- Email: [email protected]
Use of monkey muscle acetylcholine receptors for the measurement of antibody titers in myasthenia gravis
1982 CSCC ABSTRACTS coefficient of variation (CV) of 8.9% for a serum concentration of 3.0 umol/L, ahd between-baLch CV of 11.5% at 3.2 umol/L and of 10.6% at 8.4 1~mol/L. A preliminary study of 28 patients with no adrenal pathology, between 1 week and 19 years of age, gave serum DHAS results similar to those reported by Smith, M.R. et al. (Clin. Chim. Acta 65, 5-13, 1975). Further work is ongoing, and reference values will be presented for males and females in various age groups over the pediatric range.
44
u s e OF M O N K E Y M U S C L E A C E T Y L C H O L I ~ I E R E C E P T O R S F O R T H E M E A S U R E M E N T OF A N T I B O D Y T I T E R S IN M Y A S T H E N I A GRAVIS
~ L - ~ U t Hickle, D.A.G., Humohrey, J.G., Srifflth,K. and Mee, A.V. D e o a r t m e n t of C l i n i c a l B i o c h e m i s t r y , T o r o n t o G e n e r a l H o s o i t a l , T o r o n t o , O n t a r i o , MbS IL7. A s s a y of h u m a n s k e l e t a l muscle aeetyleholine r e c e p t o r a n t i b o d i e s h o l d s n r o m i s e as a d i a R n o s t i c t e s t fo~ m y a s t h e n l a g r a v i s , a n d as a l a b o r a t o r y m e a s u r e of t h e ~ e s D o n s e to t h e r a p y . This method involves a d o u b l e immunoprecipitation a s s a y . The b ~ s i c o r [ n c i n l e is s u m m a r i z e d by the f o l l o w i n g e q u a t i o n s : I,
AChR
+ 1251 ~ BGT
2.
125I ~ 8 G T A C h P
3.
1251 ~ B G T A C h R - A b
) 1251~BGTAChP
(excess) + Ab 2
+ Ab
~ 1251~80TAChR-Ab
)1251~ BGTAChP-Ab-Ab21
W h e r e A C h R = a e e t y l c h o l i n e r e c e p t o r : 125 I ~ B G T = 1 - 1 2 5 l a b e l l e d alphabunBarotoxin. Ab = A n t i b o d i e s to A C h R p r e s e n c e in h u m a n s e r u m . Ab2 = S h e e p - a n t i - h u m a n IgG. The a s s a y r e q u i r e s p r e p a r a t i o n o f A C h R , c o u n l i n K r e c e p t o r s to 125I ~ B G T , bindinK with natienr se~um a n t i b o d i e s , a n d p r e c i p i t a t i o n by a n t ~ - h u m a n IgG a n t i bodies. The tire? o f a n t i b o d i e s is d i r e c t l y p r o p o r t i o n a l to the a c t i v i t y o f p ~ e c i o ~ t a t e (I~5 I ~ 8~T AChR-Ab-Ab2). Detailed methodology, normal ~anKe clinical s e n s i t i v i t y and s p e c i f i c i t y and s o m e c l i n i c a l r e s u l t s w i l l be p r e s e n t e d .
45 STABILITY OF IN-HOUSE REFERENCE ~ T E R I A L S IN ENZYME ASSAYS IN ROUTINE CLINICAL CHF~IISTRY. Chau,A.K.-K.,and Hoore~R.W.,Biochemlstry Oepartment,Sunnybrook Medical Centre,2075 Bayview Avenue,Toronto,Ontario H4N 3 ~ . The intention of this study was to devise an in,house reference material for use with the enzyme assays routinely available in Clinical Chemistry laboratories. The stability of enzyme activity in a normal human serum pool,abnormal human serum pools and a contrived animal enzyme mixture,with and without stabilizers,at different storage temperatures,was assessed over a period of six months. The enzyme activities followed were those of AST,ALT,ALP,CGT,LD and CK. Storage temperatures were 40 and -200. The contrived enzyme pool was the mixture suggested by Gruber and others in J.Clin. Chem. Clin.giochem. I_~5,565,575and 579 (1977). The only pools in which all the enzyme activities were statistically stable were,for the normal pooi,20% dimethylsulphoxide at -200 and 15% ethylene glycol at -20C,for the contrived pool, 20% dimethylsulphoxide at 40 and -200 and ethylene glycol at -200. None of the pools having high enzyme activity were stable. Similar lack of stability has been noted by ourselves and others in commercially available lyophilized and in liquid serum pools,particularly in those having high enzyme activity. Should high enzyme activity in the reference material be needed,the most promising source,from the evidence of this study,is the contrived enzyme pool in =he presence of dimethyl sulphoxide.
46
EVALUATION OF A SERUM ESTRADIOL KIT AND ITS APPLICATION TO THE MONITORING OF OVULATION INDUCTION BY FERTILITY DRUGS. Greenway, D.C. , Mak-Fan, L. and Turkington, V.E. Dept. of Lab Medicine, Ottawa General Hosi,itaL Ottawa, Ontario. KIH 8L6 We evaluated a radioiI~nunoassay kit for serLLm estradJo| (Nordiclab International, P.O. Box 1356, Huntington, P.Q.) with the intention of replacing 24 hr. urinary estrogen deterlBinations for the daily monitoring of infertile womenundergoing ovulation induction by hLunan menopausal gonadotropins (h~, Pergonal). The kit features serum-based standards rangina from 0.055-5.51 nmol/L, an 1251-tracer, short incubation time, and a polyethylene glycol (PEG) separation technique. Small voluraes (200 el) of st&ndaxds, controls and patients' sara are extracted with 1.5 ml diethyl ether, and a 1.0 ml aliquot of this extract is evaporated to dryness. The dried extract is resuspended in tracer-antibody solution and incubated at 37°C for 1 hour. PEG is added, and following centrifugation, the radioactivity in the bound fraction is counted. Total turnaround time is 3-4 hr. The intra-assay precision was 6.4~ 8.1
97 / P9
and 7.2% at levels of 0.233, 1.13 and 3.45 nmol/L respectively. Between-run precision was 11.6 and 9.8% at 0.244 and 2.21 nmol/L respectively. Mean recoveries from serum pools spiked with 0.367, 1.84 and 3.67 nmol/L estradiol were 101.9, IO2.1 and i06.2%. Linearity was observed when volumes of sara ranging from 50 to 700 ul were extracted and assayed. Sensitivity was found to be 0.050 nmol/L. We have found the Mordiclab method especially suitable for the monitoring of ovulation induction by hMG. Eleven women have been monitored so far over 16 treatment cycles, and three pregnancies have ensued. The availability of results within 4 hr. has provided the gynecologist with a more current indication of follicular development and has allowed for a better adjustment of hMG doses than was provided previously by 24 hr urlnary estrogen assays.
q7 MODEL SYSTEMFOR NEPHELOMETRIC ASSAY OF Clq PRECIPITABLE IMMUNECOMPLEXES (IC). Levinson, S.S., and Goldman J., Department of Laboratory Medicine, Sinai Hospital, Detroit, MI 48235. IC are involved in the pathogenesis of many rheumatic disseases; removal of IC by plasma exchange may improve prognosis (Cardella, C.I. (1979) J. Rheumatology 6, 606). Nephelometric assay would simplify IC measurement by eliminating the need f o r highly purified radioiodinated Clq and other components commonly used now that l i m i t performance to only a few centers. Heat aggregated gamma globu]in (IIAGG) has been shown to exh i b i t many properties that are indistinguishable from endogenous IC (Marder, R.J., st. al. (1978) J. Inmlunology 12__~_I, 613). In a double beam sPectrophotometer, the reaction of HAGGwith Clq occurs rapidly and reaches equilibrium within lO minutes. Maximum reaction occurs between sodium concentrations of 150-160 ,~.I/L in the reaction mixture regardless of whether the sodium is in the form of chloride, phosphate or EDTA. Standard curves generated from HAGGshow a high degree of l i n e a r i t y between 12-750 ug/ml. The a c t i v i t y of the reaction plateaus between Clq concentrations of 50-lOO ug/ml, simplifying c r i t e r i a needed to ensure good reproducibility between different lots. Purified or more crude preparations of Clq can be used interchangeably. HAGG is added to serum and extracted with 2.5% polyethylene glycol. Absorbance of the resuspended precipitate is s u f f i ciently high so that HAGGcan be assayed down to 12 ug/m! We assayed sera of several patients known to have high levels of [C (positive) and normal pooled sera. All cases of positive sera exhibited absorbances s u f f i c i e n t l y high to differentiate them from the pooled sera with lower absorbances. Simple recovery of the precipitate from the reaction mixture a f t e r nephelometric assay may lend i t s e l f to investigation of the Ig composition by EIA. Such composition may be related to d i f f e r int features of the disease (Levinski, J.R., and Barratt, M.T. 1979) Lancet 24, 1100).
48
CENTRIFUGAL ASSAY FOR DETERMINING HEMOGLOBIN LEVELS IN GASTRIC JUICE. Stryd~R.P., Gilbertson,T.J., Reele,S.B. and Gumblston,T.J., Experimental Medleal and Clinical Laboratory Research and Bronson Clinical lnvestlgational Unit, The Upjohn Company, Kalamazoo, Michigan 49001 A method for the analysis of hemoglobin levels was developed for the CenttifiChem@ 500 Analyzer. The sensitivity o f the assay was increased over that of previously published methods utilizing the peroxidase reaction, with a lower detectable Ilmlt of 0.0155 umol/L. A much larger volume of samples can be analyzed in a shorter period of time and a I00 ul aliquot of specimen is tequlred for analysis. The developed method has been used to determine the hemoglobin levels of gastric juice samples. Recovery studies from a subject, ingesting known amounts of his o~rn blood, showed an average recovery of 100.8% over a hemoglobin range of .048& umol/L to &.84 umol/L. Precision studies analyzing hemoglobin levels in saline gastric washes Showed a mean with-in run CV of 2.6% and a with-in day CV of 2.9%. Precision studies analyzing hemoglobin levels in mannitol gastric washes produced a mean with-in run CV of 3.8% and a mean with-ln day CV of 3.8%. The method was successfully applied to the measurement of aspirln-induced gastric bleeding. Solutions of EDTA Or aspirin exhibi~ no interference with the analysis.
q9 KINETIC ENZYb~TIC bIETHOD FOR THE MEASUREMENT OF LACTIC ACID. R. Vieth, Dept. of Biochemistry, Sunnybrook IIospital Medical Center, Toronto and the Department of Clinical Biochemistry, University of Toronto. The amount of lactate in biological samples can be measured based on the activity of a fixed quantity of lactate dehydrogenase (LD) in the reagent. The following method is easy to perform, fast and the reagent cost is low. The method requires a spectrophotometer able to measure timed absorbance changes at 340 nm to 3 decimal places at 30°C. I used a Gilford Stasar lit c/~nected to a Syva timer/printer unit as is often used for ~IIT~assays.
44
u s e OF M O N K E Y M U S C L E A C E T Y L C H O L I ~ I E R E C E P T O R S F O R T H E M E A S U R E M E N T OF A N T I B O D Y T I T E R S IN M Y A S T H E N I A GRAVIS
~ L - ~ U t Hickle, D.A.G., Humohrey, J.G., Srifflth,K. and Mee, A.V. D e o a r t m e n t of C l i n i c a l B i o c h e m i s t r y , T o r o n t o G e n e r a l H o s o i t a l , T o r o n t o , O n t a r i o , MbS IL7. A s s a y of h u m a n s k e l e t a l muscle aeetyleholine r e c e p t o r a n t i b o d i e s h o l d s n r o m i s e as a d i a R n o s t i c t e s t fo~ m y a s t h e n l a g r a v i s , a n d as a l a b o r a t o r y m e a s u r e of t h e ~ e s D o n s e to t h e r a p y . This method involves a d o u b l e immunoprecipitation a s s a y . The b ~ s i c o r [ n c i n l e is s u m m a r i z e d by the f o l l o w i n g e q u a t i o n s : I,
AChR
+ 1251 ~ BGT
2.
125I ~ 8 G T A C h P
3.
1251 ~ B G T A C h R - A b
) 1251~BGTAChP
(excess) + Ab 2
+ Ab
~ 1251~80TAChR-Ab
)1251~ BGTAChP-Ab-Ab21
W h e r e A C h R = a e e t y l c h o l i n e r e c e p t o r : 125 I ~ B G T = 1 - 1 2 5 l a b e l l e d alphabunBarotoxin. Ab = A n t i b o d i e s to A C h R p r e s e n c e in h u m a n s e r u m . Ab2 = S h e e p - a n t i - h u m a n IgG. The a s s a y r e q u i r e s p r e p a r a t i o n o f A C h R , c o u n l i n K r e c e p t o r s to 125I ~ B G T , bindinK with natienr se~um a n t i b o d i e s , a n d p r e c i p i t a t i o n by a n t ~ - h u m a n IgG a n t i bodies. The tire? o f a n t i b o d i e s is d i r e c t l y p r o p o r t i o n a l to the a c t i v i t y o f p ~ e c i o ~ t a t e (I~5 I ~ 8~T AChR-Ab-Ab2). Detailed methodology, normal ~anKe clinical s e n s i t i v i t y and s p e c i f i c i t y and s o m e c l i n i c a l r e s u l t s w i l l be p r e s e n t e d .
45 STABILITY OF IN-HOUSE REFERENCE ~ T E R I A L S IN ENZYME ASSAYS IN ROUTINE CLINICAL CHF~IISTRY. Chau,A.K.-K.,and Hoore~R.W.,Biochemlstry Oepartment,Sunnybrook Medical Centre,2075 Bayview Avenue,Toronto,Ontario H4N 3 ~ . The intention of this study was to devise an in,house reference material for use with the enzyme assays routinely available in Clinical Chemistry laboratories. The stability of enzyme activity in a normal human serum pool,abnormal human serum pools and a contrived animal enzyme mixture,with and without stabilizers,at different storage temperatures,was assessed over a period of six months. The enzyme activities followed were those of AST,ALT,ALP,CGT,LD and CK. Storage temperatures were 40 and -200. The contrived enzyme pool was the mixture suggested by Gruber and others in J.Clin. Chem. Clin.giochem. I_~5,565,575and 579 (1977). The only pools in which all the enzyme activities were statistically stable were,for the normal pooi,20% dimethylsulphoxide at -200 and 15% ethylene glycol at -20C,for the contrived pool, 20% dimethylsulphoxide at 40 and -200 and ethylene glycol at -200. None of the pools having high enzyme activity were stable. Similar lack of stability has been noted by ourselves and others in commercially available lyophilized and in liquid serum pools,particularly in those having high enzyme activity. Should high enzyme activity in the reference material be needed,the most promising source,from the evidence of this study,is the contrived enzyme pool in =he presence of dimethyl sulphoxide.
46
EVALUATION OF A SERUM ESTRADIOL KIT AND ITS APPLICATION TO THE MONITORING OF OVULATION INDUCTION BY FERTILITY DRUGS. Greenway, D.C. , Mak-Fan, L. and Turkington, V.E. Dept. of Lab Medicine, Ottawa General Hosi,itaL Ottawa, Ontario. KIH 8L6 We evaluated a radioiI~nunoassay kit for serLLm estradJo| (Nordiclab International, P.O. Box 1356, Huntington, P.Q.) with the intention of replacing 24 hr. urinary estrogen deterlBinations for the daily monitoring of infertile womenundergoing ovulation induction by hLunan menopausal gonadotropins (h~, Pergonal). The kit features serum-based standards rangina from 0.055-5.51 nmol/L, an 1251-tracer, short incubation time, and a polyethylene glycol (PEG) separation technique. Small voluraes (200 el) of st&ndaxds, controls and patients' sara are extracted with 1.5 ml diethyl ether, and a 1.0 ml aliquot of this extract is evaporated to dryness. The dried extract is resuspended in tracer-antibody solution and incubated at 37°C for 1 hour. PEG is added, and following centrifugation, the radioactivity in the bound fraction is counted. Total turnaround time is 3-4 hr. The intra-assay precision was 6.4~ 8.1
97 / P9
and 7.2% at levels of 0.233, 1.13 and 3.45 nmol/L respectively. Between-run precision was 11.6 and 9.8% at 0.244 and 2.21 nmol/L respectively. Mean recoveries from serum pools spiked with 0.367, 1.84 and 3.67 nmol/L estradiol were 101.9, IO2.1 and i06.2%. Linearity was observed when volumes of sara ranging from 50 to 700 ul were extracted and assayed. Sensitivity was found to be 0.050 nmol/L. We have found the Mordiclab method especially suitable for the monitoring of ovulation induction by hMG. Eleven women have been monitored so far over 16 treatment cycles, and three pregnancies have ensued. The availability of results within 4 hr. has provided the gynecologist with a more current indication of follicular development and has allowed for a better adjustment of hMG doses than was provided previously by 24 hr urlnary estrogen assays.
q7 MODEL SYSTEMFOR NEPHELOMETRIC ASSAY OF Clq PRECIPITABLE IMMUNECOMPLEXES (IC). Levinson, S.S., and Goldman J., Department of Laboratory Medicine, Sinai Hospital, Detroit, MI 48235. IC are involved in the pathogenesis of many rheumatic disseases; removal of IC by plasma exchange may improve prognosis (Cardella, C.I. (1979) J. Rheumatology 6, 606). Nephelometric assay would simplify IC measurement by eliminating the need f o r highly purified radioiodinated Clq and other components commonly used now that l i m i t performance to only a few centers. Heat aggregated gamma globu]in (IIAGG) has been shown to exh i b i t many properties that are indistinguishable from endogenous IC (Marder, R.J., st. al. (1978) J. Inmlunology 12__~_I, 613). In a double beam sPectrophotometer, the reaction of HAGGwith Clq occurs rapidly and reaches equilibrium within lO minutes. Maximum reaction occurs between sodium concentrations of 150-160 ,~.I/L in the reaction mixture regardless of whether the sodium is in the form of chloride, phosphate or EDTA. Standard curves generated from HAGGshow a high degree of l i n e a r i t y between 12-750 ug/ml. The a c t i v i t y of the reaction plateaus between Clq concentrations of 50-lOO ug/ml, simplifying c r i t e r i a needed to ensure good reproducibility between different lots. Purified or more crude preparations of Clq can be used interchangeably. HAGG is added to serum and extracted with 2.5% polyethylene glycol. Absorbance of the resuspended precipitate is s u f f i ciently high so that HAGGcan be assayed down to 12 ug/m! We assayed sera of several patients known to have high levels of [C (positive) and normal pooled sera. All cases of positive sera exhibited absorbances s u f f i c i e n t l y high to differentiate them from the pooled sera with lower absorbances. Simple recovery of the precipitate from the reaction mixture a f t e r nephelometric assay may lend i t s e l f to investigation of the Ig composition by EIA. Such composition may be related to d i f f e r int features of the disease (Levinski, J.R., and Barratt, M.T. 1979) Lancet 24, 1100).
48
CENTRIFUGAL ASSAY FOR DETERMINING HEMOGLOBIN LEVELS IN GASTRIC JUICE. Stryd~R.P., Gilbertson,T.J., Reele,S.B. and Gumblston,T.J., Experimental Medleal and Clinical Laboratory Research and Bronson Clinical lnvestlgational Unit, The Upjohn Company, Kalamazoo, Michigan 49001 A method for the analysis of hemoglobin levels was developed for the CenttifiChem@ 500 Analyzer. The sensitivity o f the assay was increased over that of previously published methods utilizing the peroxidase reaction, with a lower detectable Ilmlt of 0.0155 umol/L. A much larger volume of samples can be analyzed in a shorter period of time and a I00 ul aliquot of specimen is tequlred for analysis. The developed method has been used to determine the hemoglobin levels of gastric juice samples. Recovery studies from a subject, ingesting known amounts of his o~rn blood, showed an average recovery of 100.8% over a hemoglobin range of .048& umol/L to &.84 umol/L. Precision studies analyzing hemoglobin levels in saline gastric washes Showed a mean with-in run CV of 2.6% and a with-in day CV of 2.9%. Precision studies analyzing hemoglobin levels in mannitol gastric washes produced a mean with-in run CV of 3.8% and a mean with-ln day CV of 3.8%. The method was successfully applied to the measurement of aspirln-induced gastric bleeding. Solutions of EDTA Or aspirin exhibi~ no interference with the analysis.
q9 KINETIC ENZYb~TIC bIETHOD FOR THE MEASUREMENT OF LACTIC ACID. R. Vieth, Dept. of Biochemistry, Sunnybrook IIospital Medical Center, Toronto and the Department of Clinical Biochemistry, University of Toronto. The amount of lactate in biological samples can be measured based on the activity of a fixed quantity of lactate dehydrogenase (LD) in the reagent. The following method is easy to perform, fast and the reagent cost is low. The method requires a spectrophotometer able to measure timed absorbance changes at 340 nm to 3 decimal places at 30°C. I used a Gilford Stasar lit c/~nected to a Syva timer/printer unit as is often used for ~IIT~assays.