Antibodies against toluene diisocyanate protein conjugates three methods of measurement

Antibodies against toluene diisocyanate protein conjugates Three methods Roy Patterson, Chicago,

of measurement

M.D., Kathleen

E. Harris, B.S., and C. Raymond

Zeiss, M.D.

Ill.

With the use of canine antisera against toluene diisocyanate (TDI)-dog serum albumin (DSA), techniques for meusuring antibody against TDI-DSA were evaluated. The use of an ammonium sulfate precipitation assay showed suggestive evidence of antibody binding but high levels oj TDI-DSA precipitation in the absence of antibody limit any usefulness of this technique. Doubleantibody co-precipitation techniques will measure total antibody or Ig class antibody against ‘251-TDI-DSA. These techniques are quantitative. The polystyrene tube radioimmunoassay is u highly sensitive method of detecting and quantitatively estimating IgG untibody. The enzyme linked immunosorbent assay is a rapidly adaptuble method for the quuntitative estimation of IgG, IgA, and IgM against TDI-homologous proteins. All these techniques were compared and results are demonstrated by using the same serum sample .for undysis. (J ALLERGYCWNIMMUNOL 72.~576480, 1983.)

Measurement of antibodies against TDI-protein conjugates has been a topic of interest because of the importance of TDI antibodies in producing airway reactions in workers exposed to TDI. The problem is complex and correlations of immune response with disease have been difficult, in part because some human TDI airway responses are not immunologic in origin (for extensive review see ref. 1). We have recently reported the induction of both airway responses and antibody responses in dogs, using intrabronchial TDI delivered as an aerosol.2 The responses of dogs resembled those of exposed human workers to some extent and particularly in relation to the variable responses that occur in both species. One finding of value was the demonstration of significant levels of canine antibodies against TDI-dog serum proteins. These are presumed to result from the reaction of TDI with proteins in the canine airway. This is followed

From the Section of Allergy-Immunology, Department of Medicine, Northwestern University Medical School, Chicago, 111. Supported by U.S.P.H.S. NIAID Grant Al 11403 and The Ernest S. Bazley Grant. Received for publication April 6, 1983; accepted June 23, 1983. Reprint requests: Roy Patterson, M.D., 303 East Chicago Avenue, Chicago, IL 6061 I.

676

by an immune response of the dogs to the TDI-selfprotein conjugate in a manner analogous to formation of trimellitic anhydride- self-protein antigens and antibody responses in humans exposed by inhalation to trimellitic anhydride. The availability of high-titered canine anti-TDI dog serum proteins permitted the analysis of techniques for measuring antibodies against TDI-homologous serum proteins with demonstration that a polystyrene tube radioimmunoassay or an ELISA are useful for measurement of anti-TDI protein antibodies of an Ig class. A double-antibody co-precipitation technique will measure total anti-TDI protein but the ammonium sulfate precipitation technique useful for measuring anti-trimellitic anhydride-albumin4 has very limited usefulness for the measurement of anti-TDI-albumin because of the altered nature of the TDI-albumin conjugate. The antisera tested were from animals immunized by TDI alone to the airway analogous to humans sensitized by inhalation of TDI. The results may thus differ from those of animal experiments in which animals are immunized with TDI-heterologous protein conjugates. The results described below are from multiple experiments with each technique, using serum samples from dogs mounting an immune response to .TDI-DSA after receiving intrabronchial

Antibodies

VOLUME 72 NUMBER 6

AhDmkirior~.s

CPM: USA: ELISA: TDI:

assay

TDI.” To make comparisons of techniques, all results are shown on two serum samples from the same dog prior to immunization (control) and at week 37 after receiving multiple intrabronchial challenges with TDI . MATERIALS Sera

AND METHODS

Serum samples used in these studies were obtained from three healthy adult pound dogs immunized by intrabronchial administration of TDI as described previously.Z

Immunization

procedures

Briefly, dogs were immunized with TDI every 2 wk for 4 mo and every 4 wk thereafter for 6 mo by using 1 mg/kg TDI delivered by aerosol through a cuffed endotracheal tube with an in-line nebulizer in a Bird Mark VII (Bird Corp., Ltd., Richmond, Calif .). It had been determined previously that in this system approximately 20% ? 10 of the material aerosolized is retained in the airway. For these challenges the number of breaths were varied to achieve the desired concentration. The immunization also constituted an airway challenge in subsequent experiments. TDI was used to potentially simulate airway exposure of humans. The sera were stored at -20” C until use. Sera were obtained prior to initiation of TDI immunization and these were used as controls.

Preparation

diisocyanate

677

mined and assumed to be the same in either the conjugated or unconjugated form. The molecular ratio of TDI to DSA was calculated and, in the conjugates used, the range was from 8 to 11.

LLWI

Counts per minute Dog serum albumin Enzyme-linked immunosorbent Toluene diisocyanate

against toluene

of TDI-DSA

Fifty milligrams of TDI (Aldrich Chemical Co., Milwaukee, Wise .) as a liquid was added dropwise to 50 mg of chromatographically pure DSA (Cappel Laboratories, Inc., Cochranville, Pa.) in 10 ml of 9% sodium bicarbonate. The mixture was stirred for at least 30 min at room temperature and then dialyzed extensively against O.lM sodium bicarbonate and distilled water. Sufficient concentrated Tris saline buffer, pH 7.6, was added to make the preparation isotonic. The TDI-DSA was then sterilized with a 0.2 Frn filter (Millipore Corp., Bedford, Mass.). To document that TDI had conjugated with DSA, absorbance spectra of the conjugate and the unconjugated DSA were run and compared. The molecular ratio of TDI to DSA was determined as follows. The differential between spectra was found to be maximal at 240 nm. The epitope density was calculated by using the difference in absorbance of TDI-DSA and unconjugated DSA at equivalent DSA concentrations. The extinction coefficient of TDI was deter-

Radioiodination TDI-DSA ( 100 pg) or dog IgG (100 pg) (Cappel Laboratories) was labeled with ‘?;I by using chloramine T.’ according to the method described by Gleich et al.” The “‘ITDI-DSA was 98% precipitable with 10% trichloroacetic acid. The specific activity of the ‘““I-TDI-DSA was 21.000 CPMing. The “>I-dog IgG was 98% precipitable with 10% trichloroacetic acid and the specific activity was 30,000 CPMing.

Double-antibody

co-precipitation

A modification of techniques originally used to detect IgE was used to assay for IgG or IgG, IgA, and 1gM antibodies .?, ” One-tenth milliliter of various dilutions of a serum are incubated with 0.1 ml of r2”1-TDI-DSA (approximately 500,000 CPM) at 37” C for ‘/2 hr and then overnight at 4” C. The next day 0.1 ml of 1: 5 rabbit anti-dog IgG or a mixture of rabbit anti-dog IgG, IgA, and 1gM to a final concentration of 1:5 was added and the tubes were incubated for 48 hr at 4” C. Finally, 0.3 ml of goat anti-rabbit IgG was added and the tubes were incubated for ‘/2 hr at 37” C and then 3 hr at room temperature. Precipitates formed and were washed three times with phosphate-buffered saline. The washed precipitates were then counted in a gamma counter and the counts in sera thought to have antibody were compared with the counts bound by an unimmunized control serum.

Polystyrene

tube assay

The polystyrene tube radioimmunoassay was performed as previously described.g Briefly, polystyrene tubes were coated with TDI-DSA in O.lM sodium bicarbonate overnight at 4” C. The tubes were washed and 1% bovine serum albumin in phosphate-buffered saline was added for 1 hr at room temperature. The tubes were again washed and dilutions of dog serum were added and then incubated at 4” C overnight. The tubes were again washed, rabbit anti-dog IgG (Miles Laboratories, Inc., Elkhart, Ind.) was added, and the mixture was incubated overnight at 4” C. Tubes were washed, Y-dog IgG (200,000 CPM/ml) was added, and the mixture was incubated overnight at 4” C. The next day the tubes were washed and counted. The antibody activity is indicated by the CPM of “‘l-dog IgG bound.

Enzyme-linked

immunosorbent

assay

The ELISA assay used for detection of antibody against TDI has been described previously.’ Briefly, the following reagents were added in sequence to Immulon micro-ELISA plates (Dynatech Laboratories, Inc., Alexandria, Va.) with appropriate washes and incubation periods according to the methods of Voller et al.‘O and Sepulveda et al.]‘: TDI-DSA, dilutions of dog serum containing antibody or control

680

Patterson

J. ALLERGY CLIN. IMMUNOL. DECEMBER 1983

et al.

6. 38%

d

“‘0

body not available for canine IgE. The co-precipitation and polystyrene tube techniques have been used for human IgE assays and should be applicable to detection of human IgE antibodies against TDIhuman serum proteins. REFERENCES I. Bernstein

o--

100

%

$

too

1000

100

1000

10000

2.

0.

(-2.

35%

In E 8 t 0.

10000

33%

3.

4.

50 OQb “‘.....O

Q “‘--0

-LA

5.

0

100

1000

10000

ng TDI-DSA

100

1000

10000

Added

6.

FIG. 4. Co-precipitation serum trations

of 1251-TDA-DSA with normal dog (0) or dog anti-TDA-DSA (0) at different concenof ammonium sulfate.

The polystyrene tube radioimmunoassay gives a quantitative estimate of antibody activity as compared with control or other positive serum samples, using the technique described with layering of TDI-protein, IgG antibody, anti-&G and ‘2”I-IgG. The technique is highly sensitive in detecting antibody in a serum dilution of 10m4. This sensitivity results from the amplification of the reaction as the result of the sequential addition of the four reactants. The ELBA technique is a semiquantitative technique largely using commercially available reagents and no radioactivity. It thus has wide applicability and may be used for measurement of IgG, IgA, and IgM antibodies against TDI-proteins. No in vitro studies of IgE antibodies against TDIDSA were carried out, although the dogs immunized showed respiratory and cutaneous reactivity to TDIDSA consistent with antibody reactions due to IgE antibody.’ Such in vitro assays require anti-IgE anti-

7

8

9

10

11.

12.

13.

IL: Isocyanate-induced pulmonary diseases: a current perspective. J ALLERGY CLIN IMMUNOL 70:24. 1982 Patterson R, Zeiss CR. Harris KE: Immunologic and respiratory responses to airway challenges of dogs with toluene diisocyanate. J ALLERGY CLIN IMMUNOL 71:604, 1983 Patterson R. Zeiss CR, Pruzansky JJ: Immunology and immunopathology of trimellitic anhydride pulmonary reactions. J ALLERGY CLIN IMMUNOL 70:19, 1982 Zeiss CR, Levitz D. Chacon R, Wolkonsky P, Patterson R, Pmzansky JJ: Quantitation and new antigenic determinant specificity of antibodies induced by inhalation of trimellitic anhydride in man. Int Arch Allergy Appl lmmunol 61:380. 1980 Greenwood FC. Hunter WM. Clover JS: The preparation of ‘:“I-labeled human growth hormone of high specific radioactivity. Biochem J 89:114. 1963 Gleich GJ. Averbech AK, Swedlund HA: Measurement of IgE in normal and allergic serum by radioimmunoassay. J Lab Clin Med 77:690. 1971 Zeiss CR, Pruzansky JJ, Levitz D, Wang J: The quantification of IgE antibody specific for ragweed antigen E (AgE) on the basophil surface in patients with ragweed pollenosis. Immunology 35:237. 1978 Zeiss CR, Pruzansky JJ, Patterson R, Roberts M: A solid phase radioimmunoassay for the quantitation of human reaginic antibody against ragweed antigen E. J Immunol 110:414, 1973 Patterson R, Roberts M, Zeiss CR. Pruzansky JJ: Human antibodies against trimellityl proteins: comparison of specificities of IgG, IgA and IgE classes. Int Arch Allergy Appl Immunol 66:332, 1981 Voller A, Bidwell DE, Bartlett A: Enzyme immunoassays in diagnostic medicine: therapy and practice. Bull WHO 5355, 1976 Sepulveda R. Longbottom JL, Pepys J: Enzyme linked immunosorbent assay (ELISA) for IgG and IgE antibodies to protein and polysaccharide antigens ofAspergi/lusfumiRarus. Clin Allergy 9:359. 1979 Zeiss CR, Metzger WJ, Levitz D: Quantitative relationships between IgE antibody and blocking antibodies specific for antigen E in patients given immunotherapy with ragweed antigen E. Clin Exp Immunol 28:250, 1977 Lidd D, Farr RS: Primary interaction between ‘:“I-labeled ragweed pollen and antibodies in the sera of humans and rabbits. J ALLERGY 33:45. 1962

VOLUME 72 NUMBER 6

Antibodies

Anti Dog

TIN-DSA A Week

against

toluene

M

37

Pre-immunization Serum Dog A Week o

diisocyanate

Anti Dog

679

TDI-DSA A Week

37

0 .._...0 ~~rr;~munization Dog

s -

A

Week

0

0..“.(.......o,,. “........o ,,_, .......0 .,..,....... 0 ....... . .0

1 01

20

iO-

Reciprocal

0’ 104

104

Serum FIG. 2. Polystyrene body

against

80

tube

TDI-DSA.

101

point titer serum is serum.

Dilutions for 1.

IgG

sulfate

of Serum

5120

20480

Dilution

for IgG antibody against TDI-DSA. The end is 5120, where the optical density (0.0.) of test at least twice the optical density of control

anti-

1: 1280 and IgM titers as high as 1: 320 have been observed. Ammonium

1280

FIG. 3. ELISA

101

radioimmunoassay Same sera as in Fig.

320

co-precipitation

Multiple attempts to develop an ammonium sulfate co-precipitation technique were made. An example of the best results are shown in Fig. 4. A continuing problem was the precipitation of significant amounts of the iZsI-TDI-DSA with normal dog serum. This occurred with various TDI-DSA conjugates and various concentrations of ammonium sulfate (as shown in Fig. 4). Although the antiserum resulted in a higher degree of precipitation of ‘*“I-TDI-DSA, suggesting the possible use of the ammonium sulfate technique as an immunoassay, the minimal difference from control levels indicates that there would likely be considerable difficulty in establishment of a quantitative, reliable immunoassay. DISCUSSION The ammonium sulfate co-precipitation technique has proved highly useful for measurement of total antibody activity of human antiserum against trimellityl-human serum albumin. Our expectation that the same technique would be as effective in measuring anti-TDI-DSA was not met as shown in Fig. 4. The high percentage of ““I-TDI-DSA precipitated at 33%

to 40% saturated ammonium sulfate suggests that the solubility of albumin is altered by conjugation with TDI. TDI,,-DSA is shown in Fig. 4, and since that the average ligand concentration per DSA molecule is 10, it seems likely that the TDI-DSA molecules precipitated at these concentrations of ammonium sulfate would be those with a higher number of TDI ligands per molecule of DSA. Thus not only are the control levels (which would be subtracted as background) high but also the differential precipitation of varying populations of TDI-DSA molecules could produce erratic results. The results demonstrate three methods for the detection of antibody against TIN-homologous serum albumin, other homologous proteins, or even autologous proteins. These techniques should be applicable to antibody studies of humans exposed to TDI. The double-antibody co-precipitation technique is useful for detection of total antibody against TDIhomologous protein when the precipitating antibodies are anti-IgG, anti-IgA, and anti-IgM. This same technique can be used for measuring antibody activity of antibodies of individual Ig classes as IgG antibody in Fig. 1. The technique is quantitative and antibody activity can be expressed as micrograms of InsITDI-protein bound or be calculated as antibody by measuring binding in antigen excess. The only disadvantage is the requirement for radioactive isotope labeling of the TDI-protein.

682

J ALLERGY CLIN. IMMUNOL DECEMEFR 1983

Mekori et al.

Ahhwvi~rtions

CU: PBS

used

Chronic urticaria Phosphate-buffered

saline

pan T cells,!’ was obtained from Antibodies, Inc., Davis, Calif. Biotinylated horse anti-mouse IgG and avidin-horseradish peroxidase conjugates were obtained from Vector Laboratories, Burlingame, Calif. Immutqwrmidusr .stuining. A labeled avidin-biotin method with minor modification at the last stage was used.“‘. I’ Briefly, acetone-fixed cryostat sections were rehydrated in PBS for 3 min. Sections were incubated sequentially with the following reagents at appropriate dilutions for the times shown and rinsed with PBS after each incubation: (1) murine monoclonal antibody, 15 min; (2) biotinylated horse anti-mouse IgG, 15 min; (3) avidinhorseradish peroxidase, 15 min; (4) substrate 3-amino-9ethyl-carbozole and hydrogen peroxide in acetate buffer (pH = 5.2). 10 min. Then sections were rinsed with water, counterstained 5 set with Gill’s hematoxylin (Fisher Scientific Co., Fair Lawn, N. J.), rinsed again with water, and mounted with Aquamount (Lemer Laboratories, New Haven, Conn.). Monoclonal antibody staining was periodically checked with lymphoid tissues to ensure proper staining patterns. Blocking with nonspecific serum or peroxide was unnecessary because of very low nonspecific binding and endogenous peroxidase activity. Only sections containing at least 75 mononuclear cells were counted. Cells counted ranged up to 200 in individual sections. Tissue sections were scored independently by two observers. The number of positive cells (membrane rim red-brown staining with various monoclonal antibodies) was determined. and the percentage of positively-stained cells calculated.

RESULTS All the urticarial lesions had mild edema of the dermis with nonnecrotizing perivascular infiltrate of mononuclear cells. The five biopsy specimens from unaffected skin lacked significant lymphocytic infiltrate. In all biopsy specimens from urticarial lesions, most of the mononuclear cells in the perivascular dermis were stained with anti-T101 (>90%) and anti-T4 antibodies (mean 68%) (Fig. 1). Only occasional cells were stained with either anti-B1 or antiM02, which suggested that only a few B cells and monocytes/macrophages were present in the skin lesions. This suggests that most of the infiltrating mononuclear cells were T cells possessing the helperinducer phenotype. In contrast, a smaller number of mononuclear cells stained with anti-T8 antibodies (mean 19%) indicated that T cells with suppressorcytotoxic phenotype formed a minor component of

the mononuclear cell infiltrate. The relative proportion of these two T cell subsets varied considerably from case to case with the T4+ :T8+ ratio varying from 2: 1 to 9: 1 (Fig. 2). A variable number of cells (mean 65%) in the perivascular cellular infiltrate were stained with antiI2 antibody (la+ cells). In addition, la+ cells were also seen occasionally in the epidermis attributed to the Langerhans cells. Since Ia antigen is expressed by macrophages and activated T12 and B cells,‘:: and since cells stained with anti-B1 and anti-MO2 were sparse in the skin lesions of our patients, it seems likely that a large proportion of the mononuclear cells in these lesions coexpressed the T4 and the la antigens. A double staining technique was not used to clarify this issue. DISCUSSION CU is often frustrating for both patient and physician. It is a common disease, and yet its pathogenesis is still not well understood. Several findings characterize this disease: (1) A specific etiology could be identified in no more than 20% of cases.I. ” Thus most patients appear to have an “idiopathic ” disorder. (2) Vasoactive amines, in particular, histamine, have been considered to be the primary mediators involved in the production of the lesion. The origin of these amines is skin mast cells, stimulated to degranulate by both immunologic and nonimmunologic factors. Ia* l3 The average tissue histamine content in the skin of patients with CU was found to be significantly higher than that in normal controls, another bit of evidence suggesting that local histamine elevations may be important in the pathogenesis of this disease in many patients. l6 (3) More than 90% show a nonnecrotizing infiltration of lymphocytes surrounding small venules in the more superficial dermis without deposition of immunoglobulins or complement .I Recently, with various staining techniques, Natbony et al.” defined the type of cellular infiltrates that characterize CU. These authors found a significant increase in the numbers of lymphocytes and mast cells in the involved areas without evident damage to the vessel walls. The cause of this cellular infiltrate is unknown, and the mechanism by which mast cell degranulation occurs is similarly unclear. In our histologic study of 42 patients with CU,lH a predominant lymphomonocytic infiltrate was within postcapillary venules in 11 (26%) and in a perivascular location only in 18 (43%). The latter patients were similar to those studied by Natbony et al.li and the patients in this study. To further phenotype the dermal mononuclear infiltrate in the present study of CU, T and B cell antigens were detected with monoclonal antibodies by use