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A double-sandwich ELISA test for Glm(3) phenotyping of bloodstains with a mouse monoclonal antibody
ORIGINAL PAPERS
Forensic Science Society 1987
A double-sandwich ELISA test for Glm(3) phenotyping of bloodstains with a mouse monoclonal antibody Ch FRANCOIS-GERARD and B HOSTE
Blood Group Laboratory, University of Lit?ge, 41 rue Dos Fanchon, B-4020 Li2ge, Belgium Abstract ELISA procedures have been developed for the Gm(3) allotypic marker of human IgGl immunoglobulins, using a mouse monoclonal anti-Gm(3) antibody. The test has a high sensitivity since the monoclonal reagent, even diluted 11 32,000, still detects the Gm(3) antigen in serum samples diluted 1/128,000 to 1/2,000,000. On account of its sensitivity and its objective reading, the test is well adapted for bloodstain typing. The addition of a parallel test for IgG makes it possible to distinguish negative results due to Gm(-3) phenotypes from those due to a lack of IgG material. Gm(3) and IgG were detected in bloodstains more than 5 years old. Key Words: Double-sandwich ELISA test; Glm(3) allotypic marker; Monoclonal antibody; Bloodstains.
Journal of the Forensic Science Society 1987; 27: 31-38 Received 23 November 1985
Introduction Because human immunoglobulins are very polymorphic and stable as far as their antigenic properties are concerned, Gm allotyping is very useful in paternity work as well as in bloodstain analysis. The most usual method is the inhibition of haemagglutination by polyclonal anti-Gm reagents [I]. Recently, a mouse monoclonal anti-Gm(3) antibody has become available (Seward Laboratory, England) that can be used both in haemagglutination and in enzyme-immunoassay. Fletcher and colleagues [2] and Newall [3] have developed a single-sandwich ELISA procedure for the Glm(3) phenotyping of bloodstains. We present here a new ELISA test characterised by a double-sandwich-like antigen-antibody reaction. Because of its specificity and greater sensitivity, this test constitutes a method of choice for small volume samples and bloodstain analysis. 31
Materials and method Materials Human sera phenotyped for Gm allotypic markers were obtained from the Centraal Laboratorium van de Bloedtransfusie dienst van het Nederlandse Rode Kruis (Amsterdam, The Netherlands) and from the Groupe de Recherche U 7 8 de 1'Inserm sur la gCnCtique des protCines humaines (Bois-Guillaume, France). Normal sera were collected from healthy blood donors of the Transfusion Centre of Libge.
Bloodstains phenotyped for Gm(3) in the course of this study were experimental bloodstains on cotton cloth as well as forensic casework bloodstains. They were extracted overnight at 37OC with phosphate buffered saline containing 1% (wlv) bovine serum albumin and 1% (w/v) sodium azide (100 p1 buffer per 10 mm2 stain, i.e., 0.3 mg dried blood). All tests were performed in polystyrene immunoplates with 96 flat-bottomed wells (Nunc no 4-39454, Gibco). Mouse monoclonal anti-Gm(3) was purchased from Seward Laboratory (BAM clone Tm 14) and rabbit immunoglobulins anti-human IgG (RAH-IgG) (A090), peroxidaseconjugated RAH-IgG (P 214) and peroxidase-conjugated rabbit immunoglobulins anti-mouse Ig (RAM-Ig) (P 260) were obtained from Dako. The peroxidase substrate used was ABTS (2,2'-Azino-di-3-ethylbenzthiazoline sulphonate 6, no 102 946 Boehringer, Mannheim). Optical densities at 405 nm were measured in the plates with a spectrophotometer (Multiskan, Titertek, Flow Laboratories). Method In all ELISA methods for Gm(3) phenotyping previously published, bloodstain extracts were directly coated onto the plates. Schematically, our test looks like a double sandwich, with a preliminary step before introduction of the sample (Table 1).
Polystyrene immunoplates were coated overnight at 4OC with RAH-IgG (y-chain specific) on the basis of 75 p1 per well of the appropriate dilution in basic buffer (Na2C03 1-59g; NaHC03 2.938; NaN3 0.2g; H,O 1 litre, pH9.6). The plates were then washed three times with buffer (NaH2P04. 2H20 0-59 g; K2HP04. 3H20 3.67 g; NaCl 8.76 g; H 2 0 1 litre; pH 7.4 containing 20 ml/l of calf serum and 0.5% (v/v) Tween 20). After drying, plates were ready to receive the samples to be tested. Fifty microlitres of 1/2,000 prediluted normal serum (or plasma), or undiluted bloodstain extracts, were added in twofold dilutions and kept overnight at 4°C. A positive control of purified human IgGl at a known concentration (2-2.10~~ pg/ml) was added to each plate in twofold dilutions. A 1/2,000 prediluted human serum known to be Gm(3) negative was used as negative 32
TABLE 1 Schematic presentation of the double-sandwich ELISA procedure adapted to Gm(3) phenotyping ELlSA steps 1. 2. 3. 4. 5.
Coated antibody Unknown antigen First antibody Second antibody-E Substrate
Reagents
Rabbit Ig anti-human IgG Human serum or bloodstain extract Mouse monoclonal anti-Gm(3) Rabbit Ig anti-mouse Ig-peroxidase ABTS
control and optical blanks were obtained from wells treated similarly to the samples but without human IgG material. After three further washings, 50 p1 of the appropriate dilution of anti-Gm(3) mouse monoclonal antibody were added to each well and incubated for 2 h at 37°C. After three washings, fixation of the monoclonal antibody on positive samples was revealed by using 50 pl of peroxidase-conjugated RAM-Ig at a 11250 dilution. This reagent was left for 1h at 37°C and the excess removed by four washings with running water. Fifty microlitres of substrate solution (ABTS: 75 mg1100 ml of phosphate-citrate buffer-Na2HP0, . 2H20 20.5 g, citric acid 8.9 g, H 2 0 1litre, pH 5.6 containing 4 mlll 0.3% H202) were added to each well. The green colour appearing within half an hour was consistent with positive samples whereas negative samples remained uncoloured. All results were expressed as the last optical density values ( A sample - A coefficient of enzyme-immunoassays is and positive controls were run on each
sample dilution still giving positive blank > 0.100). Since the variation not negligible, appropriate negative plate.
Results Determining optimal dilutions of each reagent was the first and the most important part of this work. Optimal dilution of coated antibody Various dilutions (114,000-1120,000) of RAH-IgG were coated and then incubated in the presence of decreasing amounts of purified human IgG (2-2.10~~ pglml). The antigen-antibody complex was revealed by the use of peroxidase-conjugated RAH-IgG at a dilution of 11250 as recommended by the manufacturer.
Tridimensional graphical illustration of optical densities measured at 405 nm allowed the selection of an optimal dilution as the one still giving highest absorbancy values while using as little reagent as possible. From this experiment it appeared that a 118,000-1/10,000 dilution of the RAH-IgG
was still able to detect IgG concentrations as low as 2nglml. This standardization assay must be performed with every new batch of reagent. Optimal dilution of mouse monoclonal anti-Gm(3) Similarly, increasing dilutions of monoclonal anti-Gm(3) were tested in the presence of decreasing amounts of Gm(3) positive purified human IgGl (2-2-10-~yglml). The Gm(3)-anti-Gm(3) complexes formed were revealed by adding peroxidase-conjugated RAM-Ig diluted 11250. Figure 1 shows that in these experimental conditions the monoclonal reagent may be diluted 1/30,000 (log dil= 4-3) while retaining a high sensitivity. This dilution was adopted for all ELISA tests performed in the course of the study.
--
FIGURE 1 Tridimensional graphical illustration of optical densities measured at 405 nm.
It must be pointed out that the coated antibody specificity is vital to the efficiency of the test. Indeed, this first antibody must be devoid of any cross-reactivity either with the mouse monoclonal anti-Gm(3) or with the
enzyme-conjugated anti-mouse rabbit immunoglobulins. When necessary, the first coated antibody must be affinity purified. In order to test for the sensitivity of this indirect ELISA test, it was compared with haemagglutination inhibition, the method classically used for Gm phenotyping of human sera [4]. In these experiments (Table 2), the sensitivity is expressed in terms of the last serum dilution still significantly detected by the anti-Gm(3).
TABLE 2 Compared sensitivity of different methods used for Gm(3) phenotyping Method Double-sandwich ELISA
Reagent and working dilution
Last serum dilutions still detected
Monoclonal anti-Gm(3)
1/128,000-1/2,000.000*
1/30,000 Haemagglutination inhibition Haemagglutination inhibition
Monoclonal anti-Gm(3)
l/8,m
1/8,00(J Polyclonal anti-Gm(3)
1/1,Ooo
111-118
* The range of dilutions is related to individual variations of IgGl levels in serum.
The double-sandwich ELISA appears to be much more sensitive than haemagglutination even when this latter method is performed with the monoclonal reagent. Extracts of 0.3 mg dried blood in 100pl yielded positive results up to the 114,000 dilution so that, in most cases, Gm(3) antigen will be detected even in small size bloodstains. The Gm(3) specificity of the monoclonal was confirmed in ELISA by looking for Gm(3) antigen in 26 phenotyped panel sera as well as in more than 80 samples previously analyzed for Gm(3) by the classical method. No discordance was observed. Monoclonal reagents antithetic to the Gm(3) specificity are not yet commercially available. Interpretation of negative results in the course of bloodstain analyses is therefore a problem. Indeed, it is not possible to distinguish negative samples due to Gm(-3) phenotypes from those due to a lack of IgGl molecules in the extract. In order to solve this question, the amount of IgG available in the extract was determined at the same time. This test consisted of a single sandwich test identical to the first one described in this paper for the search for optimal dilution of the coated antibody. RAH-IgG is coated on the plates, the sample is added in twofold dilutions and IgGs are revealed by the addition of peroxidase-conjugated RAH-IgG. A positive control made of purified human IgGl at a known concentration (2-2.10~~ pg/ml) is added to each plate in twofold dilutions. By analyzing replicates of Gm(3) negative sera, it was calculated that the mean absorbancy value of negative controls (2 + 2a) remains in the range 35
TABLE 3 IgG and Gm(3) stability in bloodstains by ELISA double-sandwich Log, last positive dilution BloodAge stain (months)
control ZgG *
Gm(3)t
Gm(3) phenotype
* Rabbit immunoglobulins anti-human IgG/sample/peroxidaseconjugated rabbit immunoglobulins anti-human IgG. t Rabbit immunoglobulins anti-human IgG/sample/mouse monoclonal anti-Gm(3)/peroxidase-conjugated rabbit immunoglobulins anti-mouse IgG.
0.02 to 0-08. For safety reasons, a threshold value of A > 0.100 was used for the interpretation. Table 3 shows the results obtained with a series of experimental bloodstain extracts dating from 3 months to 6 years. Gm(3) negative results in bloodstains could be clearly interpreted as Gm(-3) phenotype when the IgG titre was high, as in samples 2, 4, 5, 17. Gm(3) negative results remained doubtful only when there was little or no IgG as in sample 31. It is 36
worth noting that Gm(3) antigen remains detectable even in samples more than 5 years old, and is as stable as IgG, at least for the period studied. Indeed, in a series of 27 bloodstains dating up to 5 years, both IgG and Gm(3) levels were proved to be positively correlated (r2= 0.932). The stability of Gm(3) antigen appears to be excellent in various storage conditions; whole bloods including corpse blood, stored at 4OC for as long as 7 months; sera stored at -20°C for as long as 44 years and frozen and thawed several times during this period; experimental and casework bloodstains stored at room temperature for as long as 2 to 5 years. All these biological materials were still Gm(3) typable. In casework, interference may occur, due to the presence of IgG from non-blood material. Garments, especially dirty, yielded weak or no reaction, whereas our ELISA test detected relatively high levels of Gm(3) marker in several non-blood human fluids (Table 4). Possible contamination of the bloodstain by secretion must therefore be kept in mind when results are interpreted. These preliminary results show the applicability of this test to the typing of semen and other body fluids.
TABLE 4 IgG and Gm(3) detection by ELISA in non-blood human fluids Dilutions of the extracts detected
Fresh bloodstains Non-blood controls 10 dirty cloth samples 2 semen stains 1 nasal mucus stain 1 saliva stain 1 faeces stain
>I11024
>I11024
0-1116 1132-11512 11256 118 0
0-1116 1116-11512 11256 1/16 0
* Rabbit immunoglobulins anti-human IgG/sample/peroxidaseconjugated rabbit immunoglobulins anti-human IgG. t Rabbit immunoglobulins anti-human IgG/sample/anti-Gm(3) monoclonal/peroxidase-conjugated rabbit immunoglobulins antimouse IgG.
Discussion It is important for laboratory immunologists to take into consideration the new generation of reagents that are monoclonal antibodies. Many examples have already been published in which monoclonal reagents bring advantageous improvements in methods previously based on polyclonal sera. To our knowledge, the Seward monoclonal anti-Gm(3) is the first commercially available anti-Gm specificity, but it is foreseeable that in the near future, other anti-immunoglobulin allotypic specificities will be produced. 37
The double-sandwich test described here is much more sensitive than the single sandwich previously developed [2]. Several explanations justify this observation. Because of the y-chain specificity of the coated antibody, IgGs are the only proteins of the sample to be attached to the solid phase, and especially IgGl that constitutes the major component of IgGs. Furthermore, it is probable that IgG molecules captured by means of a polyclonal antibody are more accessible to the monoclonal. Indeed, with anti-IgG coated antibodies acting as a spacer group, IgG molecules are more randomly distributed on the well surface, with an amplification effect as a consequence. This work also confirms the advantages of ELISA tests. The optical densities of an ELISA test are measured by the use of a spectrophotometer. This constitutes an important advantage over manual haemagglutination inhibition, the interpretation of which is much more subjective. The threshold of sensitivity is much greater than that observed with polyclonal anti-Gm(3) or even with the same monoclonal used in haemagglutination inhibition test. In addition, the measurement of IgG in the same bloodstain extract in order to make sure that IgG molecules are present in sufficient amounts, allows identification of Gm(3) negative phenotypes without any risk of error. The practical consequence of these improvements is that small blood or human fluid samples can be typed. In forensic work, this point is sometimes crucial. The indirect or double layer ELISA method makes use of only small amounts of expensive reagents which can be much diluted. Finally, 96-well immunoplates are better suited for the handling of large series of analyses. Acknowledgements We thank Mrs C Opret-Meunier and Mrs MR Dessaintes-Mathy for skilful technida~assistance.
References 1. Gmbb R. The genetic markers of human immunoglobulins. Berlin, Heidelberg, New York: Springer-Verlag, 1970. 2. Fletcher SM, Dorrill MJ and Dolton P. Bloodstain allotyping: an ELISA method for Glm(3). Report of the 10th International Congress of the Society for Forensic Haematogenetics 1983; 221-226. 3. Newall P. Advantages of monoclonal antiserum and ELISA in the identification of bloodstains. Report of the 10th International Congress of the Society for Forensic Haematogenetics 1983; 227-232. 4. Hoste B, Brocteur J and Andrt A. Indefinite storage of dried Gm and Km antigens (Examination of bloodstains 33 years old). Forensic Science 1978; 11: 109-113.
Forensic Science Society 1987
A double-sandwich ELISA test for Glm(3) phenotyping of bloodstains with a mouse monoclonal antibody Ch FRANCOIS-GERARD and B HOSTE
Blood Group Laboratory, University of Lit?ge, 41 rue Dos Fanchon, B-4020 Li2ge, Belgium Abstract ELISA procedures have been developed for the Gm(3) allotypic marker of human IgGl immunoglobulins, using a mouse monoclonal anti-Gm(3) antibody. The test has a high sensitivity since the monoclonal reagent, even diluted 11 32,000, still detects the Gm(3) antigen in serum samples diluted 1/128,000 to 1/2,000,000. On account of its sensitivity and its objective reading, the test is well adapted for bloodstain typing. The addition of a parallel test for IgG makes it possible to distinguish negative results due to Gm(-3) phenotypes from those due to a lack of IgG material. Gm(3) and IgG were detected in bloodstains more than 5 years old. Key Words: Double-sandwich ELISA test; Glm(3) allotypic marker; Monoclonal antibody; Bloodstains.
Journal of the Forensic Science Society 1987; 27: 31-38 Received 23 November 1985
Introduction Because human immunoglobulins are very polymorphic and stable as far as their antigenic properties are concerned, Gm allotyping is very useful in paternity work as well as in bloodstain analysis. The most usual method is the inhibition of haemagglutination by polyclonal anti-Gm reagents [I]. Recently, a mouse monoclonal anti-Gm(3) antibody has become available (Seward Laboratory, England) that can be used both in haemagglutination and in enzyme-immunoassay. Fletcher and colleagues [2] and Newall [3] have developed a single-sandwich ELISA procedure for the Glm(3) phenotyping of bloodstains. We present here a new ELISA test characterised by a double-sandwich-like antigen-antibody reaction. Because of its specificity and greater sensitivity, this test constitutes a method of choice for small volume samples and bloodstain analysis. 31
Materials and method Materials Human sera phenotyped for Gm allotypic markers were obtained from the Centraal Laboratorium van de Bloedtransfusie dienst van het Nederlandse Rode Kruis (Amsterdam, The Netherlands) and from the Groupe de Recherche U 7 8 de 1'Inserm sur la gCnCtique des protCines humaines (Bois-Guillaume, France). Normal sera were collected from healthy blood donors of the Transfusion Centre of Libge.
Bloodstains phenotyped for Gm(3) in the course of this study were experimental bloodstains on cotton cloth as well as forensic casework bloodstains. They were extracted overnight at 37OC with phosphate buffered saline containing 1% (wlv) bovine serum albumin and 1% (w/v) sodium azide (100 p1 buffer per 10 mm2 stain, i.e., 0.3 mg dried blood). All tests were performed in polystyrene immunoplates with 96 flat-bottomed wells (Nunc no 4-39454, Gibco). Mouse monoclonal anti-Gm(3) was purchased from Seward Laboratory (BAM clone Tm 14) and rabbit immunoglobulins anti-human IgG (RAH-IgG) (A090), peroxidaseconjugated RAH-IgG (P 214) and peroxidase-conjugated rabbit immunoglobulins anti-mouse Ig (RAM-Ig) (P 260) were obtained from Dako. The peroxidase substrate used was ABTS (2,2'-Azino-di-3-ethylbenzthiazoline sulphonate 6, no 102 946 Boehringer, Mannheim). Optical densities at 405 nm were measured in the plates with a spectrophotometer (Multiskan, Titertek, Flow Laboratories). Method In all ELISA methods for Gm(3) phenotyping previously published, bloodstain extracts were directly coated onto the plates. Schematically, our test looks like a double sandwich, with a preliminary step before introduction of the sample (Table 1).
Polystyrene immunoplates were coated overnight at 4OC with RAH-IgG (y-chain specific) on the basis of 75 p1 per well of the appropriate dilution in basic buffer (Na2C03 1-59g; NaHC03 2.938; NaN3 0.2g; H,O 1 litre, pH9.6). The plates were then washed three times with buffer (NaH2P04. 2H20 0-59 g; K2HP04. 3H20 3.67 g; NaCl 8.76 g; H 2 0 1 litre; pH 7.4 containing 20 ml/l of calf serum and 0.5% (v/v) Tween 20). After drying, plates were ready to receive the samples to be tested. Fifty microlitres of 1/2,000 prediluted normal serum (or plasma), or undiluted bloodstain extracts, were added in twofold dilutions and kept overnight at 4°C. A positive control of purified human IgGl at a known concentration (2-2.10~~ pg/ml) was added to each plate in twofold dilutions. A 1/2,000 prediluted human serum known to be Gm(3) negative was used as negative 32
TABLE 1 Schematic presentation of the double-sandwich ELISA procedure adapted to Gm(3) phenotyping ELlSA steps 1. 2. 3. 4. 5.
Coated antibody Unknown antigen First antibody Second antibody-E Substrate
Reagents
Rabbit Ig anti-human IgG Human serum or bloodstain extract Mouse monoclonal anti-Gm(3) Rabbit Ig anti-mouse Ig-peroxidase ABTS
control and optical blanks were obtained from wells treated similarly to the samples but without human IgG material. After three further washings, 50 p1 of the appropriate dilution of anti-Gm(3) mouse monoclonal antibody were added to each well and incubated for 2 h at 37°C. After three washings, fixation of the monoclonal antibody on positive samples was revealed by using 50 pl of peroxidase-conjugated RAM-Ig at a 11250 dilution. This reagent was left for 1h at 37°C and the excess removed by four washings with running water. Fifty microlitres of substrate solution (ABTS: 75 mg1100 ml of phosphate-citrate buffer-Na2HP0, . 2H20 20.5 g, citric acid 8.9 g, H 2 0 1litre, pH 5.6 containing 4 mlll 0.3% H202) were added to each well. The green colour appearing within half an hour was consistent with positive samples whereas negative samples remained uncoloured. All results were expressed as the last optical density values ( A sample - A coefficient of enzyme-immunoassays is and positive controls were run on each
sample dilution still giving positive blank > 0.100). Since the variation not negligible, appropriate negative plate.
Results Determining optimal dilutions of each reagent was the first and the most important part of this work. Optimal dilution of coated antibody Various dilutions (114,000-1120,000) of RAH-IgG were coated and then incubated in the presence of decreasing amounts of purified human IgG (2-2.10~~ pglml). The antigen-antibody complex was revealed by the use of peroxidase-conjugated RAH-IgG at a dilution of 11250 as recommended by the manufacturer.
Tridimensional graphical illustration of optical densities measured at 405 nm allowed the selection of an optimal dilution as the one still giving highest absorbancy values while using as little reagent as possible. From this experiment it appeared that a 118,000-1/10,000 dilution of the RAH-IgG
was still able to detect IgG concentrations as low as 2nglml. This standardization assay must be performed with every new batch of reagent. Optimal dilution of mouse monoclonal anti-Gm(3) Similarly, increasing dilutions of monoclonal anti-Gm(3) were tested in the presence of decreasing amounts of Gm(3) positive purified human IgGl (2-2-10-~yglml). The Gm(3)-anti-Gm(3) complexes formed were revealed by adding peroxidase-conjugated RAM-Ig diluted 11250. Figure 1 shows that in these experimental conditions the monoclonal reagent may be diluted 1/30,000 (log dil= 4-3) while retaining a high sensitivity. This dilution was adopted for all ELISA tests performed in the course of the study.
--
FIGURE 1 Tridimensional graphical illustration of optical densities measured at 405 nm.
It must be pointed out that the coated antibody specificity is vital to the efficiency of the test. Indeed, this first antibody must be devoid of any cross-reactivity either with the mouse monoclonal anti-Gm(3) or with the
enzyme-conjugated anti-mouse rabbit immunoglobulins. When necessary, the first coated antibody must be affinity purified. In order to test for the sensitivity of this indirect ELISA test, it was compared with haemagglutination inhibition, the method classically used for Gm phenotyping of human sera [4]. In these experiments (Table 2), the sensitivity is expressed in terms of the last serum dilution still significantly detected by the anti-Gm(3).
TABLE 2 Compared sensitivity of different methods used for Gm(3) phenotyping Method Double-sandwich ELISA
Reagent and working dilution
Last serum dilutions still detected
Monoclonal anti-Gm(3)
1/128,000-1/2,000.000*
1/30,000 Haemagglutination inhibition Haemagglutination inhibition
Monoclonal anti-Gm(3)
l/8,m
1/8,00(J Polyclonal anti-Gm(3)
1/1,Ooo
111-118
* The range of dilutions is related to individual variations of IgGl levels in serum.
The double-sandwich ELISA appears to be much more sensitive than haemagglutination even when this latter method is performed with the monoclonal reagent. Extracts of 0.3 mg dried blood in 100pl yielded positive results up to the 114,000 dilution so that, in most cases, Gm(3) antigen will be detected even in small size bloodstains. The Gm(3) specificity of the monoclonal was confirmed in ELISA by looking for Gm(3) antigen in 26 phenotyped panel sera as well as in more than 80 samples previously analyzed for Gm(3) by the classical method. No discordance was observed. Monoclonal reagents antithetic to the Gm(3) specificity are not yet commercially available. Interpretation of negative results in the course of bloodstain analyses is therefore a problem. Indeed, it is not possible to distinguish negative samples due to Gm(-3) phenotypes from those due to a lack of IgGl molecules in the extract. In order to solve this question, the amount of IgG available in the extract was determined at the same time. This test consisted of a single sandwich test identical to the first one described in this paper for the search for optimal dilution of the coated antibody. RAH-IgG is coated on the plates, the sample is added in twofold dilutions and IgGs are revealed by the addition of peroxidase-conjugated RAH-IgG. A positive control made of purified human IgGl at a known concentration (2-2.10~~ pg/ml) is added to each plate in twofold dilutions. By analyzing replicates of Gm(3) negative sera, it was calculated that the mean absorbancy value of negative controls (2 + 2a) remains in the range 35
TABLE 3 IgG and Gm(3) stability in bloodstains by ELISA double-sandwich Log, last positive dilution BloodAge stain (months)
control ZgG *
Gm(3)t
Gm(3) phenotype
* Rabbit immunoglobulins anti-human IgG/sample/peroxidaseconjugated rabbit immunoglobulins anti-human IgG. t Rabbit immunoglobulins anti-human IgG/sample/mouse monoclonal anti-Gm(3)/peroxidase-conjugated rabbit immunoglobulins anti-mouse IgG.
0.02 to 0-08. For safety reasons, a threshold value of A > 0.100 was used for the interpretation. Table 3 shows the results obtained with a series of experimental bloodstain extracts dating from 3 months to 6 years. Gm(3) negative results in bloodstains could be clearly interpreted as Gm(-3) phenotype when the IgG titre was high, as in samples 2, 4, 5, 17. Gm(3) negative results remained doubtful only when there was little or no IgG as in sample 31. It is 36
worth noting that Gm(3) antigen remains detectable even in samples more than 5 years old, and is as stable as IgG, at least for the period studied. Indeed, in a series of 27 bloodstains dating up to 5 years, both IgG and Gm(3) levels were proved to be positively correlated (r2= 0.932). The stability of Gm(3) antigen appears to be excellent in various storage conditions; whole bloods including corpse blood, stored at 4OC for as long as 7 months; sera stored at -20°C for as long as 44 years and frozen and thawed several times during this period; experimental and casework bloodstains stored at room temperature for as long as 2 to 5 years. All these biological materials were still Gm(3) typable. In casework, interference may occur, due to the presence of IgG from non-blood material. Garments, especially dirty, yielded weak or no reaction, whereas our ELISA test detected relatively high levels of Gm(3) marker in several non-blood human fluids (Table 4). Possible contamination of the bloodstain by secretion must therefore be kept in mind when results are interpreted. These preliminary results show the applicability of this test to the typing of semen and other body fluids.
TABLE 4 IgG and Gm(3) detection by ELISA in non-blood human fluids Dilutions of the extracts detected
Fresh bloodstains Non-blood controls 10 dirty cloth samples 2 semen stains 1 nasal mucus stain 1 saliva stain 1 faeces stain
>I11024
>I11024
0-1116 1132-11512 11256 118 0
0-1116 1116-11512 11256 1/16 0
* Rabbit immunoglobulins anti-human IgG/sample/peroxidaseconjugated rabbit immunoglobulins anti-human IgG. t Rabbit immunoglobulins anti-human IgG/sample/anti-Gm(3) monoclonal/peroxidase-conjugated rabbit immunoglobulins antimouse IgG.
Discussion It is important for laboratory immunologists to take into consideration the new generation of reagents that are monoclonal antibodies. Many examples have already been published in which monoclonal reagents bring advantageous improvements in methods previously based on polyclonal sera. To our knowledge, the Seward monoclonal anti-Gm(3) is the first commercially available anti-Gm specificity, but it is foreseeable that in the near future, other anti-immunoglobulin allotypic specificities will be produced. 37
The double-sandwich test described here is much more sensitive than the single sandwich previously developed [2]. Several explanations justify this observation. Because of the y-chain specificity of the coated antibody, IgGs are the only proteins of the sample to be attached to the solid phase, and especially IgGl that constitutes the major component of IgGs. Furthermore, it is probable that IgG molecules captured by means of a polyclonal antibody are more accessible to the monoclonal. Indeed, with anti-IgG coated antibodies acting as a spacer group, IgG molecules are more randomly distributed on the well surface, with an amplification effect as a consequence. This work also confirms the advantages of ELISA tests. The optical densities of an ELISA test are measured by the use of a spectrophotometer. This constitutes an important advantage over manual haemagglutination inhibition, the interpretation of which is much more subjective. The threshold of sensitivity is much greater than that observed with polyclonal anti-Gm(3) or even with the same monoclonal used in haemagglutination inhibition test. In addition, the measurement of IgG in the same bloodstain extract in order to make sure that IgG molecules are present in sufficient amounts, allows identification of Gm(3) negative phenotypes without any risk of error. The practical consequence of these improvements is that small blood or human fluid samples can be typed. In forensic work, this point is sometimes crucial. The indirect or double layer ELISA method makes use of only small amounts of expensive reagents which can be much diluted. Finally, 96-well immunoplates are better suited for the handling of large series of analyses. Acknowledgements We thank Mrs C Opret-Meunier and Mrs MR Dessaintes-Mathy for skilful technida~assistance.
References 1. Gmbb R. The genetic markers of human immunoglobulins. Berlin, Heidelberg, New York: Springer-Verlag, 1970. 2. Fletcher SM, Dorrill MJ and Dolton P. Bloodstain allotyping: an ELISA method for Glm(3). Report of the 10th International Congress of the Society for Forensic Haematogenetics 1983; 221-226. 3. Newall P. Advantages of monoclonal antiserum and ELISA in the identification of bloodstains. Report of the 10th International Congress of the Society for Forensic Haematogenetics 1983; 227-232. 4. Hoste B, Brocteur J and Andrt A. Indefinite storage of dried Gm and Km antigens (Examination of bloodstains 33 years old). Forensic Science 1978; 11: 109-113.