lramunochemistry,1976,Vol. 13, pp. 963-966. PergamonPress. Printedin Great Britain
LASER LIGHT SCATTERING SPECTROSCOPIC IMMUNOASSAY IN THE AGGLUTINATION-INHIBITION M O D E FOR H U M A N CHORIONIC G O N A D O T R O P I N (hCG) A N D H U M A N LUTEINIZING H O R M O N E (hLH) G U S T A V K. V O N S C H U L T H E S S , 1 R I C H A R D J. C O H E N 1'2 a n d G E O R G E B. B E N E D E K 1 XDepartment of Physics, Center for Materials Science and Engineering, Massachusetts Institute of Technology; and 2Harvard-M.I.T. Program for Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, U.S.A. (First received 5 April 1976; in revised form 28 June 1976) Al~tract--A laser light scattering immunoassay was used to measure concentrations of human chorionic gonadotropin (hCG) and human luteinizing hormone (hLH) in urine. In this assay quasi-elastic light scattering spectroscopy is used to detect the inhibiting effect of hCG and hLH on an hCG-hLH co-specific rabbit antibody, which acts to agglutinate hCG coated latex spheres. The results obtained with this assay are quantitatively accurate and the sensitivity of the assay is found to be about 0.015 i.u. (U.S.P. standard)/ml of hCG or hLH equivalent. This sensitivity permitted the measurement of the pituitary hLH stimulus for ovulation in the menstrual cycle. The agglutination-inhibition mode permits one to assay for a wide variety of molecules including haptenes.
INTRODUCTION
The purpose of this c o m m u n i c a t i o n is to demonstrate that the laser light scattering i m m u n o a s s a y described in the preceding paper (von Schulthess et al., 1976) a n d proposed initially by C o h e n a n d Benedek (1975) m a y be successfully used in the inhibition mode. In this mode antigen is assayed for by measuring its blocking effect on antibodies which act as agglutinator for latex particles coated with antigen. The agglutination-inhibition mode has definite advantages over the direct m o d e (von Schulthess et al., 1976) in that it eliminates the requirement of large antigen size a n d the need for more t h a n one haptenic group per antigen. We have examined the applicability of the assay in the agglutination-inhibition m o d e by assaying for h u m a n chorionic g o n a d o t r o p i n (hCG) a n d h u m a n luteinizing h o r m o n e (hLH). This assay has potentially i m p o r t a n t clinical applications for the early a n d rapid detection of n o r m a l a n d ectopic pregnancies, and the detection of the pituitary stimulus for ovulation in the menstrual cycle. MATERIALS AND EXPERIMENTAL PROCEDURES
An agglutination-inhibition assay requires antigen coated carrier particles, antiserum and the inhibiting antigen being tested. The latex particle stock solution covalently* coated with hCG was obtained through the courtesy of Dr, H. Hager (Hoffmann-La Roche Company, Nutley, N J) and is commercially available as part of a pregnancy test kit (under the trademark names "Percentex" and "Pregnosis"). In order to obtain a latex particle concentration of about 5 #g/ml, which we previously have found suitable (von Schulthess et al., 1976), it was necessary to dilute the latex sphere stock solution by a factor of 400. Two hCG-hLH cospecific rabbit antisera were obtained through the generosity of Dr. H. Hager and Dr. * U.S. patent No. 3,857,931. 963
T. Kosasa (Peter Bent Brigham Hospital, Boston). To calibrate the assay a U.S.P. standard containing 1 i.u. of hCG/ml dissolved in normal male urine was used. Normal male urine was also used as a control and as a diluent in all experiments. Test samples consisted of urine samples from a pregnant and a normally menstruating woman. Before use, the urine samples and the diluent were processed in the following manner: the pH was adjusted to 8.2, the urine was spun for 5 min at 10,000 rev/min and filtered through a 0.2 #m Nuclepore filter. In order to conduct the assay in the inhibition mode we must determine the antiserum dilution which agglutinates the spheres so as to reduce the mean diffusion constant D by a factor between 2 and 3. For a discussion of the quasi-elastic light scattering technique for measuring D see von Schulthess et al. (1976) and the references listed there. Assuming that the latex spheres are present in monomeric form prior to the addition of antibody, a factor of 2 or 3 in diffusion coefficient or diameter implies the formation of polymers consisting of 23 or 33 latex spheres after addition of antibody. It was found that by diluting the originally provided rabbit antisera by a factor between 15,000 and 20,000 such aggregates were obtained. In addition, a kinetic experiment showed that the agglutination reaction requires 3-4 hr to attain equilibrium. Determination of hCG and hLH concentrations were performed as follows. Serial dilutions (successive dilution factor of two) of the 1 i.u./ml hCG standard or of the test samples, containing either hCG or hLH, were prepared to serve as the inhibiting antigen in the assay. Aliquots (100 #1) of these serial dilutions were incubated with 10 pl aliquots of equally diluted antiserum for 40 rain at room temperature. During this phase the antigen in the sample would block some antibody thus preventing it from taking part in the agglutination of the hCG coated latex spheres. In this agglutination step 10#l aliquots of diluted latex stock suspension were added so that the final reaction mixtures contained a 1:400 dilution of the latex stock suspension and a 1 : 15,000 dilution of antiserum. After incubation at room temperature for 4 hr the average diffusion constant of each of the samples was determined and plotted as a function of the dilution factor.
964
G.K. VON SCHULTHESS, R. J. COHEN and G. B. BENEDEK
.15C
o
*/ t~
'r
/
t00
/
o
¢, /
e
~
~
STANDARD
---
PREGNANT WOMAN (t27 DAYS AFTER LMP)
o
.050
I DILUTION
NUMBER
Fig. 1. hCG assay in urine samples. The mean diffusion constant D is plotted vs the serial dilution number. The standard denoted by the solid line consists of male urine to which has been added 1 i.u. hCG/ml. The dashed line represents the results obtained from a urine sample of a pregnant women (117 days after last menstrual period). From the shift between the unknown sample and the standard curve the hCG concentration can be quantitated, and is found to be 22.5 i.u. (U.S.P. standard)/ml of hCG.
RESULTS
Figure 1 shows a plot of the average diffusion constant /~ of the diluted samples as a function of n, the serial dilution number: the dilution factor f is related to n by f = 2-". At low hCG or hLH concentrations the aggregation process occurs and gives a value of/~ equal to that of the uninhibited control. With increasing antigen concentration an increasing number of antibodies will not be able to cross-link latex spheres because either one or both of the antibody binding sites will already be occupied by antigen. Eventually, at very high antigen concentrations the agglutination of the spheres is completely inhibited and one measures a D characteristic of a, latex suspension to which neither inhibiting antigen nor antiserum has been added. In Fig. 1 we represent as a solid line the agglutination-inhibition curve for the standard sample. The smallest amount of hCG required to produce a detectable inhibition of the agglutination occurs at n = 6 which corresponds to 0.0t6 i.u./ml (U.S.P. standard) of hCG. We also show as a dashed line in Fig. 1 the agglutination-inhibition curve of an unknown urine sample taken from a pregnant woman (117 days after her last menstrual period). The shift n between the unknown and standard curves is An = 4.5. We therefore calculate that the concentration of hCG in this unknown sample is 2a~ x I i.u./ml = 22.5 i.u./ml. The error +__ein the measurement of An produces a relative uncertainty (rc/c) in concentration given by (6c/c) = e In 2. In our experiments e = 0.25 and therefore (6c/c) ~ 17%, regardless of the value of c. In a second experiment we used the assay to measure hLH levels in the morning urine of a normally menstruating woman (average cycle 32 days). The urine was collected over a period of 9 days starting at the 12th day after onset of menstruation. The
hLH levels were measured using the shift of the agglutination-inhibition curves with respect to the standard in the same way as described in the preceding paragraph. Since in this case the antigen concentrations are much lower than 1 i.u./ml hCG equivalent one finds agglutination-inhibition curves shifted to the right of the standard. In our samples the hLH concentrations were so small, that some of them could produce noticeable inhibition of the agglutination only in almost undiluted form, or not all. The deduced hLH concentrations are shown in Fig. 2. During the days 15-19 of the cycle the characteristic hLH peak associated with ovulation is demonstrated. In samples taken before and after days 15-19 the concentration of hLH was insufficient to produce any detectable inhibition of the agglutination.
DISCUSSION
The data presented in this communication and the preceding paper (von Schulthess et al., 1976) show that the laser light scattering immunoassay permits a quantitative determination of very small antigen concentrations in sample volumes down to 1 #1. Using a conversion factor of 1 mg hCG = 20,000 i.u. hCG (van Hell, 1972) we calculate that the sensitivity of this assay is about 1 ng/ml of hCG. The sensitivity (0.016 i.u. hCG/ml) of the laser light scattering immunoassay for hCG is about 50 times greater than the sensitivity (1-2 i.u. hCG/ml) of the commercially available slide agglutination tests (e.g. "Percentex" and "Pregnosis" by Roche, Nutley, N J; "Gravindex" by Ortho Diagnostics, Raritan, NJ; "Dap-Test" by Princeton Laboratories, Princeton, N J). In fact, the sensitivity of the laser light scattering immunoassay for hCG is similar to the value of 0.008 i.u. hCG/ml obtained using a RIA as reported
Laser Light Scattering Spectroscopic Immunoassay
80
965
I l l !
g I I I
hCG
lO'aI.U. 50 (U.S.P.) equivalent
I I I I
/o I
20
f,' --o- -
o-
--~-
I
I
1
12
13
14
°'-~'o
-
- ¢ Sensitivity
Level
/ I
I
I
I
I
I
15 16 17 18 19 20 DAY AFTER LMP
Fig. 2. hLH levels in the urine of a normally menstruating woman. The procedure used to determine the concentrations of the hLH in the sample is the same as in Fig. 1.
by Kosasa et al. (1974). However, these authors used a hCG fl-subunit specific antibody. In the case of hLH, Schalch et al. (1968) report a sensitivity of 0.2 ng hLH/ml in their radioimmunoassay for hLH. This value is again of a similar order of magnitude as the sensitivity of 1 ng/ml, which we find in our assay, if one can assume that the co-specific antibody we used binds hLH equally well as hCG. As a confirmation of the quantitative validity of the inhibition mode of the LIA we observe that the peak of the curve in Fig. 2 occurs at approx 0.08 i.u. hCG equivalent/ml (U.S:P. standard). This value is in good agreement with the value obtained by RIA (e.g. Kosasa et al., 1974; Schalch et al., 1968). The agglutination inhibition mode has significant practical advantages over the direct assay mode: (1) In the direct assay mode the antigen serves to cross-link antibody coated particle~. To do this the antigen must be of sufficient size (/> 100A) and have at least two accessible haptenic sites. In the inhibition mode, the antigen only serves to inactivate the antibody which acts as the agglutinator of the antigencoated particles. For the antigen to do this it need not satisfy any size or valency requirements. In fact, preliminary experiments show that morphine can be detected in this inhibition mode using the laser light scattering immunoassay. The sensitivity for the detection of the haptene morphine was about 1 ng/ml. (2) In the inhibition mode the degree of agglutination is a monotonically decreasing function of the inhibiting antigen concentration. Thus in the inhibition mode, unlike the direct mode (von Schulthess et al., 1976) there is no ambiguity as to whether the antigen concentration in the unknown samples is either too high or too low to be measured. (3) In the inhibition mode for the detection of antigens the carrier particle is coated with antigen. Frequently the antigen has a greater surface charge density than the corresponding antibody, in this case antigen-coated carrier particles are rendered more stable against non-specific agglutination (see von Schulthess et al., 1976). To apply this assay to measurements of hLH and hCG in serum samples, it is necessary to eliminate non-specific effects, due to the gamma globulin
moiety present in serum, which suppresses the agglutination (Bell, 1969). This non-specific effect may be overcome by pretreatment of the sample serum to remove gamma globulins (Hager, 1976). Otherwise, one might suppress the non-specific effects by using carrier particles less attractive to the serum proteins and the gamma globulins in particular. In using this assay as a method of detecting ectopic or early normal pregnancies, it may be advantageous to work with an hCG specific antibody. Such antibodies have been successfully prepared and are in fact in clinical use (Kosasa et al., 1974). In conclusion, the laser light scattering immunoassay run in the inhibition mode described here seems well suited for research and rapid multisample clinical use. The assay has a sensitivity comparable with the radioimmunoassay, does not require the use of any radioactive reagents, and can be performed in a few hours time. Moreover, no separation of bound from unbound antigen is required. Acknowledgements--The authors wish to thank Dr. H. Hager for providing the hCG coated latex spheres and the co-specific rabbit antiserum against hCG-hLH and appreciate the many useful discussions with him. The authors also acknowledge helpful discussions with Prof. H. Eisen. Furthermore we would like to thank Dr. T. Kosasa for providing one of the hCG-hLH cospecific rabbit antibodies and two anonymous donors for providing theirurine samples. Gustav von Schulthess is grateful for financial support from an M.I.T. Health Sciences Fund Fellowship and from the Steo Foundation of Zurich, Swit: zerland. Richard Cohen would like to express thanks for financial support from an Insurance Medical Scientist Scholarship, sponsored by the Massachusetts Mutual Life Insurance Co. This investigation was supported in part by Public Health Service Grant 5P01 HL14322-04 from the National Heart and Lung Institute (U.S.A.) to the Harvard-M.I.T. Program in Health Sciences and Technology and in part by National Science Foundation (U.S.A.) Grant DMR 72-03027A05 to the Center for Materials Science and Engineering, M.I.T.
REFERENCES
Bell J. L. (1969) J. clin. Path. 22, 79.
966
G.K. VON SCHULTHESS, R. J. COHEN and G. B. BENEDEK
Cohen R. J. & Benedek G. B. (1975) Immunochemistry 12, 349. Hager H. (1976) private communication. Hell H. (1972) Human urinary gonadotropins, purification and characterization. Ph.D. Thesis, University of Groningen, The Netherlands.
Kosasa T. S., Levesque L. A., Goldstein D. P. & Taymor M. L. (1974) Am. J. Obstet. "Gynecol. 119, 784. Schalch D. S., Parlow A. F., Boon R. C. & Reichlin S. (1968) J. clin. Invest. 47, 665. von Schulthess G. K., Cohen R. J., Sakato N. & Benedek G. B. (1976) Immunochemistry 13, 955.
LASER LIGHT SCATTERING SPECTROSCOPIC IMMUNOASSAY IN THE AGGLUTINATION-INHIBITION M O D E FOR H U M A N CHORIONIC G O N A D O T R O P I N (hCG) A N D H U M A N LUTEINIZING H O R M O N E (hLH) G U S T A V K. V O N S C H U L T H E S S , 1 R I C H A R D J. C O H E N 1'2 a n d G E O R G E B. B E N E D E K 1 XDepartment of Physics, Center for Materials Science and Engineering, Massachusetts Institute of Technology; and 2Harvard-M.I.T. Program for Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, U.S.A. (First received 5 April 1976; in revised form 28 June 1976) Al~tract--A laser light scattering immunoassay was used to measure concentrations of human chorionic gonadotropin (hCG) and human luteinizing hormone (hLH) in urine. In this assay quasi-elastic light scattering spectroscopy is used to detect the inhibiting effect of hCG and hLH on an hCG-hLH co-specific rabbit antibody, which acts to agglutinate hCG coated latex spheres. The results obtained with this assay are quantitatively accurate and the sensitivity of the assay is found to be about 0.015 i.u. (U.S.P. standard)/ml of hCG or hLH equivalent. This sensitivity permitted the measurement of the pituitary hLH stimulus for ovulation in the menstrual cycle. The agglutination-inhibition mode permits one to assay for a wide variety of molecules including haptenes.
INTRODUCTION
The purpose of this c o m m u n i c a t i o n is to demonstrate that the laser light scattering i m m u n o a s s a y described in the preceding paper (von Schulthess et al., 1976) a n d proposed initially by C o h e n a n d Benedek (1975) m a y be successfully used in the inhibition mode. In this mode antigen is assayed for by measuring its blocking effect on antibodies which act as agglutinator for latex particles coated with antigen. The agglutination-inhibition mode has definite advantages over the direct m o d e (von Schulthess et al., 1976) in that it eliminates the requirement of large antigen size a n d the need for more t h a n one haptenic group per antigen. We have examined the applicability of the assay in the agglutination-inhibition m o d e by assaying for h u m a n chorionic g o n a d o t r o p i n (hCG) a n d h u m a n luteinizing h o r m o n e (hLH). This assay has potentially i m p o r t a n t clinical applications for the early a n d rapid detection of n o r m a l a n d ectopic pregnancies, and the detection of the pituitary stimulus for ovulation in the menstrual cycle. MATERIALS AND EXPERIMENTAL PROCEDURES
An agglutination-inhibition assay requires antigen coated carrier particles, antiserum and the inhibiting antigen being tested. The latex particle stock solution covalently* coated with hCG was obtained through the courtesy of Dr, H. Hager (Hoffmann-La Roche Company, Nutley, N J) and is commercially available as part of a pregnancy test kit (under the trademark names "Percentex" and "Pregnosis"). In order to obtain a latex particle concentration of about 5 #g/ml, which we previously have found suitable (von Schulthess et al., 1976), it was necessary to dilute the latex sphere stock solution by a factor of 400. Two hCG-hLH cospecific rabbit antisera were obtained through the generosity of Dr. H. Hager and Dr. * U.S. patent No. 3,857,931. 963
T. Kosasa (Peter Bent Brigham Hospital, Boston). To calibrate the assay a U.S.P. standard containing 1 i.u. of hCG/ml dissolved in normal male urine was used. Normal male urine was also used as a control and as a diluent in all experiments. Test samples consisted of urine samples from a pregnant and a normally menstruating woman. Before use, the urine samples and the diluent were processed in the following manner: the pH was adjusted to 8.2, the urine was spun for 5 min at 10,000 rev/min and filtered through a 0.2 #m Nuclepore filter. In order to conduct the assay in the inhibition mode we must determine the antiserum dilution which agglutinates the spheres so as to reduce the mean diffusion constant D by a factor between 2 and 3. For a discussion of the quasi-elastic light scattering technique for measuring D see von Schulthess et al. (1976) and the references listed there. Assuming that the latex spheres are present in monomeric form prior to the addition of antibody, a factor of 2 or 3 in diffusion coefficient or diameter implies the formation of polymers consisting of 23 or 33 latex spheres after addition of antibody. It was found that by diluting the originally provided rabbit antisera by a factor between 15,000 and 20,000 such aggregates were obtained. In addition, a kinetic experiment showed that the agglutination reaction requires 3-4 hr to attain equilibrium. Determination of hCG and hLH concentrations were performed as follows. Serial dilutions (successive dilution factor of two) of the 1 i.u./ml hCG standard or of the test samples, containing either hCG or hLH, were prepared to serve as the inhibiting antigen in the assay. Aliquots (100 #1) of these serial dilutions were incubated with 10 pl aliquots of equally diluted antiserum for 40 rain at room temperature. During this phase the antigen in the sample would block some antibody thus preventing it from taking part in the agglutination of the hCG coated latex spheres. In this agglutination step 10#l aliquots of diluted latex stock suspension were added so that the final reaction mixtures contained a 1:400 dilution of the latex stock suspension and a 1 : 15,000 dilution of antiserum. After incubation at room temperature for 4 hr the average diffusion constant of each of the samples was determined and plotted as a function of the dilution factor.
964
G.K. VON SCHULTHESS, R. J. COHEN and G. B. BENEDEK
.15C
o
*/ t~
'r
/
t00
/
o
¢, /
e
~
~
STANDARD
---
PREGNANT WOMAN (t27 DAYS AFTER LMP)
o
.050
I DILUTION
NUMBER
Fig. 1. hCG assay in urine samples. The mean diffusion constant D is plotted vs the serial dilution number. The standard denoted by the solid line consists of male urine to which has been added 1 i.u. hCG/ml. The dashed line represents the results obtained from a urine sample of a pregnant women (117 days after last menstrual period). From the shift between the unknown sample and the standard curve the hCG concentration can be quantitated, and is found to be 22.5 i.u. (U.S.P. standard)/ml of hCG.
RESULTS
Figure 1 shows a plot of the average diffusion constant /~ of the diluted samples as a function of n, the serial dilution number: the dilution factor f is related to n by f = 2-". At low hCG or hLH concentrations the aggregation process occurs and gives a value of/~ equal to that of the uninhibited control. With increasing antigen concentration an increasing number of antibodies will not be able to cross-link latex spheres because either one or both of the antibody binding sites will already be occupied by antigen. Eventually, at very high antigen concentrations the agglutination of the spheres is completely inhibited and one measures a D characteristic of a, latex suspension to which neither inhibiting antigen nor antiserum has been added. In Fig. 1 we represent as a solid line the agglutination-inhibition curve for the standard sample. The smallest amount of hCG required to produce a detectable inhibition of the agglutination occurs at n = 6 which corresponds to 0.0t6 i.u./ml (U.S.P. standard) of hCG. We also show as a dashed line in Fig. 1 the agglutination-inhibition curve of an unknown urine sample taken from a pregnant woman (117 days after her last menstrual period). The shift n between the unknown and standard curves is An = 4.5. We therefore calculate that the concentration of hCG in this unknown sample is 2a~ x I i.u./ml = 22.5 i.u./ml. The error +__ein the measurement of An produces a relative uncertainty (rc/c) in concentration given by (6c/c) = e In 2. In our experiments e = 0.25 and therefore (6c/c) ~ 17%, regardless of the value of c. In a second experiment we used the assay to measure hLH levels in the morning urine of a normally menstruating woman (average cycle 32 days). The urine was collected over a period of 9 days starting at the 12th day after onset of menstruation. The
hLH levels were measured using the shift of the agglutination-inhibition curves with respect to the standard in the same way as described in the preceding paragraph. Since in this case the antigen concentrations are much lower than 1 i.u./ml hCG equivalent one finds agglutination-inhibition curves shifted to the right of the standard. In our samples the hLH concentrations were so small, that some of them could produce noticeable inhibition of the agglutination only in almost undiluted form, or not all. The deduced hLH concentrations are shown in Fig. 2. During the days 15-19 of the cycle the characteristic hLH peak associated with ovulation is demonstrated. In samples taken before and after days 15-19 the concentration of hLH was insufficient to produce any detectable inhibition of the agglutination.
DISCUSSION
The data presented in this communication and the preceding paper (von Schulthess et al., 1976) show that the laser light scattering immunoassay permits a quantitative determination of very small antigen concentrations in sample volumes down to 1 #1. Using a conversion factor of 1 mg hCG = 20,000 i.u. hCG (van Hell, 1972) we calculate that the sensitivity of this assay is about 1 ng/ml of hCG. The sensitivity (0.016 i.u. hCG/ml) of the laser light scattering immunoassay for hCG is about 50 times greater than the sensitivity (1-2 i.u. hCG/ml) of the commercially available slide agglutination tests (e.g. "Percentex" and "Pregnosis" by Roche, Nutley, N J; "Gravindex" by Ortho Diagnostics, Raritan, NJ; "Dap-Test" by Princeton Laboratories, Princeton, N J). In fact, the sensitivity of the laser light scattering immunoassay for hCG is similar to the value of 0.008 i.u. hCG/ml obtained using a RIA as reported
Laser Light Scattering Spectroscopic Immunoassay
80
965
I l l !
g I I I
hCG
lO'aI.U. 50 (U.S.P.) equivalent
I I I I
/o I
20
f,' --o- -
o-
--~-
I
I
1
12
13
14
°'-~'o
-
- ¢ Sensitivity
Level
/ I
I
I
I
I
I
15 16 17 18 19 20 DAY AFTER LMP
Fig. 2. hLH levels in the urine of a normally menstruating woman. The procedure used to determine the concentrations of the hLH in the sample is the same as in Fig. 1.
by Kosasa et al. (1974). However, these authors used a hCG fl-subunit specific antibody. In the case of hLH, Schalch et al. (1968) report a sensitivity of 0.2 ng hLH/ml in their radioimmunoassay for hLH. This value is again of a similar order of magnitude as the sensitivity of 1 ng/ml, which we find in our assay, if one can assume that the co-specific antibody we used binds hLH equally well as hCG. As a confirmation of the quantitative validity of the inhibition mode of the LIA we observe that the peak of the curve in Fig. 2 occurs at approx 0.08 i.u. hCG equivalent/ml (U.S:P. standard). This value is in good agreement with the value obtained by RIA (e.g. Kosasa et al., 1974; Schalch et al., 1968). The agglutination inhibition mode has significant practical advantages over the direct assay mode: (1) In the direct assay mode the antigen serves to cross-link antibody coated particle~. To do this the antigen must be of sufficient size (/> 100A) and have at least two accessible haptenic sites. In the inhibition mode, the antigen only serves to inactivate the antibody which acts as the agglutinator of the antigencoated particles. For the antigen to do this it need not satisfy any size or valency requirements. In fact, preliminary experiments show that morphine can be detected in this inhibition mode using the laser light scattering immunoassay. The sensitivity for the detection of the haptene morphine was about 1 ng/ml. (2) In the inhibition mode the degree of agglutination is a monotonically decreasing function of the inhibiting antigen concentration. Thus in the inhibition mode, unlike the direct mode (von Schulthess et al., 1976) there is no ambiguity as to whether the antigen concentration in the unknown samples is either too high or too low to be measured. (3) In the inhibition mode for the detection of antigens the carrier particle is coated with antigen. Frequently the antigen has a greater surface charge density than the corresponding antibody, in this case antigen-coated carrier particles are rendered more stable against non-specific agglutination (see von Schulthess et al., 1976). To apply this assay to measurements of hLH and hCG in serum samples, it is necessary to eliminate non-specific effects, due to the gamma globulin
moiety present in serum, which suppresses the agglutination (Bell, 1969). This non-specific effect may be overcome by pretreatment of the sample serum to remove gamma globulins (Hager, 1976). Otherwise, one might suppress the non-specific effects by using carrier particles less attractive to the serum proteins and the gamma globulins in particular. In using this assay as a method of detecting ectopic or early normal pregnancies, it may be advantageous to work with an hCG specific antibody. Such antibodies have been successfully prepared and are in fact in clinical use (Kosasa et al., 1974). In conclusion, the laser light scattering immunoassay run in the inhibition mode described here seems well suited for research and rapid multisample clinical use. The assay has a sensitivity comparable with the radioimmunoassay, does not require the use of any radioactive reagents, and can be performed in a few hours time. Moreover, no separation of bound from unbound antigen is required. Acknowledgements--The authors wish to thank Dr. H. Hager for providing the hCG coated latex spheres and the co-specific rabbit antiserum against hCG-hLH and appreciate the many useful discussions with him. The authors also acknowledge helpful discussions with Prof. H. Eisen. Furthermore we would like to thank Dr. T. Kosasa for providing one of the hCG-hLH cospecific rabbit antibodies and two anonymous donors for providing theirurine samples. Gustav von Schulthess is grateful for financial support from an M.I.T. Health Sciences Fund Fellowship and from the Steo Foundation of Zurich, Swit: zerland. Richard Cohen would like to express thanks for financial support from an Insurance Medical Scientist Scholarship, sponsored by the Massachusetts Mutual Life Insurance Co. This investigation was supported in part by Public Health Service Grant 5P01 HL14322-04 from the National Heart and Lung Institute (U.S.A.) to the Harvard-M.I.T. Program in Health Sciences and Technology and in part by National Science Foundation (U.S.A.) Grant DMR 72-03027A05 to the Center for Materials Science and Engineering, M.I.T.
REFERENCES
Bell J. L. (1969) J. clin. Path. 22, 79.
966
G.K. VON SCHULTHESS, R. J. COHEN and G. B. BENEDEK
Cohen R. J. & Benedek G. B. (1975) Immunochemistry 12, 349. Hager H. (1976) private communication. Hell H. (1972) Human urinary gonadotropins, purification and characterization. Ph.D. Thesis, University of Groningen, The Netherlands.
Kosasa T. S., Levesque L. A., Goldstein D. P. & Taymor M. L. (1974) Am. J. Obstet. "Gynecol. 119, 784. Schalch D. S., Parlow A. F., Boon R. C. & Reichlin S. (1968) J. clin. Invest. 47, 665. von Schulthess G. K., Cohen R. J., Sakato N. & Benedek G. B. (1976) Immunochemistry 13, 955.
