample

Ell

Control-BSA-FITC

112.34

0

BSA anti-Id

52.27

53

BSA anti-Id pre-incubated with non-immune Ig

58.61

48

BSA anti-Id pre-incubated with BSA Id

112.02

0

1.70

0

Negative, microspheres only

% Inhibition2

1 FLI = mean fluorescent intensity collected in log scale; equivalent to mean channel number seen

*

el sewhere. % inhibition = percent inhibition of antigen binding to Id coated microspheres caused by anti-Id and was calculated by subtracting the negative FLl from each result then dividing the difference of the maximum FLl minus the unknown nl by the maximum FLl and then multiplying the quotient by 100. The assay for determining FLl was the flow microsphere inhibition immunoassay (FMII).

make the BSA-FITC dilution (Figure lb). The same result did not occur with the AB2 tested, and in fact the NRS proved to interfere when used in dilutions of any other reagents except the BSA-FITC. Thereafter, 1%NRS was added only to the BSA-FITC working dilution, so as to keep the increased sensitivity in the BSA test and not cause any interference in the anti-BSA Id test. HOMOGENEITY OF BSA AND ANTI-BSA ID INHIBITION OF BSA-FITC BINDING CURVES.

The reduction in the fluorescent intensity associated with the blocking of binding sites by either BSA or anti-BSA Id was used to determine the percent inhibition. BSA gave 2 95% inhibition when used as the blocking ligand at concentration of 333 pg/ml against binding of BSA-FITC to Id coated microspheres. The protein concentrations for the anti-BSA Id samples after concentration to 1 mi ranged from 0.130-1.367 mg/ml. The anti-BSA Id samples used as blocking Iigands gave 4144% inhibition against binding of BSA-FITC to Id coated microspheres. Six anti-Id, each from a different bird, were chosen from a group of over 20 isolates and were used in the remainder of this study.

575


TABLE 11


Means, standard error of the mean, and confidence intervals of data for BSA and anti-BSA Id used in validation of the FMII. Percent Inhibition3

Reagent1

Dil

Con$

BSA

1 2 3 4

0.110 0.037 0.012 0.004

82.12 54.06 21.08 5.77

1 2 3 4

6.650 2.210 0.738 0.246

1

2 3 4

990

CIS

SEM4 1.04 1.62

-

0.94

79.23 49.57 18.94 3.17

48.88 26.72 11.06 6.64

0.61 1.14 1.42 1.39

47.18 - 50.58 23.54 - 29.90 7.11 - 15.01 2.78 - 10.00

30.750 10.250 3.420 1.140

69.68 47.42 23.36 13.80

0.80 0.99 0.67 0.74

67.46 - 71.90 44.67 - 50.17 21.50 - 25.22 11.75 - 15.00

1 2 3 4

29.500 9.830 3.270 1.090

65.46 35.94 15.78 10.38

1.30 1.37 1.17 1.68

61.85 32.14 12.55 5.72

-

69.07 39.74 19.01 15.00

991

1 2 3 4

48.500 16.160 5.340 1.800

56.78 35.58 18.46 10.22

0.71 1.93 1.05 1.37

54.80 30.21 15.55 6.42 -

58.76 40.95 21.37 14.00

983

1 2 3 4

68.35 22.78 7.59 2.53

80.12 55.66 28.56 14.40

0.46 0.71 1.45 1.31

78.83 - 81.41 53.69 - 57.63 24.54 - 32.58 10.78 - 18.00

937

1 2 3 4

76.36 61.56 35.40 15.21

0.65 0.80 1.90 2.17

74.54 59.35 30.12 9.18

552

946

6.50 2.16 0.722 0.24

0.77

-

85.01 58.55 23.22 8.30

78.18 63.77 40.68 21.00

1 BSA=bovine serum albumin; other numerical designates are anti-Id isolate designations. 2 The amount of protein (in wg) added from each sample to the test volume. 3 mean percent inhibition for 5 replications SEM=standarderror of the mean 5 Confidence intervals were determined for each dilution based on a t -value of 2.776 (two-sided confidence interval at 0.95 for 4 degrees of freedom); ( mean - t.95(SEM) ;mean + t.95(SEM)

576

BROWN AND AINSWORTH


100 7

c

0

Q)

I

FIGURE 3

-

Mean percent inhibition of binding by BSA-FJTC to Id coated microspheres induced by the test ligands as determined by FMII from replications of dilution series for each of the anti-Id antibodies 552 , 946 , 990 , .........+........ 991 , - - *-. 983 , 937 and bovine serum BS* ; plotted as pg of ligand in the test volume versus albumin (BSA) percent inhibition of BSA-FITC. I

-

Figure 2a shows a FACS generated histogram comparing the unblocked microspheres with maximum FLl to the anti-BSA Id blocked microspheres showing the reduction in fluorescent intensity because the binding of BSA-FITC to the microspheres has been blocked. Preincubation of the anti-BSA Id with non-immune IgG did not effect the ability of the anti-Id to block the antigen-Id reaction (Figure 2b). The resuits for test of the specificity of the FMII to detect paratope reactive IgG samples are shown in Table I. The avian BSA model using the FMII was shown to demonstrate both specificity of binding for BSA and its anti-Id image with no cross reactions with non-immune IgG of the same origin and the inhibition curve did not change when anti-Id was preincubated with non-immune IgG (Table I). In addition, the anti-BSA Id was no longer able to block after preincubation with BSA Id. Together these results are highly suggestive, although not definitive, of paratope directed reaction (Table I). Table 11shows the percent inhibition determined for each anti-BSA Id as well as a BSA solution for five replicates of a dilution series used to validate the FMII. Mean percent of inhibition ranged from 6 to 82% for BSA and 6 to 80% for



517

the anti-BSA Id group. The confidence intervals for each dilution did not overlap even as the percent of inhibition approached lower levels. Figure 3 graphically depicts the means for the replications for each dilution as the amount of ligand versus percent of inhibition for both BSA and anti-BSA Id. The slopes for all the lines are similar, and it was determined that valid statistical comparisons for BSA would be from 4 to 110 ng/test and for antiBSA Id from 240 ng to 68 pg/test. The test for equality of slopes and intercepts as well as the test for homogeneity for replicates within ligands were not significantly different. DISCUSSION The reactions for BSA and anti-BSA Id inhibition of BSA-FITC binding to Id coated microspheres were reproducible as indicated by obtaining the same line equation for each replication. To ensure that the anti-BSA Id was reacting specifically with the BSA Id and not in a non-specific fashion with 1) the microspheres or 2) with allotypic markers present on the antibody coated onto the microspheres, a series of experiments incorporating various combinations of reactants was performed. The results of these experiments indicated that the anti-Id was reacting only with an idiotypic marker associated with the antigen binding region of the Id. Preincubation of anti-Id with non-immune serum did not cause an appreciable change in the percent of inhibition of antigen binding whereas preincubation with Id reduced the percent of inhibition to zero. Addition of non-immune IgG did not show inhibition. The explanation for the 5% decrease in percent of inhibition with non-immune Ig was due to nonspecific binding occurring because of the similar occurrence with NRS with antiId. We have described a useful technique employing flow cytometry to detect paratope binding anti-idiotypic antibodies. The FMII was reliable based on the interassay reproducibility and on the fact that equal line equations were generated for a given ligand in multiple experiments. Care must be taken that each reagent is optimized, in our case BSA Id coated microspheres and BSAFITC. In our hands the lower limit of an anti-Id concentration that would result in detectable percent of inhibition was 240 ng/test (1.33 X 10 -12M),whereas the lower limit for BSA was 5 ng (2.79 X 10 -14M). If the anti-Id lines were shifted to the left, they would shadow the BSA curve. The slopes are very similar indicating similar dynamic ranges although the anti-Id are at a higher concentration and therefore appear to show a lower sensitivity than the putative antigen. However, the anti-Id isolated concentration was the limiting factor for

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the upper range for the test in that total inhibition was not >95%at the starting concentration before dilution. Note that BSA gave 95 percent inhibition in this assay at concentrations > 300 n g / d (16 ng per test). The FMII has many advantages when compared to other assays for detecting paratope binding to anti-Id. The assay can be performed in approximately 60 min, radionuclide usage and disposal are eliminated, and fluoresceinated antigen and Id coated microspheres have a much longer shelf life (>6 mo.) than radionuclides commonly used. The test also did not require any washing steps to remove unreacted analyte, which agrees with results reported in earlier manuscripts (25-28). Another advantage of using flow cytometry is that data can be collected on large numbers of particles (microspheres) within seconds. In this study, information was collected on 5,000 microspheres per sample to ensure statistically valid data was gathered. Although we have validated the FMII using a specific antigen, BSA, the FMII could be easily adapted to other antigens. We have used the method in idiotypic studies on an extract of Pastewella rnuftocida and found it to work as well (data not shown). The one drawback of the FMII is the need for an expensive instrument to analyze samples. However, most research institutions have organized flow cytometry facilities that only require paying for instrument time thus eliminating the necessity to purchase an instrument. The cell sorting feature is not a necessary feature to perform the FMII and a standard flow cytometer, such as a FACScan, would suffice. In summary, the FMII is a uncomplicated assay that provides information on the presence of paratope binding anti-Id based on the ability of anti-Id to block the binding of fluoresceinated antigen to its complementary Id. ACKNOWLEDGEMENTS

We thank J. Brazil and J. May for technical assistance. We also thank B. Boyd for flow cytometry operation and C. Ebyle for providing the statistical analysis. This research was supported in part by National Institutes of Health Grant # 1 R15 AI26264-01 and the Mississippi Agriculture and Forestry Experiment Station. Contribution No. J-7653 from the Mississippi Agriculture and Forestry Experiment Station. REFERENCES 1.

2.

N. K. Jeme, Annals. Immunol., 125c,373-389, (1974). 7 5-24, (1984). N. K. Jerne, Immunol. Rev., -9


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Enzyme immunoassay for detecting Brucella antibodies in cow's milk.

Antiidiotypic antibodies against anti-cGMP polyclonal antibodies.

Microsphere-based immunoassay for the detection of azaspiracids.

Replacing antibodies with aptamers in lateral flow immunoassay.

Novel strategies to enhance lateral flow immunoassay sensitivity for detecting foodborne pathogens.

New enzyme immunoassay for detecting cryptococcal antigen.

An enzyme-immunoassay method for detecting circulating immune complexes by inhibition of polyclonal rheumatoid factor.

A rapid inhibition micro ELISA for detecting antibodies to Plasmodium falciparum sporozoites in human blood.

Cellphone-based detection platform for rbST biomarker analysis in milk extracts using a microsphere fluorescence immunoassay.

Microparticle-enhanced nephelometric immunoassay with microsphere-antigen conjugates.

Antiidiotypic antibodies against anti-TPO antibodies in sera of patients with autoimmune thyroid disorders.

Establishment of immunoassay for detecting HPV16 E6 and E7 RNA.

Relationship between naturally occurring human antibodies to casein and autologous antiidiotypic antibodies: implications for the network theory.

Process monitoring by flow-injection immunoassay. Evaluation of a sequential competitive binding assay.

Comparative Evaluation of Tubex TF (Inhibition Magnetic Binding Immunoassay) for Typhoid Fever in Endemic Area.

6-Plex microsphere immunoassay with imaging planar array detection for mycotoxins in barley.

Multiplex chemiluminescent immunoassay for screening of mycotoxins using photonic crystal microsphere suspension array.

A simple method for detecting antibodies to rubella.

Flow cytometric microsphere-based immunoassay as a novel non-radiometric method for the detection of glutamic acid decarboxylase autoantibodies in type 1 diabetes mellitus.

Simultaneous detection and quantification of six equine cytokines in plasma using a fluorescent microsphere immunoassay (FMIA).

Perinatally derived antiidiotypic antibodies from the antipolyfructosan immune response. Molecular analysis of idiotype regulation.

Solid-phase enzyme immunoassay for immunoglobulin M antibodies to cytomegalovirus.

Sandwich enzyme immunoassay for serum integrins using monoclonal antibodies.

An immunoassay for histamine based on monoclonal antibodies.