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Conjugation Methods in Immunofluorescence Roger McKinney, Leroy Thacker and G. Ann Hebert J DENT RES 1976 55: A38 DOI: 10.1177/002203457605500117011 The online version of this article can be found at: http://jdr.sagepub.com/content/55/1_suppl/A38

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Conjugation Methods in Immunofluorescence ROGER McKINNEY, LEROY THACKER, and G. ANN HEBERT Center for Disease Control, Public Health Service, US Department of Health, Education, and Welfare, Atlanta, Georgia 30333, USA

Since the immunofluorescent (IF) procedures were first used1l2 and fluorescein isothiocyanate3 (FITC) was introduced as a fluorescent label for immune serum globulins, numerous conjugation methods have been advocated. Initially, Riggs et a13 added an acetone solution of FITC to an immune globulin fraction containing acetone and carbonate-bicarbonate buffer. The reaction mixture was held at 4 C to avoid protein denaturation. Marshall, Eveland, and Smith4 added FITC as a dry powder to immune serum globulin and avoided the use of organic solvents. Clark and Shepard5 introduced a dialysis method of labeling in which FITC in a buffered aqueous solution was allowed to diffuse into the immune serum globulin through a dialysis membrane. Rinderknecht6 advocated the use of FITC on celite for labeling. He claimed greater ease of weighing and handling small amounts of FITC by this method and rapid reaction with protetin. However, there is no reason to believe that FITC in this form would be more reactive than FITC in aqueous solution. Other modifications include labeling whole serum and then fractionating it on a DEAEcellulose column.7 The DEAE-cellulose removes unreacted dye and serum components other than IgG, plus some of the more heavily labeled IgG, in one operation. This method is attractive from the standpoint of the time involved but is somewhat wasteful of the labeling reagent because of the greater reactivity of FITC with albumin This investigation was performed pursuant, in part, to Interagency Agreement No. YOI-DE-3001 1 with the

National Institute of Dental Research, National Institutes of Health, US Department of Health, Education,

and Welfare, Bethesda, Md. Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or by the US Department of Health, Education, and Welfare.

and other serum components relative to the IgG fraction.8 The procedure may also be criticized because of problems in reproducibly obtaining the desired degree of antibody labeling because of the wide variation in the IgG content of immune serums. However, we have found whole serum labeling a very useful means of following the immune state of rabbits during the course of immunization. The and Feltkamp9,10 combined the findings and recommendations of several groups8,11,12 who have investigated labeling and IF staining from various aspects. They recommended isolating the IgG component of immune serum globulin by DEAE-Sephadex chromatography, labeling with a buffered aqueous solution of FITC, and finally isolating the optimally labeled fraction by further DEAE-Sephadex fractionation. Forsum13 labeled F (ab') 2 fragments of immune IgG, obtained by pepsin digestion, to avoid the reaction of the Fc fragment of IgG with the protein A antigen of Staphylococcus aureus. He then isolated the optimally labeled fraction by chromatography on Sephadex G-25 with a buffer at a pH of 4.7. Recommendations for the optimum degree of labeling with FITC to obtain the desired staining intensities with minimal nonspecific staining vary widely. Apparently, the substrate to which the conjugate is applied is of primary importance in determining the range of optimal fluoresceinprotein (F/P) ratios.14 This is by no means a complete survey of the methods of labeling with FITC that have been used or are now being used to prepare IF reagents. It is obvious, however, that the number of methods and, in some cases, contradictory recommendations are sufficient to thoroughly confuse one who wishes to use IF techniques for the first time.

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Vol 55 1976

CONJUGATION METHODS IN IMMUNOFLUORESCENCE

TABLE 1 LABELING OF RABBIT y-GLOBULIN WITH FLUORESCEIN ISOTHIOCYANATE ISOMER I: RATE AND EFFICIENCY OF LABELING WITH REACTION

CONDITIONS DESIGNED TO GIVE RAPID

CONJUGATION Conjugate

Reaction Mixture % Effi-

F/P Ratio

(Ag/mg)

25 12.5

6.25

Reaction Time

F/P Ratio (ftg/mg)

ciency of Labeling

15 min 30 min 2 hr 15 min 30 min 2 hr 15 min 30 min

14.2

57

16.0 16.4 7.7 8.4 8.6 4.1 4.3

64 66 61

2hr

4.3

67 69 66 69 69

Note: Data from McKinney, Spillane, and Pearce8; F/P, fluorescein-protein ratio. Conditions of the reaction were as follows: phosphate buffer, 0.05 M, with a pH of 9.5; protein concentration, 2.5 %; temperature, 25 C.

Some commercial preparations of FITC are of poor quality,'5 making adequate control of the degree of labeling difficult. However, relatively simple methods for determining the purity of FITC products have been available for some time.16 Furthermore, commercial FITC is available that has been

assayed and certified by the Biological Stain Commission as to the percent of FITC that it contains.17 Thus, in preparing a reaction mixture, the user of a product can make allowances for the degree of purity. A high pH, salt concentration, temperature, and concentration of reactants all favor a rapid rate of reaction of FITC with y-globulin. F/P ratios obtained and the efficiencies of reaction with respect to the FITC used are shown in Table 1 for FITC with rabbit y-globulin under defined conditions. The conditions were designed to be mild enough to minimize denaturing of antibody and yet give relatively rapid labeling. The FITC was added as a freshly prepared solution (1 mg/ml in 0.1 M Na2HPO4) so as to produce a single-phase reaction mixture without the need for stirring. With these conditions, the reaction was essentially complete in all cases within 30 minutes, and the efficiency of labeling varied over a relatively narrow range of 64 to 67%. These data indicate that labeling im-

A39

mune serum globulin to the desired F/P ratio can be quite precise when labeling conditions are well defined. In our laboratory, some variations of these conditions are used to prepare antibacterial conjugates. The different amounts of time required to obtain essentially complete reactions are shown in Table 2. Conditions were the same as those used to obtain the data in Table 1 except for variations in the protein content of the reaction mixtures. Since working with 2.5%, protein concentrations is not always convenient, a reaction time of 2½2 hours to 30 minutes is used with protein concentrations ranging from 0.5 to 2.5%, respectively. This allows maximum reaction or use of approximately 65% of the FITC. Another method for obtaining a desired degree of labeling, or F/P ratio, is to use an excess of FITC and to interrupt the reaction before all of the available FITC has been bound. To obtain reproducible results with this procedure, one must more precisely control the reaction conditions. In our experience with antibacterial conjugates, we have had to use rather high F/P ratios to obtain acceptable IF staining. Immune serum globulin preparations obtained by (NH4) 2SO4 fractionation are labeled to somewhat higher F/P ratios than IgG or purified antibody preparations to compensate for the greater reactivity of the /3- and, in some cases, the a-globulins present in these preparations. Although the degree of labeling that we use is higher than is generally recommended for IF applications, it appears to be well justified in the case of antibacterial conjugates. The result is much TABLE 2 TIME NECESSARY FOR ESSENTIALLY COMPLETE REACTION OF FITC WITH VARIOUS CONCENTRATIONS OF RABBIT y-GLOBULIN# gm/100 mg y-Globulin in the Reaction Mixture

0.5 1.0 1.5

2.0 2.5

Time Required for Essentially Complete Reaction

2½2 hr

I hr 45 min 30 min 30 min

Note: FITC, fluorescein isothiocyanate. * Conditions of the reaction were as follows: phosphate buffer, 0.05 M, with a pH of 9.5; temperature, 25 C.

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A40

McKINNEY, THACKER, AND HEBERTJJ Dent Res

TABLE 3 STAINING INTENSITIES OF FIVE STRAINS* OF S mnutans SEROTYPE C WITH C SPECIFIC ANTIBODY LABELED TO Two DIFFERENT F/P RATIOS Staining Intensitiest

Dilution Factors of Conjugate Relative to 10 mg of Antibody/ ml as Undilute

F/P Ratio of Conjugate

8

21

2

3-4t

4+

4

3-4

4+

8 16

3 2-3

4+ 4+

32

2-3

4+

64 128

2-3 1-2

3-4+ 3-4+

Note: F/P, fluorescein-protein ratio. 0 Strains GS5, 10449, PKI, B3, and B7. t Two numbers for staining intensities with a given conjugate dilution indicate slight differences in staining intensities among the strains; 4+ staining indicates brightest fluorescence.

higher titers and more intense staining. Although specific but unwanted staining (crossreaction) has also been more intense with the more heavily labeled conjugates, true nonspecific (nonimmunological) staining has not been a problem in using working dilu-

tions of conjugates. Data from Table 3 were obtained by staining five strains of Streptococcus mutans, serotype c, with conjugates of c type-specific antibody prepared by column immunosorption techniques.18 The two conjugates were made from the same antibody preparation and labeled to F/P ratios of eight and 21.

Special

The slides were examined with an Ortholux microscope with a Ploem vertical illuminator by using a 4-mm BG 38 filter, two KP490 primary filters, a 495-nm interference plate, and a K510 barrier filter. The opticsa consisted of a 95x objective with a 1.15-mm aperture and a IOx ocular. Serial twofold dilutions were made of the conjugates with a borate-buffered saline diluent (pH, 8.0) containing 0.5% bovine serum albumin (BSA). The results in Table 3 clearly show that the conjugate with the F/P ratio of eight is not sufficiently labeled to produce bright staining, whereas the conjugate with the F/P ratio of 21 was sufficiently labeled. The S mutans c to e cross-reacting antibodies, obtained as described by McKinney and Thacker,18 were labeled to F/P ratios of 10 and 20. IF staining results with these two conjugates with strains of S mutans serotypes c and e are shown in Table 4. Again, the lower level of labeling was obviously not sufficient. The conjugate with an F/P ratio of ten did not give brilliant staining even at the lowest dilution, whereas the conjugate with an F/P ratio of 20 was quite satisfactory.

Conjugates have been prepared with a systematic progression of F/P ratios by using (NH4) 2SO4-fractionated immune serum globulins against S mutans types b and e. Satisfactory IF staining titers were a

Provided by E. Leitz, Rockleigh, NJ.

TABLE 4 STAINING INTENSITIES OF FIVE STRAINS OF S mutans SEROTYPE C# AND FOUR STRAINS OF S mutans SEROTYPE et WITH c-e CROSS-REACrING ANTIBODY LABELED TO Two DIFFERENT F/P RATIOS Dilution Factors of the Conjugate Relative to 10 mg of Antibody/ ml as Undilute

Staining Intensitiest Serotype c Strains Serotype e Strains F/P Ratios F/P Ratios 10

20

10

20

2

3-4*

4+

3

4+

4

3-4

4+

3

4+

8 16

3 2-3 2-3 2-3 2

4+ 4+ 4+ 4+

3 3 3 2 2

4+ 4+ 4+ 3-4

32 64 128

Issue A

3-4

3-4

Strains GS5, 10449, PKI, B3, and B7. t Strains LM7, VIOO, ATIO, and B2. $ Two numbers for staining intensities with a given conjugate dilution indicate slight differences in staining intensities among the strains; 4+ staining indicates brightest fluorescence. Downloaded from jdr.sagepub.com at Cape Breton University Library on March 1, 2013 For personal use only. No other uses without permission.

Vol 55 1976

CONJUGATION METHODS IN IMMUNOFLUORESCENCE

IgG ratio of 20 Acg/mg in the reaction mixture. 4. Adjust the pH to 9.0 with 0.1 M Na3-

not obtained with either of these systems with conjugates having F/P ratios much below 15 ug/mg. Conjugates having F/P ratios within the range of 25 to 30 cug/mg gave satisfactory IF staining with no apparent decrease in titer at the higher level of labeling. On the basis of these observations, we routinely label anti-S mutans pure IgG preparations to an F/P ratio of approximately 20 ,ug/mg, and (NH4) 2SO4-fractionated globulins to an F/P ratio of about 25 to 30 jig/mg.

P04.

5. Allow a 2.5-hour reaction period at 25 C, then separate the conjugate from unreacted dye by gel filtration on a column of Sephadex G-50. 6. Centrifuge the conjugate to remove any insoluble material.

Alternatively, TMRI can be dissolved in dimethylformamide at a concentration of 4 mg/ml. This solution is diluted to 10 ml with 0.1 M Na2HPO4, and the appropriate volume is added to give the desired dyeIgG ratio in the reaction mixture. This procedure also works well with rhodamine B isothiocyanate (RBI), which is essentially insoluble in 0.02 M HC1.24 The ratio of absorbance (A) of the conjugate at 554 nm to absorbance at 280 nm serves as a useful guide for determining whether the labeling is satisfactory. In our experience, absorbance ratios (554/280 nm) between 0.35 and 0.55 have been found to indicate a satisfactory degree of labeling. At present, commercial sources of TMRI appear to be very limited. The data in Table 5 were obtained in an evaluation of commercial TMRI products by an infrared analysis that measures isothiocyanate content and from a BSA labeling efficiency test. Both tests were patterned after methods used to evaluate FITC.24 TMRI samples I and 2 (Table 5) were 65 and 60% pure, respectively, by infrared analysis, and had a relatively high capacity for labeling BSA, as indicated by the values in the A per milligram per liter column. These two sam-

Rhodamine Isothiocyanate Labeling Methods Rhodamine isothiocyanates have not been used as extensively as FITC for antibody labeling, and relatively few variations of labeling methods are found in the literature.3,19-24 Generally, the IgG fraction is isolated by DEAE-cellulose or DEAE-Sephadex chromatography, and dye-IgG ratios recommended for use in the labeling mixture have usually been within the range of 10 to 40 ,ug/mg. Several investigators have recommended further fractionating the conjugated IgG by DEAE-cellulose or DEAESephadex chromatography to isolate the optimally labeled fraction. The method we are currently using to label with tetramethylrhodamine isothiocyanate (TMRI) is as follows: 1. Dissolve the TMRI in 0.02 M HCI at a concentration of 2.5 mg/ml. 2. Centrifuge for ten minutes to remove any insoluble material. 3. Add the appropriate amount of freshly prepared TMRI solution to a buffered saline solution of IgG to give a dye-

BLE 5 IR AND BSA LABELING EFFICIENCY MEASUREMENTS OF COMMERCIAL TMRI SAMPLES TMRI Sample mg Weighed

for Conju-

BSA Conjugate

Sample

% by IR

gation

A at 554 nn

1*

65 60 0

11.5

0.625

12.6 15.0

0.574 0.075

2t 3i:

A41

A/mg/

0.100 0.091 0.010

liter

Note: Data from McKinney and Spillane.24 IR, infrared; BSA, bovine serum albumin; and TMRI, tetramethylrhodamine isothiocyanate. 0 Sample, purchased in 1972 from Baltimore Biological Laboratories, Cockeysville, Md, was 79% pure by infrared analysis method at the time of purchase. t Sample was purchased in 1974 from the same source as sample 1. 1: Sample was obtained from source different from that of samples 1 and 2. Downloaded from jdr.sagepub.com at Cape Breton University Library on March 1, 2013 For personal use only. No other uses without permission.

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McKINNEY, THACKER, AND HEBERT

J Dent Res Special Issue A

TABLE 6 IR AND BSA LABELING EFFICIENCY MEASUREMENTS OF COMMERCIAL RBI SAMPLES RBI Sample mg Weighed

Sample

% by IR

for Conjugation

1*#66 0 0

2t 3t 4t

6.5

BSA Conjugate _A at A/mg/ liter 558 nm

0.472 0.093 0.078

11.8

10.3 13.0 11.9

0.178

0.040

0.009 0.006 0.015

Note: Data from McKinney and Spillane.24 IR, infrared; BSA, bovine serum albumin; and RBI, rhodamine B isothiocyanate. 0 Sample was obtained by the courtesy of the British Drug House, Poole, Eng. t Samples obtained from sources different from that of sample No. 1.

ples,b were quite satisfactory for the preparation of TMRI-labeled IgG. Sample 3, purchased from a different source, had no measurable isothiocyanate content by the infrared analysis and very little reactivity with BSA, indicating that it was unsatisfactory for labeling protein. The use of a shelf-life date on commercially packaged TMRI products is highly desirable, since TMRI products have been observed to undergo apciable degradation with time even when stored in an airtight desiccator over Drierite.24 A similar set of data obtained for commercial RBI products from four different sources is shown in Table 6. Sample 1 of this setc was 66% pure by the infrared analysis. The value, 0.040, in the A per milligram per liter column for the BSA labeling efficiency test is low relative to that obtained for the two satisfactory TMRI samples. This reflects the lower specific absorbance of RBI rather than low purity or reactivity. The fluorescence emission of RBI is also lower than that of TMRI, and a higher degree of labeling is desirable with RBI in order to obtain satisfactory conjugates. RBI sample 1 was very satisfactory for the preparation of conjugates of immune IgG by using a dye-protein ratio of 40 ptg/ mg in the reaction mixture or twice the ratio that was optimal with TMRI of comparable purity.24 RBI samples 2, 3, and 4 (Table 6) were of very poor quality, as indicated by infrared analysis and BSA labeling efficiency, and were not satisfactory for use in preparing immune conjugates. b Purchased from Baltimore Biological Laboratories, Cockeysville, Md. c Obtained from the British Drug House, Poole, Eng.

The data in Tables 5 and 6 show that dye purity is a major problem in rhodamine isothiocyanate labeling. Selection of a labeling reagent of satisfactory purity is essential for the successful preparation of rhodamine

conjugates. Performance Comparison of FITC and TMRI Conjugates of the Same Antibody Preparation The TMRI conjugate was prepared as described in our general procedure by using a freshly prepared solution of TMRI in 0.02 M HCI. The same fluorescence equipment was used to evaluate slides stained with the TMRI conjugate as was used for the slides stained with the FITC conjugates. The filter systemd used for examination of the TMRI-stained slides was a 4-mm BG 38 red absorbing filter, a 2-mm BG 36 band absorption glass filter (to absorb the strong mercury line at 578 nm), an AL 546 excitation filter, a 580-nm interference plate, and a K590 barrier filter. Conjugate dilutions were made as described for the FITC conjugates. The IF staining titers of the TMRI conjugate of S mutans c specific antibody were compared with FITC conjugates of the same antibody preparation (F/P ratios 8 and 21). These are shown in Table 7. On the basis of these staining titers, the TMRI conjugate is at least equivalent in performance to the F/P ratio of 21 for the FITC conjugate and decidedly superior to the F/P ratio of eight for the FITC conjugate. The TMRI conjugate was prepared with TMRI of less than 60% purity by infrared measurement with d

E. Leitz, Rockleigh, NJ.

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CONJUGATION METHODS IN IMMUNOFLUORESCENCE

Vol 55 1976 TABLE 7

STAINING INTENSITIES OF FIVE STRAINS OF S mutans SEROTYPE C* WITH C SPECIFIC ANTIBODY LABELED TO Two DIFFERENT F/P RATIOS COMPARED WITH A TMRI CONJUGATE (ABSORBANCE RAriO 554 nm/280 nm = 0.35) OF THE SAME ANTIBODY PREPARATION

Dilution Factors of Conjugate Relative to 10 mg of Antibody/ml as

Staining Intensitiest FITC Conjugates F/P Ratio

TMRI -

Conju-

Undilute

8

21

gates

2

3-4 3-4

4+ 4+ 4+ 4+ 4+

4+ 4+ 4+ 4+ 4+ 4+ 4+

4 8 16

32 64 128 256

3+ 2-3 2-3 2-3 2-3 1-2

3-4 3-4 3-4

3-4

Note: TMRI, tetramethylrhodamine isothiocyanate; FITC, fluorescein isothiocyanate. 0 Strains GS5, 10449, PKI, B3, and B7. t Two numbers for staining intensities with a given conjugate dilution indicate slight differences in staining intensities among the strains; 4+ staining indicates brightest fluorescence.

an initial dye-antibody ratio of 20 Icg/mg. The actual labeling efficiency with respect to TMRI covalently bound to antibody is probably less than 50%, and the dye-antibody ratio of the conjugate is probably less than 6 ytg/mg. It is apparent, even from these rough approximations, that the TMRIlabeled antibody is superior to the FITClabeled antibody in IF staining on the basis of the amount of fluorochrome covalently bound to the antibody. This observation is in accord with the report of Hijmans et a125 who found that with epi-illumination using green light excitation, TMRI fluorescence was five to ten times more efficient than FITC fluorescence with blue light excitation. Since FITC covalently bound to y-globulin has a relatively high fluorescence quantum efficiency,26 the stronger specific emission of TMRI must be primarily the result of a greater amount of light actually being absorbed under the epi-illumination

conditions. Conclusions We have described methods of labeling

antibody preparations with FITC, TMRI, and RBI. The degree of labeling with FITC can be precisely controlled by using well-defined conjugation procedures and

A43

FITC of a known degree of purity. Our experience shows that relatively high F/P ratios of the order of 20 to 25 jxg/mg are desirable for antibacterial conjugates. Many commercial preparations of rhodamine isothiocyanate are of very poor quality and are unsatisfactory for use in conjugate preparation. Therefore, one should analyze the rhodamine isothiocyanate product before preparing immune conjugates. Our experience indicates that very satisfactory conjugates of immune IgG or pure antibody can be prepared with TMRI of about 60% purity by using a dye-protein ratio of 20 1zg/mg. The optimal dye-IgG ratio for labeling with RBI appears to be about two times that for labeling with TMRI because of the lower specific absorbance and fluorescence emission of RBI. Rhodamine conjugates may be preferred to FITC conjugates in certain situations where tissue autofluorescence interferes with the observation of the yellow-green emission of FITC. Furthermore, mixed rhodamine and FITC conjugates of different specificity can be used to great advantage in doublestaining techniques that allow simultaneous screening for two antigenically different organisms on a single microscope slide.

References 1. COONS, A.H.; CREECH, H.J.; JONEs, R.N.; and BERLINER, E.: The Demonstration of Pneumococcal Antigen in Tissues by the Use of Florescent Antibody, J Immunol 45: 159-170, 1942. 2. COONs, A.H., and KAPLAN, M.H.: Localization .of Antigen in Tissue Cells: II. Improvements in a Method for the Detection of Antigen by Means of Fluorescent Antibody, J Exp Med 91: 1-13, 1950. 3. RIGGS, J.L.; SEIWALD, R.J.; BURCKHALTER, J.H.;

DOWNS, C.M.; and METCALF, T.G.: Isothiocyanate Compounds as Fluorescent Labeling Agents for Immune Serum, Am J Pathol 34: 1081-1091, 1958. 4. MARSHALL, J.D.; EVELAND, W.C.; and SMITH,

C.W.: Superiority of Fluorescein Isothiocyanate (Riggs) for Fluorescent-Antibody Technic with a Modification of Its Application, Proc Soc Exp Biol Med 98: 898-900, 1958. 5. CLARK, H.F., and SHEPARD, C.C.: A Dialysis Technique for Preparing Fluorescent Antibody, Virology 20: 642-644, 1963. 6. RINDERKNECHT, H.: A New Technique for the Fluorescent Labelling of Proteins, Experientia 16: 430-431, 1960.

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McKINNEY, THACKER, AND HEBERT

7. RIGGS, J.L; LOH, P.C.; and EVELAND, W.C.: A Simple Fractionation Method for Preparation of Fluorescein-Labeled Gamma Globulin, Proc Soc Exp Biol Med 105: 655-658, 1960. 8. McKINNEY, R.M.; SPILLANE, J.T.; and PEARCE, G.W.: Factors Affecting the Rate of Reaction of Fluorescein Isothiocyanate with Serum Proteins, J Immunol 93: 232-242, 1964. 9. THE, T.H., and FELTKAMP, T.E.W.: Conjugation of Fluorescein Isothiocyanate to Antibodies: I. Experiments on the Conditions of Conjugation, Immunology 18: 865-873, 1970. 10. THE, T.H., and FELTKAMP, T.E.W.: Conjugation of Fluorescein Isothiocyanate to Antibodies: II. A Reproducible Method, Immunology 18: 875-881, 1970. 11. GOLDSTEIN, G.; SLIZYS, I.S.; and CHASE, M.W.: Studies on Fluorescent Antibody Staining: I. Non-Specific Fluorescence with FluoresceinCoupled Sheep Anti-Rabbit Globulins, J Exp Med 114: 89-110, 1961. 12. WOOD, B.T.; THOMPSON, S.H.; and GOLDSTEIN, G.: Fluorescent Antibody Staining: III. Preparation of Fluorescein-Isothiocyanate Labelled Antibodies, J Immunol 95: 225229, 1965. 13. FORSUM, U.: Characterization of FITC-Labelled F (ab') 2 Fragments of IgG and a Rapid Technique for the Separation of Optimally Labelled Fragments, J Immunol Methods 2: 183-195, 1972. 14. PITTMAN, B.; HEBERT, G.A.; CHERRY, W.B.; and TAYLOR, G.C.: The Quantitation of Nonspecific Staining As a Guide for Improvement of Fluorescent Antibody Conjugates, J Immunol 98: 1196-1203, 1967. 15. CHERRY, W.B.; MCKINNEY, R.M.; EMMEL, V.M.; SPILLANE, J.T.; HEBERT, G.A.; and PITTMAN, B.: Evaluation of Commercial Fluorescein Isothiocyanates Used in Fluorescent Antibody Studies, Stain Technol 44: 179-186, 1969. 16. MCKINNEY, R.M.; SPILLANE, J.T.; and PEARCE, G.W.: A Simple Method for Determining the Labeling Efficiency of Fluorescein Isothiocyanate Products, Anal Biochem 14: 421428, 1965. 17. Certification of Fluorescein Isothiocyanate,

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announcement, Stain Technol 44: No. 4, 1969. McKINNEY, R.M., and THACKER, L.: Improvement in Specificity of Immunofluorescent Reagents for Identifying Streptococcus mutans by DEAE-Cellulose-Bacterial Cell Column Immunosorption Methods, J Dent Res (Special Issue A) 55: A50-A57, 1976. SMITH, M.L.; CARSKI, T.R.; and GRIFFIN, C.W.: Modification of Fluorescent-Antibody Procedures Employing Crystalline Tetramethylrhodamine Isothiocyanate, J Bacteriol 83: 1358-1359, 1962. CEBRA, J.J., and GOLDSTEIN, G.: Chromatographic Purification of Tetramethylrhodamine-Immune Globulin Conjugates and Their Use in the Cellular Localization of Rabbit y-Globulin Polypeptide Chains, J Immunol 95: 230-245, 1965. AMANTE, L.; ANCONA, A.; and FORNI, L.: The Conjugation of Immunoglobulins with Tetramethylrhodamine Isothiocyanate: A Comparison Between the Amorphous and the Crystalline Fluorochrome, J Immunol Methods 1: 289-301, 1972. BRANDTZAEG, P.: Conjugates of Immunoglobulin G with Different Fluorochromes: I. Characterization by Anionic-Exchange Chromatography, Scauid J Immnunol 2: 273-290, 1973. BERGQUIST, M.R., and NILSSON, P.: The Conjugation of Immunoglobulins with Tetramethylrhodamine Isothiocyanate by Utilization of Dimethylsulfoxide (DMSO) as a Solvent, J Immunol Methods 5: 189-198, 1974. McKINNEY, R.M., and SPILLANE, J.T.: An Approach to Quantitation in Rhodamine Isothiocyanate Labeling, Ann NY Acad Sci 254: 55-64, 1975. HIJMANS, W.; SCHUIT, H.R.E.; TEIKO, Y.; and SCHECHTER, I.: An Immunofluorescence Study on the Synthesis of Antibodies of Single Specificity in Separate Cells After the Administration of an Immunogen with Double Specificity, Eur J Immunol 2: 1-4, 1972. CHEN, R.F.: Fluorescent Protein-Dye Conjugates: II. Gamma Globulin Conjugated with Various Dyes, Arch Biochem Biophys 133: 263-276, 1969.

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Conjugation methods in immunofluorescence.

Journal of Dental Research http://jdr.sagepub.com/ Conjugation Methods in Immunofluorescence Roger McKinney, Leroy Thacker and G. Ann Hebert J DENT R...
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