Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56966—Animal cells
  • G01N33/56977—HLA or MHC typing

Definitions

• the invention is related to methods to determine the ability of candidate moieties to bind to specific major histocompatibility complex glycoproteins. Successful candidates are useful as therapeutics in conditions mediated by particular MHC glycoproteins.
• MHC glycoproteins play an essential role in B-lymphocyte and T-lymphocyte responses.
• the MHC glycoproteins are divided into two types, Class I and Class II, where each of these classes appears to play a substantially different role.
• the structure of the Class I and Class II glycoproteins is different. Despite the differences, in each case it is found that an antigen to which the immune system will respond is degraded intracellularly and a fragment of the antigen is expressed on the cell surface associated with an MHC glycoprotein.
• Each MHC glycoprotein has polymorphic regions, which are associated with a groove, and have some degree of specificity as to the peptide which binds in the groove. Thus, each MHC glycoprotein has only a specific repertoire of relatively low molecular weight moieties to which it is capable of binding. It is the identification of this repertoire of low molecular weight moieties to which the invention described below is directed.
• the MHC glycoprotein-peptide complex is presented to a T-cell receptor which specifically recognizes the fragment in conjunction with the MHC glycoprotein to which it is bound.
• the T-cell becomes stimulated and secretes lymphokines, with resulting expansion of the lymphocytes. Since these fragments are critical to the activation of lymphocytes, peptides or small molecular weight organic molecules may be devised or discovered which will play a role in enhancing or diminishing the activation of specific lymphocytes. In this way, B- and T-lymphocyte activation may be controlled to enhance or suppress a particular immune response.
• dispersed soluble MHC glycoproteins are treated with a detectable agonist in the presence of a competitor candidate moiety under conditions wherein the agonist is known to form a complex with the MHC glycoprotein.
• the resulting complex is separated from the reaction mixture, and the effect of the candidate moiety on the agonist included in the complex is determined.
• MHC glycoprotein is captured on an assay plate which plate is, for example, derivatized to anti-MHC glycoprotein antibody or other reagent with affinity for the MHC glycoprotein and used in place of the soluble MHC in the competitive assay.
• isolated MHC glycoprotein is preloaded with a homogeneous peptide preparation to provide, therefore, a homogeneous population of already-coupled MHC glycoprotein.
• dissociation of the preloaded peptide is the rate- determining step in the association of alternate moieties, this approach permits control of the reaction rate to assure uniform competition between the detectable agonist and the candidate.
• dilution of the performed MHC complex results in rapid dissociation of the preloaded peptide, thus creating an "empty pocket" (i.e., an available domain or binding site) for binding to the agonist or candidate.
• the homogeneous, preloaded peptide is preferably chosen to be comparatively readily released by the MHC glycoprotein, thus shortening the time of the assay.
• isolated MHC glycoprotein is preloaded with a labeled peptide whose rate of dissociation from the MHC glycoprotein is known to be influenced by the presence of other peptide. The ability of test compounds to accelerate the dissociation or displacement of the labeled agonist from the MHC glycoprotein is determined.
• a useful means to detect the formation of complex with the agonist and/or candidate moiety is to determine the effect of the complex on T- cell stimulation.
• the invention is directed to a method to determine the affinity of a test compound to an MHC glycoprotein, which method comprises combining in a reaction mixture cell-free dispersed MHC glycoprotein, a soluble agonist capable of binding to said MHC glycoprotein to form a complex and capable of being detected when in said complex, and said test compound, under conditions wherein the test compound and the agonist compete for binding to the MHC glycoprotein; separating MHC glycoprotein-bound agonist from unbound agonist; and detecting the amount of agonist bound in the complex as a function of the concentration of test compound in the reaction mixture.
• the invention is directed to a method to determine the affinity of a test compound for a specific MHC glycoprotein, which method comprises treating a solid support to which the MHC glycoprotein is coupled with a reaction mixture containing a soluble agonist capable of binding to the MHC glycoprotein to form a complex and capable of being detected when in said complex and said test compound, under conditions wherein the test compound and said agonist compete for binding to the MHC glycoprotein; removing said reaction mixture from the solid support; and detecting the amount of agonist bound to the solid support as a function of the concentration of the test compound in the reaction mixture.
• the invention is directed to a method for determining the affinity of a test compound to an MHC glycoprotein, which method comprises treating an MHC glycoprotein preloaded with a first agonist with reaction mixture containing the test compound and a second agonist capable of binding to said MHC glycoprotein to form a complex and capable of being detected when in said complex under conditions wherein the test compound and second agonist compete for binding to said MHC glycoprotein; wherein said MHC glycoprotein is preloaded with a preloading agonist and diluted prior to the addition of the reaction mixture; and detecting the amount of second agonist bound in the complex as a function of the concentration of test compound in the reaction mixture.
• the invention is directed to a method for determining the affinity of a test compound to an MHC glycoprotein, which method comprises combining in a reaction mixture the test compound and said MHC glycoprotein which has been preloaded with a labeled agonist, whose dissociation rate has been shown to be affected by the presence of other peptides and comparing the dissociation rate of the preloaded complexes in the presence and absence of the test compound to detect any accelerated off rate by the test compound.
• the invention is directed to a method to detect the presence of a moiety in an MHC glycoprotein complex which method comprises contacting said complex with a culture of T-cells primed with said moiety and detecting the presence, absence or amount of proliferation of said T-cells.
• Figure 1 shows a typical inhibition curve wherein unlabeled HA 307-319 competes with labeled HA 307-319 for binding to DR4Dw4.
• Figure 2 shows a binding curve for labeled HA 307-319 using DR4Dw4 captured from a crude lysate by antibody-coated microtiter plates.
• Figures 3A, 3B and 3C show "off rates" determined for various test peptides after preloading onto DR4Dw4 MHC glycoprotein.
• Figures 4A and 4B show a binding curve and an inhibition curve, respectively, for RMBP 90-102 peptide with respect to preloaded and control MHC glycoprotein.
• Figures 5A and 5B show dissociation curves for, respectively, biotinylated RMBP 90-102 and biotinylated HSP 3-14 in the absence and presence of HA 307-319.
• Figure 6 shows a displacement curve of biotinylated HSP 3-14 by RMBP 90-102.
• Figure 7 shows a binding curve for HA 307-319 peptide using sensitized T-cell proliferation as an assay for bound peptide.
• Methods and compositions are provided for determining the binding affinity of a candidate moiety for a specific MHC glycoprotein using competition between detectable agonist and the candidate of interest using several efficient assay strategies.
• competition is effected between a detectable agonist and the candidate.
• the effect of at least one concentration, and preferably varying concentrations, of the candidate moiety on binding of the detectable agonist is then determined.
• the effect of at least one concentration, and preferably varying concentrations of the candidate moiety, on the dissociation rate of the preloaded MHC/labeled agonist complex is determined.
• detectable "agonist” is meant a peptide or other low molecular weight compound known to be capable of binding to the specific MHC glycoprotein being tested.
• useful agonists for DR alleles are peptides derived from hemagglutinin such as that represented by positions 307-319 (HA 307-319) (Int Immunol (1990) 2.:443) .
• Suitable agonists that have been found to bind MHC glycoprotein products of DR alleles include, but are not limited to, HADP 3.2, RRFAAAYAARAAA; HADP 3.6, RRFAAQYAARAAA; RMBP 90-102, HFFKNIVTPRTPA; HSP 3-14 (R5 K13), RVRRGLTVAVKG; HSP 3-14 (K5 K13), RVKRGLTVAVKG; HSP 3-14 (K5 A13) , RVKRGLTVAVAG and shortened versions thereof.
• Useful agonists may include the peptides shown above and equivalents in which the amino and carboxyl charges eliminated. Some agonists are allele specific such as pertussis toxin 31-43, ragweed 3 50-62 and flu matrix 18-30.
• the agonist is generally labeled in a manner so as to permit its convenient detection when complexed to MHC. However, if detection is effected in a manner which is dependent on the nature only of the agonist, as described with respect to the T-cell proliferation assay for determination of complexed peptide, no extraneous labeling is needed. Thus, the agonist need only be detectable when in the complex.
• the label may be in various forms.
• various labels may be employed, such as radioisotopes, biotin, fluorescers, chemiluminescers and the like.
• the choice of the label will be primarily directed to convenience, sensitivity, minimizing background, minimizing interference with binding of the agonist to the MHC glycoprotein, and the like.
• the agonists will be at a concentration of about 0.1-50 times the concentration of the MHC glycoprotein.
• a particularly preferred label is biotin.
• streptavidin which is in turn labeled with a wide variety of labels may be used for detection.
• the streptavidin may be labeled with radioisotopes, fluorescers, chemiluminescers, enzymes, colloidal particles, or the like. As indicated above, a variety of considerations will dictate which label will be employed.
• the concentration of candidate moiety of interest will vary depending upon the concentration of agonist present in the medium, and the relative affinities of the candidate and agonist. Usually, the amount of the candidate will not differ by more than about 100-fold from the amount of agonist present in the medium. '
• the various components e.g., MHC glycoprotein, agonist and test compound
• the temperature is at about 37°C.
• the time for reaching equilibrium will be at least about 0.5 hours, more usually about 12 hours, and will generally not exceed about 48 hours. While a rate determination can be used, where a plurality of samples are employed and each sample analyzed for the amount of complex formation, it will usually be sufficient to do a single determination at varying concentrations of candidate.
• Suitable candidate moieties include any small molecular weight material which is thought to block the MHC glycoprotein of interest.
• these candidates are peptides of on the order of 5 or more amino acids or are small organic molecules which mimic the conformation of such peptides.
• a wide variety of candidates is generally known in the currently active field of rational drug design. There is no theoretical limitation on the range of candidates, and any putatively active compound may be employed.
• a soluble MHC glycoprotein in solution is used in the complex formation reaction.
• the labeled agonist complexed with the MHC glycoprotein is separated from free agonist and the amount of complexed agonist determined as a measure of the affinity of the candidate to the MHC glycoprotein.
• the method provides for a rapid, simple and accurate technique for screening large numbers of candidates and obtaining relevant values for the affinity of the candidate to MHC solubilized glycoproteins which is thought to correlate to the affinity found when said MHC glycoproteins are in their natural locations in cellular membranes.
• a solution is prepared of the MHC glycoprotein, where the glycoprotein may be the naturally occurring dimeric glycoprotein freed of the cell membrane or a soluble glycoprotein which lacks the transmembrane region.
• the latter can be prepared in a variety of ways, using recombinant techniques, where the genes encoding the alpha and beta chains of a Class II MHC glycoprotein or the alpha chain of the Class I MHC glycoprotein may be truncated by removal of all or a portion of the transmembrane region.
• the transmembrane sequence may be replaced with a region capable of linking to a lipid. See, for example, Caras et al., Science (1987) 238:1280: Tykocinski et al., Proc Natl Acad Sci USA (1988) 85:3555.
• the lipid may then be removed by an appropriate esterase, e.g., phosphatidyl inositol-specific phospholipase C. - !! •
• the concentration of the MHC glycoprotein will generally be in the range of about 0.01 to 50 ⁇ M, more usually about 0.1 to 1 ⁇ M. This range is convenient and is not critical, since in some experiments it may be desirable to use either higher or lower concentrations, depending upon the affinity of the mixtures to be bound, the concentration of moiety employed, and the like.
• the medium will generally be buffered at about physiologic pH, pH 4.5-8, preferably about 5-6.5, with a buffer concentration of about 10 to 200 mM.
• Other additives may include salt, to a concentration of about 10 to 20 mM, or nonionic surfactants.
• the nonionic surfactants will generally be present in a concentration of about 0.1 to 2%. If a peptide agonist is used, the peptide will generally be at least about 3 amino acids and not more than about 30 amino acids, preferably being from about 3 to 16 amino acids, more preferably from about 5 to 15 amino acids.
• the complex may be separated in a variety of ways.
• the complex may.be separated from free agonist by gel filtration, gel electrophoresis in a nonreducing SDS polyacrylamide gel, or by binding the complex to a plate coated with antibodies or other affinity reagent (ligand) specific for the MHC glycoprotein.
• affinity reagent particularly antibody separation is used, preferably on a plate, more particularly on a multi-well plate.
• the agonist is labeled with biotin
• labeled avidin one can obtain a plurality of labels bound to a single agonist- MHC glycoprotein complex.
• fluorescent labels particularly lanthanide chelates, more particularly europium chelates, or enzymes, particularly horseradish peroxidase.
• fluorescent labels may be quantitated in accordance with conventional procedures, there being numerous fluorimeters for detecting fluorescence from lanthanide chelates and numerous spectrophotometers for detecting peroxidase substrates which result in chromophores.
• the MHC glycoprotein of interest is first captured on a solid support and the remaining components of the lysate washed free of the adsorbed MHC glycoprotein on the support.
• the coupled support is then treated with a reaction mixture containing the agonist capable of detection when complexed with the MHC glycoprotein and the competing candidate moiety.
• the nature of the labeling of the agonist, if needed, and of the concentrations of the competing substances in the reaction mixture is similar to that described above with respect to the use of solubilized MHC glycoprotein.
• the derivatized solid support is prepared by passive adsorption or by covalent coupling of an affinity reagent specific for the MHC glycoprotein of interest by standard techniques well known in the art. Typically, microtiter plates or other multiwell reaction matrices are used as solid support. This method has the advantage of removing contaminants from the crude lysate which might interfere with the binding of the candidate and/or agonist to the MHC glycoprotein. The activity of such contaminants must be minimized since binding of agonists to MHC glycoproteins occurs only at elevated temperatures where proteases and the like present in the lysates would exhibit activity.
• a reaction mixture containing the relevant amounts of agonist and candidate is incubated for a suitable time period, usually about 3- 48 hours, in the presence of the MHC complex coupled to solid support.
• a suitable time period usually about 3- 48 hours
• the solid support is removed from the reaction mixture, washed, and the agonist bound to MHC glycoprotein determined according to the nature of the label as described above.
• the first competition protocol may be used, but may be further optimized by preloading the MHC glycoprotein with a homogeneous, usually peptide agonist, which has a suitable off rate to permit the binding of the agonist/competitor combination.
• a homogeneous, usually peptide agonist which has a suitable off rate to permit the binding of the agonist/competitor combination.
• the purified MHC glycoprotein or the crude lysate is incubated with a preloading agonist, generally overnight in a suitable buffer, generally containing octylglucoside, for a time sufficient to replace the heterogeneous endogenous peptides contained in the native MHC glycoprotein with the preloading homogeneous moiety.
• the preloaded MHC complexes are then diluted and used in the assay systems described above.
• This approach provides a uniform provision of "empty" MHC glycoprotein binding domains for binding or coupling with the agonist or candidate.
• the acceleration of what is otherwise the rate-limiting step in the association of the candidate or agonist provides a shorter assay time and provides a more accurate determination of the association rate and affinity of the candidate.
• the affinities of the competitors for the unloaded MHC glycoprotein can be compared more readily. Because the MHC molecule is preloaded with a peptide with a defined dissociation rate, the assay can be run for a shorter period of time, preferably three hours.
• the MHC glycoprotein is preloaded with a homogeneous, labeled peptide agonist which has been demonstrated to be displaced in the presence of other peptides.
• the purified MHC glycoprotein is incubated with the labeled agonist, generally overnight in a suitable buffer, generally containing octylglucoside, for a time sufficient to replace the heterogeneous endogenous peptides with the preloading moiety.
• the preloaded MHC complexes are then diluted into a solution of a displacing candidate moiety for about 0.5 hour and loss of counts with and without the candidate moiety is monitored as described above.
• An additional aspect of the invention obviates the necessity to label the agonist used in the competition assays.
• T-cells which have been primed with the agonist are used to detect the presence of the agonist in the complex.
• the complex is used in a T-cell proliferation assay with the primed T-cells.
• Enhanced proliferation of the primed T-cells is a measure of the binding of the agonist to the complex.
• This method can also be used to detect any test moiety in the complex by priming the T-cells with test moiety.
• Anti-DR (LB3.1) and anti-DQ (IVD12) affinity columns were prepared using spherical cellulose resin (Amicon) and 40 mg of each of the antibodies.
• the maximum amount of solubilized Class II that combined to the columns is twice the molar concentration of the bound antibody. Therefore, 40 mg of immunoglobulin can bind at most 32 mg of Class II MHC glycoprotein. Practically, only between 10-30% of the theoretical capacity of an affinity column is allowed, which corresponds to between 3 and 9 mg of DR and DQ.
• a comparison of the L243 and LB3.1 columns was performed with an NP-40 detergent extract from 1.8 x 10 10 cells which was equally divided, loaded on each column, and eluted with either 5 or 15 minute exposure to pH 11.5 buffer. The yields are as follows:
• HA 307-319 319 of influenza hemagglutinin (HA 307-319), biotinylated at the amino terminus, was incubated with affinity- purified DR4Dw4 (2 ⁇ M) PBS/1% octylglucoside) .
• Peptide DR complexes were separated from free peptide as with the iodinated HA except 25 ⁇ l of PBS/1% octylglucoside (5% FCS) and 10 ⁇ l of the incubation mixture were added to the wells of the antibody-coated ELISA plates.
• HA 307-319 bound was quantitated by incubation with 125I streptavidin (6-30 mg/well) for an hour at 4°C followed by washing and counting.
• c) Competition for binding of biotinylated HA 307-319 to DR4Dw4 was performed as in b) with or without several HA monosubstituted peptides (e.g., lys, ser or phe at position 309) (200 ⁇ M) in PBS/1% octylglucoside.
• Detection Systems for the Binding of a Biotinylated Hemagglutinin Analog to DR4Dw4 The assay was formed as described in Example 3b using a biotinylated alanine backbone analog of HA (AAFKAAEAAAARA) at 2 ⁇ M and DR4Dw4 at 0.5 ⁇ M.
• the peptide DR complexes were quantitated using either a fluorescent europium-conjugated streptavidin, a horseradish peroxidase-conjugated streptavidin or 125I- conjugated streptavidin. Each staining reagent was titrated to obtain the maximum signal over background possible.
• the monoclonal antibody LB3.1 specific for DR Class II molecules at 2 ⁇ g/ml, 200 ⁇ l/well, was coated onto a Costar EIA-RIA plate in 50 mM Tris-HCl, pH 9.0, either overnight at 4°C or for 1 hour at 37°C.
• the plate was washed and blocked for 1 hour at room temperature with 5% FCS/PBS and then washed 3-4 times with 0.05% Tween 20/0.01% azide/PBS (wash buffer) using a Titertek plate washer. Cell lysates assessed to contain approximately 20 nM DR MHC glycoprotein was then incubated for 4 hours at 4°C on the coated plate.
• the plate was washed 3-4 times with 0.05% Tween 20/0.01% azide/PBS.
• Biotinylated HA peptide 307-319 was then added to the plate in 5% FCS/1% octylglucoside (OG) /PBS at 200 ⁇ l/well and incubated overnight at 37°C in a C0 2 incubator.
• OG octylglucoside
• the plate was washed 3-4 times with wash buffer and incubated for 4 hours at 4°C with 200 ⁇ l of europium chelated streptavidin (Pharmacia/LKB Nuclear) at 60 ng/ml. After an additional washing cycle, the plate was treated for 30 min at room temperature with 200 ⁇ l/well of enhancement solution (Pharmacia/LKB) which releases bound europium for detection in a 1234 Delfia research fluorometer (Pharmacia/LKB) .
• Binding curves proportional to concentration of the biotinylated HA 307-319 peptide are obtained when a Priess cell lysate was used (solid circles) as well as when purified DR4 MHC glycoprotein at 10 nM (open circles) or the lysate from Cos 7 cells transfected with DR4Dw4 (open squares) were used to provide the MHC glycoprotein.
• Mock transfected Cos cells used as a control showed no uptake of the labeled peptide.
• Example 6 Alternate Plate Assay Protocol The assay was conducted in a manner similar to that set forth in Example 5 except that the antibody- coupled plates were prepared as follows:
• Avid-HZTM plates (Bioprobe International) which covalently couple oxidized antibody to their surfaces were used.
• LB3.1 monoclonal antibody was first oxidized by diluting to 10 ⁇ g/ml in 50 mM acetate buffer pH 5, followed by addition of a 1/10 volume freshly prepared 10 mM sodium metaperiodate. After 30 minutes at room temperature, the reaction was stopped by addition of 1/10 volume of 20 mM ethylene glycol in acetate buffer. 115 ⁇ l of the oxidized antibody solution were added to the wells of an Avid-HZTM plate and incubated overnight at 4°C.
• the plates were then washed 4 times with wash solution (PBS/0.05% Tween 20/0.01% sodium azide) and then blocked for 1 hour at 4°C with PBS/5% FCS/0.01% sodium azide.
• 125 ⁇ l of 20 nm DR4Dw4 in PBS/0.75% octylglucoside/0.01% sodium azide (binding buffer) were added to each well and incubated for 4 hours at 4°C.
• the plates were washed 4 times with wash buffer and treated with 125 ⁇ l of biotinylated HA 307-319 peptide contained in binding buffer and incubated overnight at 37°c.
• the plates were then washed 4 times with wash buffer and incubated overnight at 4°C with 125 ⁇ l of europium chelated streptavidin (Pharmacia/LKB) at 60 ng/ml. After an additional wash cycle, the plates were treated for 1 hour at room temperature with 125 ⁇ l/well enhancement solution (Pharmacia/LKB) and read in a 1234 Delfia research fluorometer.
• Pharmacia/LKB europium chelated streptavidin
• rat myelin basic protein 90-102 (RMBP90-102) which has the sequence HFFKNIVTPRTPA
• HADP 3.2 HA derived protein 3.2
• HADP 3.6 HA derived protein 3.6
• 50 nM of labeled preloading peptide was incubated for 48 hours with 400 nM DR4Dw4 in PBS, pH 7.0 binding buffer.
• Off rates were determined by diluting the complexes 1:40 into PBS and various concentrations of unlabeled HA 309-319 at various times, followed by ca p turing the complex on antibody plates coupled to LB3.1 as described above. As shown in Figures 3A-3C, none of the tested peptides showed off rates that were affected by the presence of HA 307-319 and the dissociation rate for RMBP 90-102 was the most rapid of the three. Therefore, preloading with RMBP 90-102 was selected for us.e in the assays.
• the preloaded complexes i.e., DR4Dw4/RMBP 90-102
• DR4Dw4/RMBP 90-102 were diluted 1:100 and incubated with varying concentrations of biotinylated RMBP 90-102 or with 8.8 nM biotinylated RMBP 90-102 in the presence of varying amounts of the same peptide unlabeled.
• Controls using diluted nonpreloaded DR MHC glycoprotein were also run. Aliquots (50 ⁇ l) of the reaction mixtures were removed to the capture plate and incubated at *4°C for 4- 24 hours.
• the plates were washed as described above and treated with 125 ⁇ l europium-streptavidin at 60 ng/ml in assay buffer (calcium and magnesium-free PBS + 0.5% BSA, 20 ⁇ M diethylenetriaminepentacetic acid (DTPA) in 0.1% sodium azide) and incubated 2-24 hours at 4°C. After additional washing, the plates were enhanced and read as described above.
• assay buffer calcium and magnesium-free PBS + 0.5% BSA, 20 ⁇ M diethylenetriaminepentacetic acid (DTPA) in 0.1% sodium azide
• Figures 4A and 4B Typical results are shown in Figures 4A and 4B.
• Figure 4A shows a binding curve for biotinylated RMBP for control and preloaded DR.
• Figure 4B shows the percent inhibition obtained with varying concentrations of unlabeled RMBP. As shown above, the results are comparable for preloaded and nonpreloaded DR MHC glycoprotein.
• the preformed complexes will be diluted 1/40 and reacted with the labeled agonist and the test compounds for 3 hours.
• the complexes will then be captured as described above.
• Ligand/receptor dissociation rates should be first order, depending only on the concentration of the complex present. In studying class II/peptide dissociation rates, rates are first order in some cases but not all. To determine the dissociation rates, 400-500 nM
• DR4Ew4 was incubated with 50 nM biotinylated RMBP or 25 ⁇ m biotinylated HSP (19 kD heat shock protein 3-14 form Mycobacterium tuberculosis) overnight at 37°C as described previously. These preformed complexes were then diluted 100-fold into buffer with or without varying concentrations of HA 30-7-319.
• the dissociation rate of RMBP is not affected by the second peptide.
• the dissociation of HSP was accelerated by the presence of the other peptide. Since the dissociation rate increases with the concentration of the other peptide, this is clearly not a first order reaction.
• T-cell clones responsive specifically to HA 307-319 were prepared from an individual with HLA Class II phenotype DR4Dw4, DR7, DRw53, DQw8, DQw9 (as described in Example 1) . Briefly, peripheral blood mononuclear cells were primed in vitro with 1/100 dilution of influenza virus vaccine (Parke Davis) in RPMI supplemented with streptomycin (100 ⁇ g/ml) penicillin (100 U/ml) and 5% pooled AB serum (Whittaker, Fredricksburg, MD) .
• influenza virus vaccine Parke Davis
• streptomycin 100 ⁇ g/ml
• penicillin 100 U/ml
• 5% pooled AB serum Whittaker, Fredricksburg, MD
• T-cell blasts were cloned by limiting dilution at 1 cell per 3 wells in the presence of a feeder mixture consisting of 10 6 allogeneic PBMC per ml (30 Gy irradiated) , 10 5 cells of autologous EBV transformed B cells (50 Gy irradiated) , 1/100 dilution of influenza virus vaccine and 1 ⁇ g/ml leucoagglutinin-A
• HA 307- 319 The suspension was plated in 96 well flat- bottom microtiter plates and incubated as described above. Growing cultures were transferred to a 24-well tissue culture plate and restimulated with the same feeder mixture. Three days later, 10% IL-2 was added and the cells were frozen or further expanded by restimulation. Specificity was determined with HA 307- 319.
• DR4Dw4 glycoprotein was affinity purified from Priess EBV-B cells as described in Example 1. Briefly, the cells were grown in RPMI medium supplemented with fetal calf serum and collected by centrifugation, washed with PBS and lysed with 1% Nonidet P-40. The cellular debris was removed by centrifugation and the lysate loaded directly to a Sepharose CL-4B column connected in series with a monoclonal antibody LB3.l cellulose column prepared by coupling 40 mg of antibody to 10 ml Matrex Cellufine Formyl (Amicon) according to the manufacturer's instructions.
• the columns were washed with 20 volumes of 10 mM Tris HC1, pH 7.5, 150 mM NaCl, 0.1% deoxycholate.
• the LB3.1 cellulose column was then washed with 5 column volumes of 10 mM Tris HC1, pH 7.5, 1% OG and eluted with 50 mM glycine, pH 11.5, 1% OG.
• the fractions were adjusted to pH 7.5 with 2 M glycine, pH 2.
• the full fractions were dialyzed against 10 mM Tris HC1, pH 7.5, 1% OG and stored at 4°C.
• DR4Dw4 was incubated overnight at 37°C with HA 307-319 at varying concentrations in 1% OG/PBS. 50 ⁇ l of the mixture were added to 96-well flat-bottom ELISA plates which had been coated with LB3.1 by incubation overnight at 4°C in 5 ⁇ g/ml LB3.1 in 50 mM Tris HC1, pH 9. The mixture was incubated with the coated plates for 6 hours at 4°C and then washed twice with PBS and once with -complete medium.
• T-cell proliferation assays 3 x 10 4 T- cells were added in 200 ⁇ l of complete medium to each well (the T-cells were used 10-11 days after restimulation) . After incubation for 24 hours, 1.0 ⁇ Ci per well of tritiated thymidine were added, and the plates were incubated for another 18 hours. The samples were harvested on glass fiber filters using a semiautomatic harvester and thymidine incorporation was assessed by counting in a scintillation counter. The results of triplicate determinations are shown in Figure 7.
• T-cell proliferation can be used to determine a binding curve for the test HA 307- 319 peptide. It is evident from the above results that rapid, efficient assays are provided for screening candidate moieties for binding affinity to particular MHC glycoproteins. Thus, the methodology allows for evaluation of a wide variety of candidates and their ability to interact with MHC glycoprotein and ultimately to modulate the immune response in a host having such MHC glycoprotein.

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