CA1086306A - Immunofluorescence reagent and process for preparing same - Google Patents

Abstract

ABSTRACT
A stable labelled immunofluorescence reagent which comprises a covalent conjugate of dichlorotriazinylaminofluorescein (DTAF) with a suitable protein such as an immunoglobulin. Preferably the F/P molar ratio is in the range of 3 to 4 ans over and under conjugated reagent can be simply removed by a simple fractionation by ammonium sulfate precipitation.

Description

10863~6 This invention relate~ to new and novel compositions of ~ !
labelled immunoglobulins and other proteins which are useful for detecting the presence of and identifying various microorganisms including viruses, bacteria, fungi, and the like which are present in tissues, and to processes for preparing the same.
Fluorescent labelled antibodies are known to the art, and in recent years substantially all fluorescein labelling of proteins haa been accomplished with the isothiocyanate derivative of fluorescein (FITC). This compound was first introduced in 1958 and, until now, a satisfactory alternative fluorescein labelling compound has not appeared.
FITC combines with proteins in a straightforward manner in aqueous solu-tions, reacting primarily with free amino groups to form a thiocarbamyl linkage. FITC i8 available commercially from numerous chemical supply houses in North America and Europe. In general, the appropriate purified antibody proteins are a~lowed to react with a limited quantity of FITC
under mildlyalkaline conditions. The fluorescein conjugated protein is then separated from excess reagents and its breakdown products by dialysis or, in most cases, gel filtration. In some cases, the conjugated protein is then subjected to an additional fractionation step on an ion exchange (DEAE) column to remove the unwanted over- and undercon~ugated portion of the conjugates. Attention is also directed to U.S. Patent 3,789,116 issued 29 January 1974 to Kay which describes one way in which the afore-said FITC,conjugated to appropriate antibodies,is stabilized for use as a reagent specific to Group A streptococci.
Anti-immunoglobulin as used in this specification means the immunoglubulin fraction derived from anti-serum containing antibody specificit~ directed against all the immunoglobulins of the donar species or subclasses (IgG, IgM, etc.) or fragments thereof (Fc,light chains,etc.).
The basis for the action of labelled antibody reagents and particularly fluorescent labelled reagents is that different antibodies --1-- ' 7~

, `.. ~ . .`

recognize and react with their corresponding antigen. An antibody specific to an antigen will become attached to that antigen whenever encountered by the antibody. If an antibody that is known to be specific for a particular antigen within the immunoglobulin fraction of serum is isolated from the serum or plasma of a host animal which has been stimu-lated to produce that antibody, it can be labelled or tagged by known means by conjugating the antibody with a labelling agent such as a radio-isotope or a fluorescent chemical. Then, when used diagnostically, if the counterpart antigen is present in a sample, the labelled antibody will attach itself to that antigen. The presence of the antigen can then be confirmed by detection of the labelled antibody. In the case of a fluo-rescent labelled reagent, such as an FITC labelled antibody, microscopic examination under suitable light condltions is all that is necessary. -.
In the "direct" technique of immunofluorescence purified immunoglobulins, mostly but not exclusively IgG, are extracted from whole blood serum, fluoresceinated and samples thereof are then reacted with known, different viruses, bacteria or other antigenic determinants in turn. The presence of a specific antibody is then indicated by fluorescence of the test sample.
In the indirect "sandwich" technique it is not necessary to under-take the relatively time consuming and tedious task of purifying an immunoglobulin portion of a serum sample but rather whole human serum samples which may contain specific antibodies are each exposed to cells or tissues suspected to possess antigenic determinants of, for example, bacterial or viral origin and then washed to remove excess serum. To each washed sample a mammalian (such as goat or rabbit) anti-human immuno-globulin which has been fluoresceinated (labelled) is added. If human antibodies are present on any of the cells or tissues possessing those antigenic determinants then the anti-immunoglobulin will bind to them and will fluoresce but will not bind to those cells or tissues which do not 10863(~6 have the specifically bound human antlbody present and hence will not fluoresce. It will be appreciated thac the mammalian antl-immunoglobulin employed in the indirect technique can be derived from almost any known animal. For convenience, however, it i8 usual to employ immunoglobulins isolated from rabbit or goat anti-sera and more infrequently from sheep or horse. Mouse immunoglobulins are, however, notoriously hard to label without affecting the antigen binding activity thereof.
As indicated hereinabove FITC has been used almost exclusively for labelling proteinaceous reagents. FITC would be close to the ideal compound for the covalent attachment of fluorescein molecules to antibody proteins but for the fact that commercial preparations of the compound vary widely in purity, stability, and labelling efficiency. Numerous published scientific reports have appeared since the introduction of FITC
attesting to the wide variability in the quality of the compound. FITC
is prepared from the reaction of aminofluorescein with thiophosgene.
This reaction is complex, difficult to control and results in poorly under-stood side products. Analytical studies of various commercial preparations by thin layer chromatography have been found to comprise up to 11 compon-ents, not all of them flùorescent. In addltion, phy6ical studies (e.g., infra-red spectroscopy) indicate that commercial preparations may vary from ~ -50 to 100 per cent in dye (fluorescein) content and by a similar range in isothiocyanate content. Recrystallized FITC is available, at great cost, but even this material is not uniformly effective from batch to batch.
The contaminating components of even very pure FITC preparations (e.g., 90 per cent) have been found to lnterfere wlth both the conjugation reaction and with the quality of the resulting fluorescent antibodies, often resulting in conjugates producing a high degree of non-specific, back-ground staining of cells or tissues. This background staining is a major limitation of the fluorescent antibody technique and must be kept to an absolute minimum if the procedure is to be effective in clinical and re-,~
,,, ~, . .

lOB6306 search applications. In addition to lack of purlty, FITC is not a particularly stable compound and preparations stored for only a few months frequently lose their capacity to label proteins, or then do so only poorly.
It is an object of the present invention to provide an im- i~
proved labelled immunofluorescence reagent containing a fluorescein compound which is relatively inexpens$ve, simply prepared in extremely pure form and is highly stable.
It is another object to provide an improved process for prepar- `
ing dichlorotriazinylaminofluorescein (DTAF) immunoglobulin conjugates.
Thus by one aspect of this invention there is provided a stable ~ `
labelled immunofluorescence reagent comprising a covalent con~ugate of dichlorotriazinylaminofluorescein (DTAF) with a protein which has the capacity to react with and bind specific antigenic determinants or unique structures on or in other macro molecules and cells; said conjugate having -a fluorescein/protein molar ratio in the range 3-4. ``~
By another aspect of this invention there is provided a process for producing an immunofluorescence reagent comprising (a) reacting amino-fluorescein with cyanuric acid thereby producing DTAF; (b) conjugating said DTAF with a protein selected from the group comprising protein A and immunoglobulins; (c) fractionating the product of said conjugation by mix-ing with a 45 - 60% saturated aqueous solution of ammonium sulfate, there-by precipitating a conjugate having an F/P molar ratio in the range 3-4;
and (d) separating said precipitated conjugate.
DTAF was first reported in 1968 by Soviet scientists (Barskii et al, Izv. Akad. Nauk SSSR Ser. Biol. 5,744) who utilized the compound as a fluorescent stain for localizing protein in tissue sections in ~ -addition to chemically characterizing the compound and its derivatives.
DTAF may be prepared by reacting aminofluorescein, usually Isomer I (5-aminofluorescein) as indicated below, with cyanuric chloride;

A~ `~

... , ' ., ` ~ ,~ . , , . ' .

'10863Q6 Cl Cl~l~Cl U~
[~ COOH ~fOOH

2 ~ : ' Cl)~
Dry DTAF is a bright yellow powder which can be used ~
without further purification and can be stored desiccated and preferably -cold. -Immunoglobulins can, of course, be prepared by standard techniques known to the art. Generally, but not essentially goat or rabbit IgG are the preferred immunoglobulins in the indirect immunofluor-escence (IF~ test, Other mammalian immunoglobulins such as sheep, horse or pig are effective and it is also posslble to employ avian immuno-globulins~ Commercially available protein A* is also effective for IF tests. Generally the ~mmunoglobulin i8 prepared from whole serum by salt precipitation and/or ion exchange chromatography. IgG solu-tions can be concentrated by, for example, ultra-filtration and ~ -dialysis against water or appropriate salt solutions or buffered buffers.
The conjugation of DTAF with IgG solutions is effected by mix-ing buffered aqueous solutions of DTAF with IgG solutions to yield reaction mixtures of various molar ratios. Generally the IgG is used at 10-25 mg/ml, and following incubation at a controlled temperature and pH the mixture is filtered through a filter medium such as Sephadex~3 G-25 to separate conjugated protein from unreacted reagent. The molar fluoresceintprotein (F/P) ratio is preferably in the range of 3-4 as ` estimated from the empirical formula (The and Feltkamp, 1970a, Immunology 18, 865)

3.55 O.D.495 F/P
O.D.280 - 0-38 0.D.495 - wherein O.D.280 and O.D.495 is the optical density of the conjugate at280 and 495nm respectively. As will be appreciated by those skilled in * Protein A is a protein isolated from the cell wall of staphylococcus aureus.

1~86306 .

the art, over conJugation re~ults in a reagent which tends to bind non-specifically to target tissues or cells thus causing high background fluorescence and under conjugation results in a reagent which does not fluoresce brightly enough to distinguish from the general background.
It is, therefore, of considerable importance that as much as possible of the reagent should possess an F/P ratio generally within the specified range. It will further be appreciated that it is highly desirable that any material outside the desired range should be removed if uniformity of product is to be achieved. In the case of FITC reagent this has generally proved to be time consuming and hence relatively expensive as resort mùst be had to an ion exchange fractionation technique. Further the quality of the FITC itself varies widely so that the results obtained using FITC reagents are frequently not reproducible. We have found however that removal o~ over or under conjugated DTAF-IgG reagents is -simple and straightforward and need not be conducted by ion-exchange techn$ques. It is merely necessary to fractionate the con~ugates by ammonium sulfate precipitation.
During the preparation and analysis of several DTAF:IgG con-jugates, it was frequently noted that precipitates formed when conjugate solutions were stored for severalhours or more at 4C. These precipi-tates were readily redissolved by warming or the addition of base (if not excessively overconjugated) and thus did not appear to be protein covalently cross-linked by the two chloro groups of DTAF. Moreover, after removal of the precipitates, the F/P ratios of conjugate solutions were alway~ lower than that of the original solution~. This non-random precipitation suggested that IgG molecules had different solubilities depending on the number of bound DTAF ligands.
Thispossibility was investigated by treating DTAF:IgG conjugates with different ammonium sulfate concentrations. In a representative salt fractionation experiment, a conjugate with an F/P ratio of 2.75 yields ~'' .

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lC~863~6 the F/P-S (supernatant) and F/P-R (residue) values shown in Table 1.
As the conjugate was treated with increasing concentrations of ammonium sulfate, the F/P ratio of the unprecipitated protein decreased, indicating that the most heavily conjugated IgG molecules were insolubilized at low salt concentrations while lightly and unconjugated protein precipitated at high salt concentration. Analysis of the precipitated material (F/P-R) yielded data consistent with this differential solubility. The protein precipitated by low salt was highly conjugated while that pre-cipitated by higher salt had a lower F/P. The addition of saturated ammonium sulfate solution to the conjugate (final salt concentration 50%
of saturation) quantitatively precipitated the conjugated protein.

DTAF/IgG composition of ammonium sulfate fractions % (of Saturation)Ammonium F/P-S** F/P-R***
Sulfate Solution Added*

O(H20) 2.75 NP****
- 25 2.74 NP**** :
2.49 4.~4 ~
2.29 4.62 -2.00 4.11 1.72 3.73 1.46 3.41 -1.07 3.12 100 0.00 2.75 ~ 20 * To an equal volume of conjugation solution at pH 9.
** F/P ratio of supernatant.
*** F/P ratio of residue (precipitate).
**** No precipitates.
From the evidence set forth in-Table 1 and other theoretical deductions it is suggested that addition of 35% saturated ammonium sulfate solution will precipitate all those molecules with 5 or more ligands and some with 4; 50% salt apparently insolubilized those with

4 or more ligands and most with 3 and so on.
- Thus it is evident that DTAF:IgG conjugates can be prepared, fractionated and isolated without a need for a gel filtration step. For example, if a conjugate free of the most heavily conjugate protein ~ .
c~'.

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~. .

~0863~)6 molecules (e.g. 5 or more DTAF ligands) and of under-conjugated material (e.g. IgG with one or no ligands) is desired, the conjugate is first prepared as described and then mixed with sufficient ammonium sulfate to precipitate the overconjugated material. More saturated ammonium sulfate solution is then added to the supernatant to achieve a concentration precipitating the desired portion of the conjugate while ;
leaving the under-conjugated protein in solution. This precipitate is collected, dissolved in water, and precipitated 2 or 3 times more with equal volumes of saturated ammonium sulfate solution to free the conjugate of unreacted and hydrolysed reagent. The washed precipitate is then dissolved and appropriately diluted in a buffer for immediate use in ~;
immunofluorescence procedures. A standard stock isotonic solution generally contains 5-10 mg/ml of the conjugated protein in a phosphate buffered saline solution at pH 7.2, and is generally sterili~ed by known techniques. The small amount of entrained ammonium sulfate in the final precipitate is not enough to affect the antigen-binding activity of the fractionated conjugate in most if not in all cases.
Example 1 Preparation of DTAF
The compound was prepared by reacting aminofluorescein with ' cyanuric chloride after the method of Barskii et al supra. One ;`!
g of 5-aminofluorescein (Isomer I, Sigma) was dissolved in 30 ml -anhydrous methanol and clarified by centrifugation. The solution was -~
cooled to 4C and added by drops to a 4DC solution of 0.6 g cyanuric chloride (practical grade, Eastman) in 5 ml acetone. Stirring was con-tinued for 3-5 hours at 4C and the bright yellow, precipitated product ~` was collected by vacuum filtration, washed with cold acetone (10 ml) . followed bycold petroleum ether (10 ml) and air dried in a vacuum desiccator to give an 87% yield. The only aspect of the reaction requiring special attention is the handling of solid cyanuric chloride, which is a lacrymatory irritant of the eyes and nose and should be handled in a fume hood, The dry DTAF was ground to a bright yellow pow-der in a motor for convenience in handling and used without further puri-: ,~

fication. The product was routinely stored desiccated at 4C. The molar extinction coefficient of DTAF was reported as 82,200.
Example 2 Preparation of Immunoglobulins Rabbit IgG was used for all conjugation experiments. This was prepared for whole serum by precipitation at half saturation with ammonium sulfate followed by DEAE-cellulose chromatography. IgG
solutions were concentrated by ultra-filtration and dialysed against water or unbuffered saline.
Example 3 Conjugation Procedure Buffered aqueous solutions of DTAF, prepared as in Example 1, were mixed with IgG solutions prepared as in Example 2 to yield reaction mixtures of various molar ratios. In most cases, the IgG was used at 10-25 mg/ml. These were incubated for various periods at different temperatures and pH and filtered through Sephadex ~ G-25 to separate conjugated protein from unreacted reagent. The isolated conjugates were analysed spectrophotometrically at 280 and 495 nm and the molar fluore-scein/protein (F/P) ratio was estimated from the empirically derived ~-(The and Feltkamp, 1970a) formula:
(a) O D 495 where for DTAF (a) = 3.55, (b) = 0.38. The variables were calculated using 160,000 as the molecular weight of IgG and 492 for DTAP. Optical density for a 1 mg/ml solution of IgG was taken as 1.4.
, . , Example 4 Salt Fractionation Procedure One ml aliquots of the DTAF:IgG conjugates prepared as in 30 Example 3 were thoroughly mixed with one ml of different concentrations of neutralized aqueous ammonium sulfate solutions, expressed as percent-age of saturation. The mixtures were allowed to stand 30 min at room 9_ .~ r~

~' ' .
~, . .

10863~6 temperature and then centrifuged to pellet the precipitate. The supernatants were pipetted off and the residues (if any) were re-dissolved in an equal volume (2 ml) 0.1 N NaOH. The F/P ratios of the supernatant (F/P-S) and the residue (F/P-R) separated by each salt con-centration were then determined spectrophotometrically.
Example 5 Conjugation of DTAF to Rabbit IgG
:
The covalent coupling to DTAF to rabbit IgG was examined as a function of time and of pH. Preliminary observations of this reaction indicated that it proceeded quickly and smoothly at room temperature and all subsequent conjugation procedures were carried out without tempera-ture control. Also, since the use of excessive amounts of DTAF produced overconjugated and insoluble protein, it was found that the reaction was best controlled by limiting the reagent concentration and using low molar ratios of DTAF to IgG.
~ pH
- One ml mixtures of DTAF and rabbit IgG containing one mg DTAF
, ~ . .
and 100 mg IgG (molar ratio of 3:1) were prepared in phosphate buffers, pH 4 to 9. After incubation for 1 hour, undissolved DTAF in each prepara- ~ r tion was removed by centrifugation, dissolved in 0.1 N NaOH and quantit-,:
ated spectrophotometrically. Each preparation was then isolated by gel filtration through Sephadex G-25 and the F/P ratio determined. From this ratio and the initial protein concentration, the percentage of the soluble DTAF that reacted with the protein was determined. Table 2 below indicates the increasing solubility and reactivity of DTAF with increasing `
pH. DTAF is sparingly soluble at acid pH, but is sufficiently soluble above pH 7 for most conjugation reactions. It is highly soluble at pH3 9.
The relative coupling efficiency, the amount reacted with respect to the amount in solution, also increased with pH. At pH 9, about 80% of the DTAF reacted with IgG in one hour at room temperature.

". . .
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.

Effect of pH on the solubility and reactivity of DTAF
pHSolubility Conjugation (~ g/ml)* efficiency**
4 69.6 19.4 84.0 23.7 6 166.0 26.7 7 233.8 42.6 8 521.3 69.0 9very soluble Bl.O
* Insoluble residue dissolved in 0.1 N NaOH and determined spectro- -photometrically.
** Percentage of DTAF reacted in a mixture of 1.11 mg DTAF, 100 mg IgG (3.3:1 molar ratio) in 5 ml of phosphate buffers at pH indicated.
Time Preparation of DTAF:IgG at a 3:1 molar ratio were incubated at -pH 8.0 (sodium phosphate buffer) for various periods of time before ;~
isolation and analy6is. It was found that DTAF reacts rapidly with the IgG and after only 60 min about 70% of the reagent is covalently bound. ~-Thereafter, only slightly more DTAF was bound (75g after 20 hours).
On the basis of these findings, it was determined that near optimal conditions for the reaction of DTAF with IgG were room tempera-ture at pH 9.0 for 1 hour. All subsequent reactions were carried out in 0.1 N sotium borate buffer, pH 9Ø Under these conditions, with a low DTAF:IgG molar ratio (~5:1), the coupling efficiency was normally 75-80%.
-~ The use of the DTAF Ig reagents of the present invention in -the direct IF technique will be described with reference to Examples 6-8 below.
Example 6 Demonstration of Ig Receptors on the Plasma Membrane of Mouse Spleen LymPhocytes ; A rabbit was immunized so as to produce antibody against mouse Ig by the following procedure: a group of mice were repeatedly in~ected intraperitoneally with heat-killed B. pertussis organisms. This caused the mice to produce large amounts of antibody specific for antigens of this organi~m, théreby hyperi~munizing them with B. pertussis. The mice were then bled and the serum was obtained. More B. pertu6sis particles (the heat-killed organisms~ were then exposed to the antiserum in a tube (about 10 particles per milliliter of antiserum) such that the particles became coated with mouse antibodies which reacted with antigenic structures on the particles. The B. pertussis particles coated with mouse Ig were then mixed with Freund~R Complete Adjuvant and injected into a rabbit.
This was repeated one or two more time6 and the rabbit was then bled.
This serum contained antibodies specific for B. pertussiE antigens and for mouse Ig. Since B. pertussis antigens are not found on mammalian cells, they are essentially superfluous. This serum is rabbit anti-mouse Ig.
The IgG fraction of the rabblt antiserum was then obtained by DEAE-cellulose chromatography. This fraction was then conjugated with DTAF by the procedures outlined in Example6 3 and 5 (sodium borate i~ buffer, pH 9.0). In different tests with thi6 IgG material, the final F/P ratio was between 3 and 4.
Mouse spleen cell6 were collected by removing the spleens from mice i ediately after killing and teasing apart the tissue with forceps. Mice of various inbred strains were used (e.g. DBA/2, CBA, etc.).
This teasing suspended most of the cells and the clumps were removed by passing the cell suspenslon (in medium or balanced salt solution) through a short column of glass wool. Red blood cells were then removed either by Ficoll-Isopaque density gradient sedimentation or osmotic shock with Tris-buffered ammonium sulfate. The resulting spleen cells are mostly lymphocytes, the remaining cells bein8 primarily phagocytes of various types.
For the immunofluorescence test, the suspension of spleen cells (about 5-lOxlO cells/ml) were exposed to an appropriate concentra-tion (of the order of 20 microlitres of a solution containing about 2.5 mg/ml of con~ugated protein) of the DTAF con~ugated rabbit IgG
containing antibody with anti-mouse Ig specificity, After incubation for a period of time of the order of a few minutes up to about 30 minutes at an appropriate temperature such as room temperature, the mouse cells were washed three times with phosphate-buffered saline (PBS) and then examined under the fluoregcence mlcroscope employing an optical system designed for fluorescein fluorescence. Depending on the individual mouse from which the spleens were collected, about a third to a half of the cells showed fluorescence, the remainder being unstained.
The number of positive (stalned) cells correlated with the percentage of B lymphocytes ordinarily detected from mouse spleens. Moreover, the nature of the staining was the same as that observed in many previous studies using FITC labelled rabbit anti-mouse Ig; namely, patch and cap formation was observed which was dependent upon the time and temperature .,~.
of incubation (of the fluorescent reagent with the cell~). A comparison of FITC and DTAF labelled rabbit anti-mouse Ig on the same spleen cells revealed that there was no detectable difference ln the specificity or quality of either reagent.
Example 7 Detection of Immune Complexes in Human Kidney Biopsy Tissue A goat anti-human Ig was obtained from a commercial source.
This preparation contained IgG isolated from goat antiserum to human Ig, and also some bovine serum albumin, which, according to the supplier, was mixed with the IgG in order to better maintain the anti-human Ig activlty of the IgG. The goat IgG and the albumin were separated by ammonium sulfate fractionation and the IgG was con~ugated with DTAF under the same conditions as for rabbit IgG. The labelled goat anti-human IgG
was then used to examine a kidney biopsy tissue section from a patient who had already been di2gnosed as having glomerular nephritis, the 1~86306 diagnosis being based in part on the detection of immune complexes by the use of FITC labelled goat anti-human Ig. The DTAF labelled goat anti-human Ig revealed the identical pattern and specificlty of stain-ing (i.e. the staining could be blocked by the prior exposure of the tissue to unlabelled goat anti-human Ig) as did the FITC labelled material.
Example 8 Demonstration of Ig Receptors on Mouse Spleen Lymphocytes With DTA~-Protein A
The protein A was obtained from the Pharmacia company. With-out further purification it was conjugated with DTAF by the same proce-dures used with IgG. The cells were obtained as in Example 6 and essentially the same results were obtained.
~- The use of the DTAF-Ig reagents of the present invention in the ,. I .
indirect IF technique will be described with reference to Examples 9~
below. As previously indicated, in indirect IF, the target antigen is first reacted with unfluoresceinated specific sntibody after which that antibody is in turn reacted with fluoresceinated antibody specific for it; that i~, anti-antibody or snti-Ig. This anti-Ig is called a develop-`~ ing reagent and in most applications ix generally rabbit or goat anti-body reactive against the Ig of other species (i.e. goat anti-human Ig, -rabbit anti~mouse Ig, goat anti-rat Ig, rabbit anti-chicken Ig, etc.). ;
Protein A is finding increasingly wide application as a developing re-agent and thi~ bacterial protein has the property of reacting specifically wlth the Fc portion of IgG of a variety of different species. Thus pro-tein A can be used as a developing agent in a number of test systems in-volving different species, unlike the Ig developing reagents.
Example 9 Demonstration of Mouse I~ on Mouse Spleen Lymphocytes The procedures as outlined in Example 6 were repeated except that the rabbit anti-mouse Ig used was not labelled with DTAF. The 108630~i cells were exposed to the unlabelled rabbit IgG in order that the mouse Ig "antigen" receptors were bound. The cells that ~pecifically took up the rabbit antibody (the B cells) were then developed by subsequently exposing the cells, afte~ washing, to a sheep IgG preparation that was obtained from ~heep antiserum containing anti-rabbit Ig antibody~ The sheep was lmmunized with puri~ied ~abhit IgG in ~reundl~ Complete ~djuvant.
After repeated immunizations the sheep was bled and the IgG isolated from the serum. This IgG was con~ugated with DTAF under the same conditions used with rabbit IgG and wlth protein A. The mouse cells that had already been exposed to the unlabelled rabbit anti-mouse Ig were exposed to the sheep anti-rabbit. Those cells which had taken up the rabbit IgG (~he B
cells in this case) were thus strained. The number of cellæ stained and the distribution and behavior of the label (patching and capping) was con-,, sistent with B cell numbers and behavior. This procedure was repeated with FITC labelled IgG from the same sheep for comparison. There was no detect-~able difference.
~ Example 10 - Detection of Herpes Simplex Viral AntiBens on Mammalian Cells ; Rabbit kidney cells were infected with Herpes simplex virus and the cells were exposet to unlabelled human IgG isolated from a human ~ -antiserum containing antibody specific for Herpes antigen. After IgG
~reatment the cells were washed and then exposed to the same goat anti-human Ig as used in Example 7. The DTAF labelled anti-human Ig revealed the same number of positive cells as did the FITC labelled goat anti-human Ig normally used to detect the viral antigen. Neither the DTAF
nor the FITC labelled IgG stained uninfected cells.
Example 11 Demonstration of H-2 Antigens on Mouse Spleen Cells H-2 is a particular structural component of all mouse cells.
Mice of different inbred strains, however, are often found to have variants of the H-2. For example, DBA/2 mice are said to possess the ` " 1~863~6 H-2d antigen while CBA mlce have the H-2k antigen. Mlce of other inbred strain~ have still other H-2 variants though many strains may also have the same H-2 antigenic type. When the cells of a DBA/2 mouse are injected into a CBA (or any other combination where there is an H-2 difference), the CBA will produce antibody specific for the E-2d antigen.
Similarly, the DBA/2 will make anti-H-2k following challenge with CBA
cells. One of the methods for detecting the presence of particular H-2 types on mouse cells from various strains is through the use of these anti-H-2 antisera in an indirect IF test. Therefore to test for the pre-sence of and the distribution of H-2 antigens on the spleens cells, say, t of a particular mouse, the cells are first exposed to the mouse antiserum, washed and then a rabbit or goat anti-mouse Ig is added. However, in ~ the case of spleen cells, the test is complicated by the presence of - mouse Ig on the surface of some of the spleen cells (the B lymphocytes).
In order to get around this and to demonstrate the presence of H-2 on -~
spleèn cells by indirect immunofluorescence with DTAF conjugated antibody, -;
an extra step is added. DBA/2 spleen cells.were obtained as above. These `~ were then exposed to unlabelled rabbit anti-mouse Ig and in this way all of the mouse Ig "antigens" on the B lymphocytes were bound by the rabbit antibodies. Once blocked in this way, these Ig antigens will not bind additional rabbit antisera. After washing, the cells were`next exposed to mouse anti-H-2 , an antiserum from an appropriately immunized CBA
mouse. The cells were washed again and finally the DTAF labelled rabbit anti-mouse Ig was added. This antibody bound to the mouse Ig on the H-2 determinants but not to the mouse Ig that is inherently a component of the B cell's membrane and was already blocked with rabbit antibody. When the test was performed, all of the spleen cells were observed to be fluorescent, as expected since all cells display at least some H-2 antigen on their membrane. When the same procedure was carried out with `CBA rather than DBA/2 spleen cells, however, there was no staining since ``` 1086306 the mouse antiserum used was only specific for the H-2d antlgen and the CBA cells are ~1~2k. The distribution and behavior of the H-2 antigens on spleens cells showed a distribution and behavior typical of H-2 antlgens, as studied with the use of FITC labelled antibody.
- It will be appreciated that many modifications may be made r without departing from the scope of the present invention as defined in the appended claims. For example, although reference has been made herein to the use of a stable, buffered isotonic solution of the reagent, it will be appreciated that the reagent may equally well be stored in lyophilized form. Although DTAF is stable per se, the immunoglobulin to which it is covalently conjugated may or may not be stable over long periods of time so that it may be necessary or desirable to add a , ., stabilizing agent for the immunoglobulin to the reagent. Suitable stabilizing agents include carbohydrates such as dextrose and other poly-saccharides. It may also be desirable to sterilize the reagent by flltratlon or other ceans to re=ove bacterla and the ll~e therefrotl.

Claims (15)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A stable labelled immunofluorescence reagent comprising a covalent conjugate of dichlorotriazinylaminofluorescein (DTAF) with a protein which has the capacity to react with and bind specific antigenic determinants or unique structures on or in other macro molecules and cells; said conjugate having a fluorescein/protein molar ratio in the range 3-4.

2. A reagent as claimed in claim 1 wherein said reactive protein is selected from the group comprising immunoglobulins and protein A.

3. A reagent as claimed in claim 1 or 2 wherein said reagent is dissolved in an isotonic solution.

4. A reagent as claimed in claim 1 or 2 wherein said reagent is dissolved in an isotonic solution comprising phosphate buffered saline.

5. A reagent as claimed in claim 1 or 2 wherein said reagent is lyophilized.

6. A reagent as claimed in claim 1 or 2 wherein 5-10 mg/ml, based on protein content, of said reagent is dissolved in an isotonic solution comprising phosphate buffered saline.

7. A reagent as claimed in claim 1 or 2 wherein said DTAF is Isomer I, DTAF.

8. A reagent as claimed in claim 1 or 2 including a polysaccharide stabilizing agent.

9. A process for producing an immofluorescene reagent compris-ing a covalent conjugate of dichlorotriazinylaminofluorescein (DTAF) with a protein which reacts specifically with antigens in a molar ratio fluorescein/protein of 3-4 comprising mixing an aqueous solution of said conjugate with neutralized aqueous ammonium sulfate solution thereby precipitating a conjugate having the desired molar ratio, and separating said precipitated conjugate.

10. A process as claimed in claim 9 wherein said aqueous ammonium sulfate solution has a concentration in the range 45-60%
of saturation.

11. A process as claimed in claim 10 wherein said precipitated conjugate is separated by centrifuging to thereby pellet said precipitate.

12. A process as claimed in claim 11 wherein said separated precipitate is redissolved in alkaline solution.

13. A process for producing an immunofluorescence reagent comprising:
(a) reacting aminofluorescein with cyanuric acid thereby producing dichlorotriazinylaminofluorescein (DTAF);
(b) conjugating said DTAF with a protein selected from the group comprising protein A and immunoglobulins;
(c) fractionating the product of said conjugation by mixing with a 45-60% saturated aqueous solution of ammonium sulfate, thereby precipitating a conjugate having a fluorescein/protein molar ratio in the range 3-4; and (d) separating said precipitated conjugate.

14. A process as claimed in claim 13 including the step of dissolving said precipitated conjugate in an isotonic, phosphate buffered saline solution.

15. A process as claimed in claim 13 including the step of lyophilizing said precipitated conjugate.